JP7531824B2 - How to determine when an organism died - Google Patents

Description

The present invention relates to a method for determining the time of death of an organism from its corpse, based on the degree of fragmentation of nucleic acid derived from the corpse.
In particular, the present invention makes it possible to determine the time of contamination of food with dead organisms based on the degree of fragmentation of nucleic acids derived from the dead organisms.

Food is something that is taken into the body, so naturally its safety must be ensured. For that reason, in the quality control of food production, for example, any matter that could be the cause of contamination must be thoroughly eliminated.

To implement such thorough quality control, it is necessary to accurately grasp the actual state of foreign matter contamination, and for that purpose, it is important to first verify when the foreign matter was introduced into the food, whether it was introduced during the manufacturing process or after the product was opened.

For this reason, methods have been developed and investigated for determining when foreign objects such as hair or plastic were mixed in with food (Non-Patent Documents 1 and 2), and methods have been reported that analyze the degree of penetration of inorganic compounds (e.g., sodium chloride) and organic substances (e.g., glucose) in food components into foreign objects that have been mixed in with food, and then use the results to determine when the foreign object was mixed in (Patent Documents 1 and 2).

Meanwhile, Patent Document 3 describes how, under adjusted polymerase chain reaction (hereinafter referred to as "PCR") conditions, multiple primer pairs are used to generate multiple amplification products from a template nucleic acid that has been subjected to degradation, and different production efficiencies of each of the multiple amplification products are determined, and based on these production efficiencies, the degree of damage to the template nucleic acid due to degradation, the degree of food processing, and the amount of template nucleic acid before degradation are determined. However, Patent Document 3 does not disclose anything about determining the time when a foreign object was mixed into food.

JP 2005-83804 A JP 2017-146282 A JP 2015-35977 A

"Method of Identifying Contaminated Hair" by Gen Sato, Science Forum Co., Ltd., 2000 Convertec, November 2002 issue, published by Kako Gijutsu Kenkyukai Publishing Co., Ltd., "DSC analysis of CPP film reveals history of retort heat treatment by utilizing phase transition from smectic to α crystals" (Ajinomoto Co., Inc. Production Technology Development Center)

Traditionally, the time of contamination has been determined based on the degree of penetration of food dyes or ingredients into the foreign matter (such as the penetration status and amount of penetration) through microscopic observation, or the presence or absence of enzyme activity in the foreign matter.

However, if the contaminating foreign matter is small, it may not be possible to observe or measure the degree of penetration of food dyes or ingredients. In addition, as the microorganisms grow, the activity of enzymes derived from the microorganisms increases, making it impossible to determine whether or not there is enzyme activity derived from the foreign matter, which can make it difficult to determine when the foreign matter was introduced.

In this field, there was a strong need to develop a new method that would enable the determination of when foreign matter was introduced without relying on the degree of penetration of food dyes or ingredients into the foreign matter, or the presence or absence of enzyme activity in the foreign matter.

The inventors have found that it is possible to evaluate the degree of fragmentation of nucleic acids derived from the remains of an organism, and to determine the time of death of the organism based on the degree of fragmentation. In addition, when the remains of an organism have been mixed into food as a foreign body, it has been found that it is possible to determine the time when the organism or its remains were mixed into the food, by similarly evaluating the degree of fragmentation of nucleic acids derived from the remains of the organism.

The present invention is based on these findings and includes the following inventions.
[1] A method for determining the time of death of an organism from its carcass, comprising:
assessing the degree of fragmentation of nucleic acids from the remains of the organism, and determining the time of death of the organism based on the degree of fragmentation.
[2] The method according to [1], wherein a smaller degree of fragmentation of the nucleic acid indicates that the organism has recently died.
[3] The method of [1], wherein the corpse of the organism has been present in food, and the method further comprises a step of determining the time when the organism was present in the food based on the degree of fragmentation of the nucleic acid.
[4] The method according to [3], further comprising evaluating whether or not the organism has been subjected to nucleic acid degradation during a food production process, based on the degree of nucleic acid fragmentation.
[5] The method according to [3] or [4], wherein the decomposition action is a heat treatment in the manufacturing process of a food.
[6] The method according to [5], wherein the heat treatment is carried out under conditions of 100° C. or higher.
[7] The method according to any one of [1] to [6], wherein the organism is an insect.
[8] The method according to any one of [1] to [7], wherein the degree of fragmentation of the nucleic acid is evaluated by electrophoresis.
[9] Any of the methods according to [1] to [7], wherein the degree of fragmentation of the nucleic acid is evaluated based on the production efficiency of the at least two types of amplification products, each having a different sequence length, by PCR using at least two different primer pairs under polymerase chain reaction conditions, using nucleic acid derived from the corpse of the organism as a template nucleic acid.
[10] The degree of fragmentation of the nucleic acid is evaluated based on the production efficiency of each of the at least two types of amplification products, and the evaluation is calculated according to the following formula:
ΔCt (sample) = Ct _{target A (sample)} - Ct _{target B (sample)} ... formula (4)
[Indicating the value obtained by subtracting the Ct value of target B (Ct _{target B (sample)} ) from the Ct value of target A (Ct _{target A (sample)} ) among the at least two types of amplification products]
The method of [9], which is carried out based on the ΔCt value obtained by
[11] The method of [10], wherein target A produces a long amplification product and target B produces a short amplification product, or wherein target A produces a short amplification product and target B produces a long amplification product.
[12] The method of [10], wherein target A is designed on genomic DNA and target B is designed on mitochondrial DNA, or target A is designed on mitochondrial DNA and target B is designed on genomic DNA.
[13] The degree of fragmentation of the nucleic acid is evaluated based on the production efficiency of each of the at least two types of amplification products, and the evaluation is calculated according to the following formula:
ΔΔCt=ΔCt(sample)−ΔCt(NFC) Equation (1)
[wherein ΔCt(sample) is expressed by the following formula:
ΔCt(sample)=Ct _{long chain(sample)} −Ct _{short chain(sample)} Formula (2)
and represents a value obtained by subtracting the Ct value of the short chain amplification product (Ct _{short chain (sample)} ) from the Ct value of the long chain amplification product (Ct _{long chain (sample)} ) of the at least two types of amplification products,
ΔCt(NFC) is calculated using the following formula:
ΔCt(NFC)= _{Ctlong chain(NFC)} −Ctshort _chain(NFC) ...Equation (3)
and indicates the value obtained by subtracting the Ct value of the short amplification product (Ct short ((NFC) )) from the Ct value of the long amplification product (Ct _{long (NFC) )} ) of at least two types of amplification products generated from unfragmented DNA ( _{NFC: No} Fragmentation Control) corresponding to the template nucleic acid under the same conditions as the polymerase chain reaction.
The method of [9], which is carried out based on the ΔΔCt value obtained by
[14] The method according to [13], wherein the template nucleic acid comprises at least a portion of a nucleic acid encoding 18S rRNA or mitochondrial DNA.
[15] The method according to any one of [9] to [14], wherein the organism is a Smoky-breasted cockroach, and the plurality of different primer pairs include primers having the following sequence or a mutant thereof:
Forward primer 5'-ACTAGTCGCATCCGGTATCCTC-3' (SEQ ID NO:1);
Short strand amplification reverse primer 5'-CTCAATCTCGTGCGGCTAGA-3' (SEQ ID NO:2); and long strand amplification reverse primer 5'-AAGGGCAGGGACGTAATCAA-3' (SEQ ID NO:3).
[16] The method according to any one of [9] to [14], wherein the organism is a German cockroach, and the plurality of different primer pairs include primers having the following sequence or a mutant thereof:
Forward primer 5'-TAGTCGCATCCGGCATCCTT-3' (SEQ ID NO: 4);
Short strand amplification reverse primer 5'-CTCAATCTCGTGCGGCTAGG-3' (SEQ ID NO:5); and long strand amplification reverse primer 5'-AAGGGCAGGGACGTAATCAC-3' (SEQ ID NO:6).
[17] The method according to any one of [9] to [14], wherein the organism is a Smoky-breasted cockroach, and the plurality of different primer pairs include primers having the following sequence or a mutant thereof:
Forward primer 5'-GAACATCATTGAGAATATTAATTCGTGCT-3' (SEQ ID NO: 9);
Short strand amplification reverse primer 5'-GAAAGCATGTGCAGTTACAATCAC-3' (SEQ ID NO: 10); and long strand amplification reverse primer 5'-ATGGTGGGTATAACTGTTCAACCTGTA-3' (SEQ ID NO: 11).
[18] The method according to any one of [9] to [14], further comprising the step of identifying the species of the organism based on the presence or absence of an amplification product generated from the template nucleic acid.
[19] A primer pair for 18S rDNA of the cockroach, comprising a primer having the following sequence or a mutant thereof, for use in any one of the methods of [9] to [14]:
Forward primer 5′-ACTAGTCGCATCCGGTATCCTC-3′ (SEQ ID NO:1);
The short amplification reverse primer was 5'-CTCAATCTCGTGCGGCTAGA-3' (SEQ ID NO:2); and the long amplification reverse primer was 5'-AAGGGCAGGGACGTAATCAA-3' (SEQ ID NO:3).
[20] A primer pair for 18S rDNA of the German cockroach, for use in any one of the methods [9] to [14], comprising primers having the following sequence or a mutant thereof:
Forward primer 5′-TAGTCGCATCCGGCATCCTT-3′ (SEQ ID NO:4);
The short amplification reverse primer was 5'-CTCAATCTCGTGCGGCTAGG-3' (SEQ ID NO:5); and the long amplification reverse primer was 5'-AAGGGCAGGGACGTAATCAC-3' (SEQ ID NO:6).
[21] A primer pair for mitochondrial DNA of the Smoky Brown cockroach, for use in any one of the methods [9] to [14], comprising a primer having the following sequence or a mutant thereof:
Forward primer 5′-GAACATCATTGAGAATATTAATTCGTGCT-3′ (SEQ ID NO:9);
The short amplification reverse primer was 5'-GAAAGCATGTGCAGTTACAATCAC-3' (SEQ ID NO: 10); and the long amplification reverse primer was 5'-ATGGTGGGTATAACTGTTCAACCTGTA-3' (SEQ ID NO: 11).

The present invention provides a new method for determining the time of death of an organism, as well as a new method for determining the time of contamination of food with the remains of an organism that has been present as a foreign body.

1 shows a graph of the amplification curves of the short and long amplification products obtained by real-time PCR using DNA extracted from the Smoky Brown cockroach as a template, as well as DFI and ΔΔCt values. (A) shows the results of unheated samples, (B) of samples heated at 110°C for 30 minutes, (C) of samples heated at 121°C for 8 minutes, and (D) of samples heated at 121°C for 30 minutes. 2 shows a graph of the amplification curves of the short and long amplification products obtained by real-time PCR using DNA extracted from the German cockroach as a template, as well as DFI and ΔΔCt values. (A) shows the results of not heating, (B) of heating at 110°C for 30 minutes, (C) of heating at 121°C for 8 minutes, and (D) of heating at 121°C for 30 minutes. 3 shows a graph of the amplification curves of the short and long amplification products obtained by real-time PCR using DNA extracted from the Smoky Brown cockroach as a template, as well as DFI and ΔΔCt values. (A) shows the results of not heating, (B) of heating at 70°C for 30 minutes, (C) of heating at 80°C for 30 minutes, (D) of heating at 90°C for 30 minutes, and (E) of heating at 95°C for 30 minutes. 4 shows a graph of the amplification curves of the short and long amplification products obtained by real-time PCR using DNA extracted from the German cockroach as a template, as well as DFI and ΔΔCt values. (A) shows the results of not heating, (B) of heating at 70°C for 30 minutes, (C) of heating at 80°C for 30 minutes, (D) of heating at 90°C for 30 minutes, and (E) of heating at 95°C for 30 minutes. 5 is a graph showing the amplification curves of the short and long amplification products obtained by real-time PCR using DNA extracted from the Smoky Brown cockroach or the German cockroach as a template, and shows the DFI and ΔΔCt values. (A) shows the results for the Smoky Brown cockroach, unheated, (B) shows the results for the Smoky Brown cockroach, heated in a microwave (500 W, 2 min), (a) shows the results for the German cockroach, unheated, and (b) shows the results for the German cockroach, heated in a microwave (500 W, 2 min). 6 is a graph showing the amplification curves of the short and long amplification products obtained by real-time PCR using DNA extracted from the Smoky Brown cockroach or the German cockroach as a template, and shows the DFI and ΔΔCt values. (A) shows the results of the Smoky Brown cockroach, unheated, (B) shows the results of the Smoky Brown cockroach, heated in curry sauce at 121° C. for 8 minutes, (C) shows the results of the Smoky Brown cockroach, heated in curry sauce at 121° C. for 30 minutes, (a) shows the results of the German cockroach, unheated, (b) shows the results of the German cockroach, heated in curry sauce at 121° C. for 8 minutes, and (c) shows the results of the German cockroach, heated in curry sauce at 121° C. for 30 minutes. 7 is a photograph showing the results of heat treatment evaluation by electrophoresis using PCR with only one primer pair. Each lane shows the following heat treatment samples: 1: 95°C for 30 minutes, 2: 110°C for 30 minutes, 3: 121°C for 8 minutes, 4: 121°C for 30 minutes, 5: negative control, M: marker. 8 is a photograph showing the results of PCR using only one primer pair with different amounts of template DNA, showing the case where only the primer pair for short chain amplification was used (left) and the case where only the primer pair for long chain amplification was used (right), and each lane shows a sample with the following amount of template DNA: 1: 25 ng, 2: 2.5 ng, 3: 0.25 ng, 4: 0.025 ng, 5: negative control, M: marker. 9 shows a graph showing the amplification curves of the short and long amplification products obtained by real-time PCR using DNA extracted from a cockroach heated at 121° C. for 8 minutes as a template, as well as DFI and ΔΔCt values. (A) shows the results for 25 ng of template DNA, (B) for 2.5 ng of template DNA, (C) for 0.25 ng of template DNA, and (D) for 0.025 ng of template DNA. 10 shows a graph showing the amplification curves of the short and long amplification products obtained by real-time PCR using DNA extracted from untreated cockroach (hind shank) as a template, and the ΔΔCt and ΔCt values. (A) shows the results when the 18S rDNA primer set was used, and (B) shows the results when the mitochondrial DNA primer set was used. 11 shows a graph showing the amplification curves and ΔΔCt and ΔCt values of short and long amplification products obtained by real-time PCR using DNA extracted from the cockroach (hind shank) heat-treated at 121° C. for 20 minutes as a template. (A) shows the results when the 18S rDNA primer set was used, and (B) shows the results when the mitochondrial DNA primer set was used. 12 is a graph showing the amplification curves and ΔΔCt and ΔCt values of short and long amplification products obtained by real-time PCR using DNA extracted from the smoky brown cockroach (hind shank) stored in curry at 25° C. for 3 days as a template. (A) shows the results when the 18S rDNA primer set was used, and (B) shows the results when the mitochondrial DNA primer set was used. Figure 13-1 shows a graph showing the amplification curves and ΔΔCt values for short and long amplification products obtained by real-time PCR using DNA extracted from (A) an untreated Smoky Brown cockroach corpse, and (B) a Smoky Brown cockroach corpse stored at 25°C for 1 day, (C) a Smoky Brown cockroach corpse stored at 25°C for 2 days, and (D) a Smoky Brown cockroach corpse stored at 25°C for 3 days as templates. Figure 13-2 shows a graph showing the amplification curves and ΔΔCt values for the short and long amplification products obtained by real-time PCR using DNA extracted from the corpses of Smoky Brown cockroaches stored at 25°C for (E) 4 days, (F) 5 days, (G) 6 days, (H) 7 days, and (I) 10 days at 25°C. 14 shows a photograph of the results of electrophoresis of DNA extracted from (lane 1) an untreated Smoky Brown cockroach corpse, (lane 2) a Smoky Brown cockroach corpse stored at 25° C. for 7 days, and (lane 3) a Smoky Brown cockroach corpse stored in curry at 25° C. for 3 days. (lane M) 200 bp DNA Ladder. FIG. 15 shows a graph illustrating the amplification curves and ΔΔCt values for short and long amplification products obtained by real-time PCR using DNA extracted from (A) untreated mealworm larvae corpses and (B) mealworm larvae corpses heat-treated at 121° C. for 20 minutes as templates. 16 is a photograph showing the results of electrophoresis of DNA extracted from (lane 1) untreated mealworm larvae corpses and (lane 2) mealworm larvae corpses heat-treated at 121° C. for 20 minutes. (lane M) 1 kb DNA Ladder. FIG. 17 shows a graph illustrating the amplification curves and ΔΔCt values for short and long amplification products obtained by real-time PCR using DNA extracted from (A) untreated mealworm larvae corpses, (B) mealworm larvae stored at 25° C. for 2 days, and (C) mealworm larvae stored at 25° C. for 5 days as templates. 18 shows a photograph of electrophoresis of DNA extracted from (lane 1) untreated mealworm larvae corpses, (lane 2) mealworm larvae stored for 2 days at 25° C., and (lane 3) mealworm larvae stored for 5 days at 25° C. (lane M) 1 kb DNA Ladder. 19 shows a photographic image showing the results of electrophoresis of DNA extracted from (lane 1) an untreated German cockroach corpse, (lane 2) a corpse heat-treated at 80° C. for 30 minutes, and (lane 3) a corpse heat-treated at 100° C. for 30 minutes. (Lane M) 1 kb DNA Ladder. Figure 20 shows a graph showing the amplification curves and ΔΔCt values of the short and long amplification products obtained by real-time PCR using DNA extracted from (A) an untreated German cockroach corpse, (B) a corpse heat-treated at 80°C for 30 minutes, and (C) a corpse heat-treated at 100°C for 30 minutes as templates.

In the present invention, "organism carcasses" or "organism carcasses" include any substance derived from an organism that has nucleic acid, such as, but not limited to, animal or plant carcasses or parts thereof. "Nucleic acid" includes single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA, DNA-RNA hybrids, and the like. More specifically, "organism carcasses" or "organism carcasses" in the present invention include, but are not limited to, human hair such as hair or animal hair, nails, pieces of wood, stems, roots, or stalks of plant materials, bone fragments of animal materials, insects, or parts thereof. Preferably, in the present invention, the "organism's corpse" or "organism's corpse" refers to an insect's corpse or part of an insect, and examples of insects include adults and larvae of cockroaches (Periplaneta fuliginosa), Blattella germanica, and mealworms (Neatus picipes).

In the present invention, "food" refers to dishes served in restaurants and the like, and various processed foods that can be distributed frozen, chilled, at room temperature, etc. Examples of processed foods include retort products such as curry, stew, soup, and sauce, roux products such as curry and stew, frozen foods, various spice products such as wasabi paste, mustard paste, and mustard, seasoning products such as mayonnaise and dressings, dairy products such as yogurt, butter, cheese, and ice cream, dessert products such as jelly and pudding, confectionery products such as chocolate and cookies, and beverage products such as tea, coffee, fruit drinks, and soft drinks, but are not limited to these.

The present invention relates to a method for determining the time of death of an organism from its corpse, and includes a step of evaluating the degree of fragmentation of nucleic acids derived from the organism's corpse and determining the time of death of the organism based on the degree of fragmentation.

"Nucleic acids derived from the remains of an organism" can be isolated/purified from the remains of an organism using any nucleic acid extraction method. Examples of nucleic acid extraction methods that can be used include the phenol/chloroform method, cell lysis using a surfactant or a protease enzyme, physical destruction using glass beads, repeated freezing and thawing, and combinations of these. In addition, commercially available nucleic acid extraction kits (e.g., DNeasy Blood & Tissue Kit, DNeasy Plant mini Kit, Genomic-tip 20/G, DNeasy Mericon Food Kit (all from QIAGEN), etc.) can also be used.

In the present invention, "fragmentation of nucleic acid" means that nucleic acid is broken down or decomposed by decomposition. Examples of "decomposition" include digestive reactions by digestive enzymes possessed by living organisms, digestive reactions by microorganisms living in the environment where the remains of living organisms are placed, decomposition by heating, decomposition by acid, decomposition by alkali, decomposition by physical force, etc. In particular, when the remains of living organisms have been mixed into food, the "decomposition" refers to decomposition by heating during the manufacturing process of the food, for example, decomposition by heating at 65°C or higher, preferably 70°C or higher, more preferably 80°C or higher, even more preferably 90°C or higher, particularly preferably 100°C or higher, particularly preferably 120°C or higher (e.g., 121°C), or even higher. More specifically, examples of decomposition by heating include retort sterilization (121°C, 4 minutes or more). Preferably, these heating temperatures are moist heat temperatures. If the degree of fragmentation of the nucleic acid extracted from the remains of an organism is small, it means that the nucleic acid has not been significantly affected by decomposition, and if the degree of fragmentation of the nucleic acid is large, it means that the nucleic acid has been affected by decomposition.

In one embodiment of the present invention, the "degree of nucleic acid fragmentation" can be evaluated by electrophoresis of nucleic acid extracted from the corpse and nucleic acid (unfragmented control) extracted in the same manner from an untreated organism that is the same or considered to be the same as the corpse and has just died. If the two nucleic acids show the same or nearly the same electrophoretic patterns as a result of electrophoresis, it can be determined that the organism of the corpse is highly likely to have died recently. On the other hand, if the nucleic acid extracted from the corpse is detected at a smaller size than the unfragmented control, or is not detected at all, it indicates that the nucleic acid has been decomposed, and that the organism is highly likely not to have died recently. In particular, if the organism's corpse was mixed into food, it indicates that the organism's corpse was highly likely to have been decomposed by heating during the food manufacturing process, which indicates that the organism died at least before the heat treatment during the manufacturing process, i.e., that the organism or its corpse was highly likely to have been mixed into the food during the food manufacturing process. The degree of nucleic acid fragmentation increases the longer the organism has been dead and the higher the temperature of the heat treatment.

In another embodiment of the present invention, the "degree of nucleic acid fragmentation" can be evaluated by using nucleic acid derived from the remains of an organism as a template nucleic acid, performing PCR using at least two different primer pairs to generate at least two types of amplification products with different sequence lengths, and evaluating the degree of fragmentation of the template nucleic acid based on the generation efficiency of the at least two types of amplification products.

In the present invention, a nucleic acid having any sequence can be used as the "template nucleic acid", but preferably the template nucleic acid contains a nucleic acid encoding ribosomal RNA (rRNA) or a part thereof, or consists of the nucleic acid. Nucleic acids encoding rRNA have a large number of copies per genomic DNA, and can be relatively easily detected from fragmented template nucleic acids. More preferably, the template nucleic acid contains a nucleic acid encoding one or more selected from the group consisting of 18SrRNA, ITS1, 5.8SrRNA, ITS2, 28SrRNA, and parts thereof, or consists of the nucleic acid. Even more preferably, the template nucleic acid contains a nucleic acid encoding one or more selected from the group consisting of 18SrRNA, ITS1, and parts thereof, or consists of the nucleic acid. Nucleic acids encoding 18SrRNA and ITS1 are highly conserved within a species, so that primers specific to a particular species can be designed relatively easily, and they can also be used to identify the species of a corpse. Nucleic acids encoding rRNA derived from various species are known, and their genetic information is registered in known databases such as GenBank. For example, nucleic acids encoding rRNA derived from the Smoky Brown cockroach are registered in GenBank as AF321250, DQ874171, etc., and nucleic acids encoding rRNA derived from the German cockroach are registered in GenBank as DQ874116, FJ806322, AF005243, etc. These genetic information can be used in the present invention.

In the present invention, the "template nucleic acid" includes or consists of mitochondrial DNA or a part thereof. Mitochondrial DNA is less susceptible to decomposition, such as digestion by enzymes possessed by an organism in the corpse or digestion by microorganisms living in the environment in which the organism is placed, compared to the above-mentioned nucleic acid encoding rRNA (e.g., DNA encoding 18SrRNA (hereinafter referred to as "18SrDNA")), and is therefore preferred because it allows for more accurate determination of the time of death of the organism. Mitochondrial DNA derived from various organisms is known, and its genetic information is registered in known databases such as GenBank. For example, mitochondrial DNA derived from the smoky brown cockroach is registered in GenBank as MH184372, etc. This genetic information can be used in the present invention.

In the present invention, "at least two different primer pairs" refers to two or more primer pairs designed on a template nucleic acid of the same gene so that the sequence lengths of the amplified products are different from each other. At least two different primer pairs may each have a different primer, but for example, one primer (e.g., a forward primer) may be common and the other primer (e.g., a reverse primer) may be different from each other. The sequence length of the amplified product obtained from each primer pair is not particularly limited and can be appropriately selected from the range of approximately 100 bp or more, approximately 200 bp or more, approximately 300 bp or more, approximately 400 bp or more, approximately 500 bp or more, approximately 600 bp or more, approximately 700 bp or more, approximately 800 bp or more, or approximately 900 bp or more. For example, multiple different primer pairs can be prepared, including a first primer pair that produces a short amplification product of approximately 100 bp and a second primer pair that produces a long amplification product of approximately 200 bp, approximately 300 bp, approximately 400 bp, or approximately 500 bp.

Alternatively, in the present invention, "at least two different primer pairs" means two or more primer pairs each designed on a template nucleic acid of a different gene. The sequence length of the amplification product obtained from each primer pair is not particularly limited, and can be appropriately selected from the range of about 100 bp or more, about 200 bp or more, about 300 bp or more, about 400 bp or more, about 500 bp or more, about 600 bp or more, about 700 bp or more, about 800 bp or more, or about 900 bp or more. The sequence length of the amplification product obtained from each primer pair may be the same or different. For example, the multiple different primer pairs can be a primer pair targeting a part of genomic DNA, preferably a primer pair targeting DNA encoding rRNA, more preferably a primer pair targeting 18SrDNA, and a primer pair targeting mitochondrial DNA. In addition, a combination of primer pairs targeting different regions of genomic DNA can be prepared as the multiple different primer pairs. Additionally, multiple different primer pairs can be prepared, each of which targets a different region of mitochondrial DNA.

At least two different primer pairs can be designed by known methods based on the genetic information of the template nucleic acid. They may also be designed using known primer design software (e.g., OLIGO Primer Analysis Software (Molecular Biology Insights), Beacon Designer (PREMIER Biosoft), Primer Express Software (Thermo Fisher Scientific, Inc.), etc.).

At least two different types of primer pairs that can be used in the present invention include primers that contain or consist of the following sequences, which are designed based on the genetic information of the template nucleic acid derived from the Smoky Brown cockroach or German cockroach.

(Primer pair for 18S rDNA of the cockroach)
Forward primer (common)
5'-ACTAGTCGCATCCGGTATCCTC-3' (SEQ ID NO:1);
Short strand amplification reverse primer 5'-CTCAATCTCGTGCGGCTAGA-3' (SEQ ID NO:2); and long strand amplification reverse primer 5'-AAGGGCAGGGACGTAATCAA-3' (SEQ ID NO:3).

(Primer pair for German cockroach 18S rDNA)
Forward primer (common)
5'-TAGTCGCATCCGGCATCCTT-3' (SEQ ID NO:4);
Short strand amplification reverse primer 5'-CTCAATCTCGTGCGGCTAGG-3' (SEQ ID NO:5); and long strand amplification reverse primer 5'-AAGGGCAGGGACGTAATCAC-3' (SEQ ID NO:6).

(Primer pair for mitochondrial DNA of the cockroach)
Forward primer (common)
5'-GAACATCATTGAGAATATTAATTCGTGCT-3' (SEQ ID NO:9);
Short strand amplification reverse primer 5'-GAAAGCATGTGCAGTTACAATCAC-3' (SEQ ID NO: 10); and long strand amplification reverse primer 5'-ATGGTGGGTATAACTGTTCAACCTGTA-3' (SEQ ID NO: 11).

In addition, in the present invention, oligonucleotides that have a base sequence that binds (hybridizes) under stringent conditions to a base sequence complementary to the base sequences shown in SEQ ID NOs: 1 to 11 and similarly function as primers can also be used as the primers. Here, "stringent conditions" means, for example, hybridization (e.g., about 3.0 x SSC or 2.0 x SSC, 30°C or 37°C) based on the Tm value calculated by the following formula, followed by washing under conditions that are more stringent than the hybridization conditions (e.g., about 2.0 x SSC, 30°C, 37°C, 40°C, 44°C or 48°C or higher, or 1.0 x SSC or 0.5 x SSC, 37°C or higher). It is well known in the art to select "stringent conditions" appropriate for hybridization and washing depending on the base sequence to be hybridized, etc.

Tm = 81.5 + 16.6 (log ₁₀ [Na ⁺ ]) + 0.41 (fraction G + C) - (600/N)
[Na ⁺ ]: Molar concentration of Na ⁺ (mol/L)
Fraction G+C: The percentage of G and C in the oligonucleotide
N: length of oligonucleotide (number of bases)

In this specification, such primers may be referred to as "mutants" of the primers having the base sequences shown in SEQ ID NOs: 1 to 11, and unless otherwise specified, the primers having the base sequences shown in SEQ ID NOs: 1 to 11 also include such "mutants." Such mutants may include oligonucleotides consisting of a base sequence having an addition, substitution, deletion or insertion of several bases in the base sequences shown in SEQ ID NOs: 1 to 11 (here, "several bases" means a number of bases of 4 bases or less, 3 bases or less, or 2 bases or less), and oligonucleotides consisting of a base sequence having 80% or more, more preferably 90% or more, and most preferably 95% or more identity to the base sequences shown in SEQ ID NOs: 1 to 11 when calculated using BLAST or the like (for example, default, i.e., initial setting parameters).

"PCR" may be any method that allows detection and quantification of amplified products, and is not particularly limited, but real-time PCR, digital PCR, etc. can be used.

In the present invention, the "degree of fragmentation of the template nucleic acid" can be evaluated based on the production efficiency of the at least two types of amplification products. When the PCR is real-time PCR, the "production efficiency of the amplification product" can be expressed, for example, by a threshold cycle value (i.e., Ct value) indicating the number of PCR cycles when a predetermined amount of amplification product is produced. The Ct value is selected from the exponentially amplified region in the amplification curve.

In addition, when the PCR is digital PCR, the "production efficiency of amplification products" can be expressed as the probability of generating an amplification product when PCR is performed multiple times, or the number of times an amplification product is generated when PCR is performed a specified number of times. More specifically, the production efficiency of amplification products in digital PCR can be expressed as the number of wells in which an amplification product is observed when a specified PCR is performed in multiple wells on a PCR plate (hereinafter also referred to as the "number of positive wells"), or the copy number calculated by analyzing the number or percentage of positive wells by fitting them to a Poisson distribution model.

PCR can include a probe for detecting the amplified product. The probe is not particularly limited as long as it can bind (hybridize) to the target sequence in the amplified product under stringent conditions. The probe may be labeled with a fluorescent substance (e.g., FAM ^™ , TET ^™ , VIC ^™ , HEX ^™ , NED ^™ , PET, etc.) and/or a quencher (e.g., TAMRA, ROX, etc.).

In one embodiment, the degree of fragmentation of the template nucleic acid can be evaluated based on the efficiency of producing at least two types of amplification products with different sequence lengths. When the template nucleic acid is degraded, partially fragmented nucleic acids are mixed in the template nucleic acid. Usually, nucleic acids with shorter sequence lengths are less susceptible to degradation than those with longer sequences, and tend to have a higher probability of remaining unfragmented. Therefore, for the regions amplified by the at least two different primer pairs, those with shorter sequence lengths are less susceptible to degradation than those with longer sequences, and those with longer sequence lengths are more susceptible to degradation and tend to have a lower probability of remaining unfragmented.

For this reason, even if PCR is performed under conditions that allow multiple amplification products of different sequence lengths to be produced with substantially the same production efficiency from a template nucleic acid that has not been degraded, if the template nucleic acid has been degraded, the production efficiency of amplification products replicated from long sequence regions that are susceptible to degradation will be lower than that of amplification products replicated from short sequence regions that are less susceptible to degradation, and differences in the production efficiency of each amplification product may occur depending on whether or not the template nucleic acid has been degraded.

Specifically, when the template nucleic acid is degraded, in real-time PCR, the Ct value of the long amplification product is larger than the Ct value of the short amplification product. In addition, in digital PCR, the number of wells in which the long amplification product is confirmed or the number of copies is reduced compared to the number of wells in which the short amplification product is confirmed or the number of copies.

The degree of fragmentation of the template nucleic acid can be evaluated based on the presence or absence of differences in the production efficiency of these multiple amplification products.

The fragmentation of the template nucleic acid was evaluated according to the following formula:
ΔΔCt=ΔCt(sample)−ΔCt(NFC) Equation (1)
This can be done based on the following:

In the formula, ΔCt(NFC) is represented by the following formula:
ΔCt(NFC)= _{Ctlong chain(NFC)} −Ctshort _chain(NFC) ...Equation (3)
It is represented by the formula: At least two types of amplification products with different sequence lengths are generated from unfragmented DNA (NFC: No Fragmentation Control) corresponding to the template nucleic acid by PCR using at least two different primer pairs, and two types of amplification products selected from the obtained amplification products are shown as the Ct value (Ct long chain _{(NFC) ) of the relatively long amplification product (long} amplification product) minus the Ct value (Ct _{short chain (NFC)} ) of the relatively short amplification product (short amplification product). "Unfragmented DNA corresponding to the template nucleic acid" means any form of control DNA containing a PCR target sequence that has not been subjected to any of the above-mentioned degradation actions, and DNA extracted from an organism that is the same as or is thought to be the same as the aforementioned organism that has not been subjected to degradation actions, or plasmid DNA containing a PCR target sequence can be used. For example, if the organism is a cockroach, the DNA of a cockroach immediately after death may be used as the "unfragmented DNA corresponding to the template nucleic acid", or a plasmid DNA containing a PCR target sequence in the cockroach DNA may be used as the "unfragmented DNA corresponding to the template nucleic acid".

In the formula, ΔCt (sample) is represented by the following formula:
ΔCt(sample)=Ct _{long chain(sample)} −Ct _{short chain(sample)} Formula (2)
At least two types of amplification products, each having a different sequence length, are generated from the template nucleic acid by PCR using at least two different primer pairs, and two types of amplification products are selected from the obtained amplification products, and the Ct value is expressed as the value obtained by subtracting the Ct value (Ct _{short (sample))} of a relatively short amplification product (short amplification product) from the Ct value (Ct _{long (sample} )) of a relatively long amplification product (long amplification product).

If the obtained ΔΔCt value does not exceed the standard value, it indicates that the template nucleic acid has not been decomposed or has been decomposed only slightly, and that it is highly likely that the organism has died recently. On the other hand, if the obtained ΔΔCt value exceeds the standard value, it indicates that the template nucleic acid has been decomposed, and that it is highly likely that the organism has not died recently. In particular, if the remains of an organism have been contaminated in food, if the ΔΔCt value exceeds the standard value, it indicates that it is highly likely that the remains of the organism have been decomposed by heating during the food manufacturing process.

The ΔΔCt value that is the "reference value" can be preferably set by the following method. After subjecting an organism that is the same as or is thought to be the same as the organism and that has not been subjected to any of the above-mentioned decomposition actions to a predetermined decomposition action treatment (e.g., storage for a predetermined period after death, heat treatment under the same conditions as in the manufacturing process, etc.), the nucleic acid extracted from the corpse of each organism is used as a template nucleic acid to obtain the ΔΔCt value as described above. A specific ΔΔCt value obtained from the corpse of an organism that has been subjected to this predetermined decomposition action treatment can be used as the "reference value". For example, if the ΔΔCt value obtained for the corpse of an organism that has been stored for three days after death is 1.0, if the ΔΔCt value obtained for the corpse of the organism that is the sample is equal to or less than the reference value of 1.0, it can be determined that the organism is likely to have been dead for less than three days, while if it exceeds the reference value of 1.0, it indicates that the organism is likely to have been dead for four days or more. Furthermore, if the ΔΔCt value obtained for the remains of an organism that has been heat-treated at a temperature exceeding 100°C is 2.0, and if the ΔΔCt value obtained for the remains of the organism as a specimen is equal to or greater than the reference value of 2.0, it indicates that the organism or its remains are highly likely to have been subjected to the heat treatment. The reference value may be set in advance, or may be set each time a specimen is analyzed.

Alternatively, the fragmentation of the template nucleic acid can be evaluated according to the following formula:
ΔCt (sample) = Ct _{target A (sample)} - Ct _{target B (sample)} ... formula (4)
This can be done based on the following:

In one embodiment, "target A" and "target B" in formula (4) can be "long chain" and "short chain", respectively, and each Ct value can be as defined above. As above, if the obtained ΔCt value does not exceed the reference value, it indicates that the organism is likely to have died recently. On the other hand, if the obtained ΔCt value exceeds the reference value, it indicates that the organism is likely to have not died recently, and in particular, if the organism's remains were mixed into food, it indicates that the organism's remains are likely to have been decomposed by heating during the food manufacturing process. Note that the absolute value of ΔCt (sample) does not change even if target A and target B are interchanged, so the result can be used to make an equivalent evaluation.

The ΔCt value that serves as the "reference value" can be determined in the same manner as the ΔΔCt reference value described above.

In another embodiment, "target A" and "target B" in formula (4) can be different gene targets, with "target A" referring to a target nucleic acid that is more susceptible to degradation than target B, and "target B" referring to a target nucleic acid that is less susceptible to degradation than target A. As described in detail in the Examples below, the effect of degradation varies depending on the target nucleic acid, and even when extracted from the remains of the same organism, the degree of fragmentation varies for each target nucleic acid.

When amplification products of a target nucleic acid are generated from a template nucleic acid that has not been degraded and a template nucleic acid that has been degraded, if the template nucleic acid has been degraded, the efficiency of generating amplification products from the target nucleic acid that is susceptible to degradation decreases. On the other hand, the efficiency of generating amplification products replicated from the target nucleic acid that is not susceptible to degradation does not change significantly even when the template nucleic acid has been degraded. This results in a difference in ΔCt value between cases where the template nucleic acid has been degraded and cases where it has not been degraded.

For example, mitochondrial DNA is less susceptible to degradation than genomic DNA such as 18SrDNA, and tends to remain unfragmented. Therefore, mitochondrial DNA corresponds to "target B". On the other hand, 18SrDNA corresponds to "target A". In real-time PCR, the Ct value of the 18SrDNA amplification product when the template nucleic acid is degraded is larger than the Ct value of the 18SrDNA amplification product when the template nucleic acid is degraded. Therefore, the ΔCt value, which is the value obtained by subtracting the Ct value of the mitochondrial DNA amplification product (Ct _{target B (sample) ) from the Ct value of 18SrDNA (Ct target} A (sample)), differs depending on whether the template nucleic acid is degraded or not. Note that the absolute value of ΔCt (sample) does not change even if target A and target B are interchanged, so the same evaluation can be performed using the result.

The degree of fragmentation of the template nucleic acid can be evaluated based on the presence or absence of differences in the production efficiency of these amplification products. As above, if the obtained ΔCt value does not exceed the standard value, it indicates that it is highly likely that the organism has died recently. On the other hand, if the obtained ΔCt value exceeds the standard value, it indicates that it is highly likely that the organism has not died recently, and in particular, if the organism's remains were contaminated in food, it indicates that it is highly likely that the organism's remains have been subjected to decomposition by heating during the food manufacturing process.

The ΔCt value that serves as the "reference value" can be determined in the same manner as the ΔΔCt reference value described above.

Furthermore, in the present invention, the species of the organism corpse can be identified based on the presence or absence of an amplification product generated from a template nucleic acid derived from the organism corpse.
In the present invention, a plurality of different primer pairs, preferably a primer pair for 18SrDNA, are designed based on the template nucleic acid of a specific species of organism, and therefore, when an amplification product is generated using any of the primer pairs, preferably a plurality of primer pairs, it can be determined that the corpse of the organism is the specific species of organism or a part thereof. For example, if the generation of an amplification product is observed when using the primer pair for the Smoky Brown cockroach, the corpse of the organism is determined to be a Smoky Brown cockroach or a part thereof, if the generation of an amplification product is observed when using the primer pair for the German cockroach, the corpse of the organism is determined to be a German cockroach or a part thereof, and further, if the generation of an amplification product is not observed when using either the primer pair for the Smoky Brown cockroach or the primer pair for the German cockroach, it can be determined that the corpse of the organism is not a Smoky Brown cockroach or a German cockroach, but is another insect.
The present invention will now be described in more detail with reference to examples.

(Preparation of template DNA solution)
<For Smoky Brown Cockroaches>
DNA encoding undegraded 18S rRNA of the cockroach (Accession No. DQ874171) was introduced into the plasmid DNA. The resulting recombinant plasmid DNA was cleaved with the restriction enzyme NdeI to prepare template DNA solutions containing linear recombinant plasmid DNA into which the undegraded 18S rRNA gene was inserted at ¹⁰⁵ copies/μL and ¹⁰² copies/μL, respectively.

<For German cockroaches>
DNA encoding undegraded 18S rRNA of the German cockroach (Accession No. DQ874116 or FJ806322) was introduced into the plasmid DNA. The resulting recombinant plasmid DNA was cleaved with the restriction enzyme NdeI to prepare template DNA solutions containing linear recombinant plasmid DNA into which the undegraded 18S rRNA gene had been inserted at ¹⁰ copies/μL and ¹⁰ copies/μL, respectively.

(Real-time PCR conditions)
Two primer pairs for short chain amplification and long chain amplification, as shown in Table 1, and a fluorescently labeled nucleic acid probe were prepared for the Smoky Brown cockroach and the German cockroach, respectively.

<For 18S rDNA of cockroaches>
The two primer pairs share a common forward primer (SEQ ID NO: 1). The primer pair for short chain amplification has a reverse primer represented by SEQ ID NO: 2, and the primer pair for long chain amplification has a reverse primer represented by SEQ ID NO: 3.

<For German cockroach 18S rDNA>
The two primer pairs share a common forward primer (SEQ ID NO: 4). The primer pair for short chain amplification has a reverse primer represented by SEQ ID NO: 5, and the primer pair for long chain amplification has a reverse primer represented by SEQ ID NO: 6.

For both the Smoky Brown cockroach and the German cockroach, the primer pair for short chain amplification produces an amplification product with a sequence length of approximately 100 bp from the template DNA. The primer pair for long chain amplification produces an amplification product with a sequence length of approximately 300 bp from the template DNA. Each fluorescently labeled probe was synthesized by modifying the 5' end with FAM and the 3' end with TAMRA.

The reaction solution used had the following composition. The amount of each component in the table is expressed in μL.

Real-time PCR was performed using a 7900HT Fast Real Time PCR System (Applied Biosystems) under the following conditions: 95°C for 10 minutes, followed by 45 cycles of 95°C for 30 seconds, 58°C for 10 seconds, and 72°C for 2 minutes.

(Confirmation of primer set specificity)
A reaction solution was prepared by adding 2.5 μL each of DNA derived from the Smoky Brown cockroach and DNA derived from the German cockroach instead of the template DNA plasmid solution of the PCR reaction solution prepared above. Real-time PCR was performed using the PCR conditions adjusted above.

As a result, when a primer set for the Smoky Brown cockroach was used, only DNA from the Smoky Brown cockroach was amplified, and the amplified product appeared on the graph as an amplification curve, whereas DNA from the German cockroach was not amplified, and no amplification curve appeared.

Similarly, when a primer set for the German cockroach was used, only DNA from the German cockroach was amplified, and the amplification product appeared on the graph as an amplification curve, while DNA from the Smoky Brown cockroach was not amplified, and no amplification curve appeared.

Therefore, it was confirmed that the primer set for the Smoky Brown cockroach does not cross-react with DNA from the German cockroach, and that the primer set for the German cockroach does not cross-react with DNA from the Smoky Brown cockroach, confirming the specificity of each primer set.

(Confirmation of DNA fragmentation by heating at 100° C. or higher)
DNA that was not decomposed by heating was extracted from unheated Smoky Brown cockroaches using a Blood & Tissue kit (QUIAGEN). In addition, Smoky Brown cockroaches were completely placed in water in a retort pouch and sealed, and subjected to pressurized heating treatment (Autoclave HG-50LB (Hirayama Seisakusho)) at 110°C for 30 minutes, 121°C for 8 minutes, and 121°C for 30 minutes. After heating, the Smoky Brown cockroaches were removed and water was removed. DNA was extracted from the heat-treated Smoky Brown cockroaches using a Blood & Tissue kit. DNA extraction was also performed on the unheated and heat-treated German cockroaches in the same manner. Thereafter, real-time PCR was performed using the above PCR conditions, except that the extracted DNA was used as the template DNA. From the obtained results, the ΔΔCt value at an arbitrarily set threshold of 0.256 was calculated. In addition, the obtained ΔΔCt value is calculated using the following formula:
DFI=1-(1/2) ^ΔΔCt/2
The DNA fragmentation index (DFI) was calculated based on the above. When the DFI value was 0 or less, it was corrected to DFI = 0. All cockroaches used in the experiment were frozen and stored.

As a result, the calculated ΔΔCt values and DFI values for the Smoky Brown cockroach were ΔΔCt value 0.7, DFI value 0.217 (Figure 1(A)); ΔΔCt value 3.2, DFI value 0.665 (Figure 1(B)); ΔΔCt value 2.7, DFI value 0.606 (Figure 1(C)); ΔΔCt value 8.9, DFI value 0.955 (Figure 1(D)) for unheated, 110°C 30 min; 121°C 8 min; and 121°C 30 min, respectively.

For the German cockroach, the ΔΔCt and DFI values calculated at an arbitrarily set threshold of 0.256 were as follows: unheated, 110°C for 30 min; 121°C for 8 min; and 121°C for 30 min: ΔΔCt value 1.4, DFI value 0.379 (Fig. 2(A)); ΔΔCt value 6.6, DFI value 0.881 (Fig. 2(B)); ΔΔCt value 5.0, DFI value 0.821 (Fig. 2(C)); ΔΔCt value > 17.0, DFI value > 0.996 (Fig. 2(D)).

It was confirmed that both the ΔΔCt value and the DFI value increased as a result of being subjected to a heat treatment at 100°C or higher. For the Smoky Brown cockroach, it was confirmed that the ΔΔCt value exceeded 0.7 and the DFI value exceeded 0.2 when subjected to a heat treatment. For the German cockroach, it was confirmed that the ΔΔCt value exceeded 1.4 and the DFI value exceeded 0.3 when subjected to a heat treatment. It was also confirmed that the higher the heating temperature, the larger the ΔΔCt value. Furthermore, it was confirmed that even at the same heating temperature, the longer the heating time, the larger the ΔΔCt value. In other words, it was revealed that the degree of heating during retort cooking can be estimated based on the ΔΔCt value and/or the DFI value.

(Confirmation of DNA fragmentation by heating at less than 100° C.)
Next, the extent of DNA fragmentation caused by heating at less than 100° C. was confirmed.
A 50 mL centrifuge tube was filled completely with water and heated at 70°C, 80°C, 90°C, and 95°C for 30 minutes each. After the heat treatment, the cockroach was removed from the water and DNA was extracted using a Blood & Tissue Kit. The German cockroach was also heat-treated in the same manner and DNA was extracted.

Real-time PCR was performed using the above PCR conditions, except that the extracted DNA was used as template DNA.

As a result, for the Smoky Brown cockroach, when unheated, the ΔΔCt value was 0.7 and the DFI value was 0.227, and when heated at 70°C, 80°C, 90°C, and 95°C for 30 minutes, the ΔΔCt value was 0.4, DFI value was 0.137, ΔΔCt value was 0.3, DFI value was 0.084, ΔΔCt value was 0.5, DFI value was 0.158, ΔΔCt value was 0.7, and DFI value was 0.201, respectively, which was not significantly different from the values for unheated samples, and no significant DNA fragmentation was confirmed (Figure 3). For the German cockroach, when not heated and heated at 70°C, 80°C, 90°C, and 95°C for 30 minutes, the ΔΔCt value was 0.2, the DFI value was 0.054, the ΔΔCt value was 0.4, the DFI value was 0.127, the ΔΔCt value was 0.1, the DFI value was 0.05, the ΔΔCt value was 0.5, the DFI value was 0.152, the ΔΔCt value was 0.8, and the DFI value was 0.237, respectively, with no significant differences in the values and no significant DNA fragmentation being confirmed (Figure 4).

From this, it is possible to use the ΔΔCt value and/or the DFI value as indicators to determine whether the DNA of a sample has been subjected to high-temperature heating such as retort processing, or only to relatively low-temperature heating. In other words, this method makes it possible to determine the heating history of the contaminated cockroach, and to determine whether the cockroach has been heated during the manufacturing process (retort sterilization processing) or only cooked at home (bath water).

(Confirmation of DNA fragmentation due to microwave heating)
Next, the extent of DNA fragmentation caused by microwave heating was confirmed.
The Smoky Brown cockroach was placed in a microwave-safe pouch so that it was immersed in water, sealed, and heated for 2 minutes at 500 W. After the heat treatment, it was removed from the water, and DNA was extracted using a Blood & Tissue Kit. The German cockroach was also heat-treated in the same manner, and DNA was extracted.

Real-time PCR was performed using the PCR conditions adjusted above, except that the extracted DNA was used as template DNA.

As a result, when heated at 500 W for 2 minutes, no significant difference was observed in the Ct values of the two amplification curves for the short and long amplification products, and no significant DNA fragmentation was confirmed (Figure 5).

Therefore, using the Ct values of the two amplification curves for the short and long amplification products and/or DNA fragmentation as indicators, it is possible to determine whether the DNA in a sample has been subjected to high-temperature heating such as retort processing, or only to microwave heating. In other words, this method makes it possible to determine the heating history of the contaminated cockroach, and to determine whether the cockroach has been heated during the manufacturing process (retort sterilization processing) or only cooked at home (microwave cooking).

(Estimation of when Smoky Brown Cockroaches and German Cockroaches were introduced into food)
DNA that was not decomposed by heating was extracted from unheated Smoky Brown cockroaches using a Blood & Tissue kit (QUIAGEN). In addition, Smoky Brown cockroaches were placed in commercially available retort curry sauce, sealed, and subjected to pressurized heating treatment at 121°C for 8 minutes and 121°C for 30 minutes. After heating, the Smoky Brown cockroaches were removed and the curry sauce components attached thereto were removed with sterilized water. DNA was extracted from the heat-treated Smoky Brown cockroaches using a Blood & Tissue kit. DNA was similarly extracted from unheated and heat-treated German cockroaches. Thereafter, real-time PCR was performed using the above PCR conditions, except that the extracted DNA was used as the template DNA.

As a result, as shown in Figure 6, when the Smoky Brown cockroach was not heated, the ΔΔCt value was 0.5 and the DFI value was 0.146 (Figure 6 (A)). On the other hand, when the Smoky Brown cockroach was heated, the ΔΔCt value was 2.1 and the DFI value was 0.516 after heating at 121°C for 8 minutes, and the ΔΔCt value was 10.9 and the DFI value was 0.977 after heating at 121°C for 30 minutes, both values being larger than when the Smoky Brown cockroach was not heated (Figures 6 (B) and (C)). Furthermore, it was confirmed that the ΔΔCt value and DFI value increased with increasing heating time.

Similarly, in the German cockroach, the ΔΔCt value was 0.6 and the DFI value was 0.197 when not heated (Fig. 6(a)), whereas when heated at 121°C for 8 minutes the ΔΔCt value was 6.3 and the DFI value was 0.889, and when heated at 121°C for 30 minutes the ΔΔCt value was >20.0 and the DFI value was >0.999, both values being larger (Fig. 6(b) and (c)). Furthermore, it was confirmed that the ΔΔCt and DFI values increased with increasing heating time.

Therefore, by extracting DNA from an unknown sample, performing real-time PCR, and obtaining the ΔΔCt value and/or DFI value, it is possible to determine whether the unknown sample has undergone a heating process equivalent to retort sterilization, and to what extent that heating took place.

(Determination of heat treatment using electrophoresis)
Next, we investigated a method for determining the presence or absence of heat treatment by electrophoresis using PCR with only one primer pair. Generally, template DNA is fragmented by heat treatment, so according to this method, if no PCR product is confirmed by electrophoresis, it is determined that the sample has been subjected to heat treatment.
The Smoky Brown cockroach was placed in a 50 mL centrifuge tube so that it was completely immersed in water, and heated at 95°C for 30 minutes. The Smoky Brown cockroach was placed in a retort pouch so that it was completely immersed in water, which was then sealed, and subjected to pressurized heat treatment (autoclave HG-50LB (Hirayama Seisakusho)) at 110°C for 30 minutes, 121°C for 8 minutes, and 121°C for 30 minutes. After heating, the Smoky Brown cockroach was removed and the moisture was removed. DNA was extracted from the Smoky Brown cockroach that had been subjected to the heat treatment using a Blood & Tissue kit. Then, PCR (27 cycles) was performed using only the primer pair for long chain amplification, using the extracted DNA as template DNA. After the reaction, the amplified product was confirmed by electrophoresis.

The results are shown in Figure 7. Bands were detected in samples heated at 95°C for 30 minutes, 110°C for 30 minutes, and 121°C for 8 minutes, but not in samples heated at 121°C for 30 minutes. If an attempt is made to determine the presence or absence of heat treatment based solely on the presence or absence of an amplified product, it would be impossible to distinguish between samples heated at 95°C for 30 minutes, 110°C for 30 minutes, and 121°C for 8 minutes, and there is a risk of erroneously determining that no heat treatment was performed. On the other hand, as described above, according to the present invention, it is possible to determine not only the presence or absence of heat treatment, but also the degree of heating, and therefore it is possible to distinguish and determine samples heated at 95°C for 30 minutes, 110°C for 30 minutes, and 121°C for 8 minutes.

(Effect of difference in amount of template DNA on results)
Next, the influence of differences in the amount of template DNA on the results in the method of determining the presence or absence of heat treatment using electrophoresis was confirmed.
The Smoky Brown cockroach was placed completely in water in a retort pouch, sealed, and subjected to a pressurized heat treatment at 121° C. for 8 minutes. After the heat treatment, it was removed from the water, and DNA was extracted using a Blood & Tissue Kit.

The extracted DNA was added to the reaction system in amounts of 25 ng, 2.5 ng, 0.25 ng, or 0.025 ng, and PCR (27 cycles) was performed using only the primer pair for short chain amplification or only the primer pair for long chain amplification. After the reaction, the amplified product was confirmed by electrophoresis. After the reaction, the amplified product was confirmed by electrophoresis.

The results are shown in Figure 8. In both cases, when only the primer pair for short chain amplification was used (left) and when only the primer pair for long chain amplification was used (right), bands were observed when 25 ng and 2.5 ng of DNA were used, but bands were not observed when 0.25 ng and 0.025 ng were used. When attempting to determine the presence or absence of heat treatment based solely on the presence or absence of an amplified product, if no band is detected, it is difficult to determine whether this is due to DNA fragmentation caused by heat treatment or whether a sufficient amount of DNA was not extracted from the foreign matter.

Next, according to the method of the present invention, the extracted DNA was added to the reaction system in amounts of 25 ng, 2.5 ng, 0.25 ng, or 0.025 ng, and real-time PCR was performed using the PCR conditions described above, using a primer pair for short chain amplification and a primer pair for long chain amplification.

The results are shown in Figure 9. When 25 ng of template DNA was used, the ΔΔCt value was 3.4 and the DFI value was 0.697; when 2.5 ng was used, the ΔΔCt value was 2.9 and the DFI value was 0.633; when 0.25 ng was used, the ΔΔCt value was 3.1 and the DFI value was 0.653; and when 0.025 ng was used, the ΔΔCt value was 3.3 and the DFI value was 0.659.

This confirmed that the ΔΔCt and DFI values obtained showed equivalent results even if the amount of template DNA was different. In general, foreign objects found in food are likely to be small and only a small part of the food. Therefore, there is no guarantee that a sufficient amount of DNA will be obtained, and it is difficult to adjust the DNA concentration to a constant concentration and subject it to PCR. On the other hand, according to the present invention, it is possible to subject the template DNA to PCR without adjusting the amount, and it is possible to determine whether or not it has been heat-treated based on the ΔΔCt and DFI values.

(Identification of Foreign Objects)
The primer sets used in real-time PCR can specifically amplify the DNA of the Smoky Brown cockroach and the German cockroach, respectively, so if it is possible to confirm which primer set was used to amplify the DNA, it is possible to determine what kind of sample was extracted. If an insect suspected to be a cockroach is found in food, it is possible to estimate the time of contamination and at the same time identify the insect species.

Until now, estimation of the time of contamination and identification of the insect species had to be carried out in separate processes, but with the introduction of this method, both results can be obtained in a single real-time PCR run.

(Fragmentation measurement using mitochondrial DNA primer set)
Primers and probes specific to the Smoky Brown cockroach were similarly designed on mitochondrial DNA (mtDNA) (Accession No. MH184372, etc.), which is a region different from 18S rDNA, and DNA fragmentation was measured.

(Adjustment of real-time PCR conditions)
Two mtDNA primer pairs and a fluorescently labeled nucleic acid probe shown in Table 3 were prepared for the Smoky Brown cockroach.
<For Smoky Brown Cockroaches>
The two primer pairs have a common forward primer of SEQ ID NO: 9. The first primer pair has a reverse primer of SEQ ID NO: 10, and the second primer pair has a reverse primer of SEQ ID NO: 11.
The first primer pair produces an amplification product with a sequence length of about 100 bp from the template DNA. The second primer pair produces an amplification product with a sequence length of about 300 bp from the template DNA. The fluorescently labeled probe of SEQ ID NO: 12 was synthesized by modifying the 5' end with FAM and the 3' end with TAMRA.

The reaction solution used had the following composition. The amount of each component in the table is expressed in μL.

Real-time PCR was performed under the same conditions used to amplify 18S rDNA above.

(Comparison of 18S rDNA and mitochondrial DNA primer sets)
DNA was extracted from the hind shank of a Smoky Brown cockroach (untreated Smoky Brown cockroach) that had been rapidly frozen alive and was considered to be not highly fragmented, using a Blood & Tissue kit.
On the other hand, a Smoky Brown cockroach was placed in a retort pouch so that it was completely immersed in the curry, which was then sealed, and subjected to a pressurized heating treatment at 121°C for 20 minutes (Autoclave HG-50LB (Hirayama Seisakusho)). After heating, the Smoky Brown cockroach was taken out, the curry components were removed with sterilized water, and DNA was extracted from the hind shank in the same manner.
In addition, retort curry was placed in a 50 mL centrifuge tube so that the cockroach was completely immersed, and the tube was placed in a 25°C thermostatic chamber (LU-112T, manufactured by ESPEC) for 3 days. After 3 days of storage, the cockroach was taken out, the curry components were removed with sterilized water, and DNA was extracted from the hind shank in the same manner.

Using the three types of DNA extracts prepared as described above, real-time PCR was carried out using the 18SrDNA primer set and the mtDNA primer set. Note that with the mtDNA primer set, real-time PCR was carried out without preparing the PCR reaction solution so that the production efficiency was the same, so the value was calculated as ΔCt.

Figure 10 shows the results of fragmentation of DNA extracted from untreated Smoky Brown cockroaches. The ΔΔCt measured when the 18SrDNA primer set was used was 0.1, confirming that the extracted DNA was hardly fragmented at all. On the other hand, the ΔCt measured when the same extract was used with the mtDNA primer set was 1.1. Since the reaction solution for the mtDNA primer set was not prepared to achieve the same production efficiency, this value was used as the standard value for a state in which fragmentation was not advanced, and the following comparison was made.

Next, the results of DNA fragmentation extracted from cockroaches that had been heat-treated at 121°C for 20 minutes are shown in Figure 11. In both cases, when the 18SrDNA primer set and the mtDNA primer set were used, differences were observed between the long and short amplification products, and the results exceeded the respective standard values mentioned above, confirming DNA fragmentation.

Furthermore, Figure 12 shows the results of fragmentation of DNA extracted from DNA of Smoky Brown cockroaches left in curry at 25°C for three days. When the 18SrDNA primer set was used, the results exceeded the standard value, confirming that fragmentation had progressed, whereas when the mtDNA primer set was used, the ΔCt value was shown to be the same as that of DNA derived from untreated Smoky Brown cockroaches, confirming that fragmentation had hardly progressed at all. As can be seen from the results of the mtDNA primer set, it was confirmed that the degree of fragmentation can be evaluated from the ΔCt value alone, without analyzing NFC and calculating the ΔΔCt value.

Results using the 18SrDNA primer set showed that DNA fragmentation was relatively advanced in cockroaches left in curry at 25°C for three days, but this is thought to be due to DNA fragmentation caused by the effects of the insect's own enzymes and microorganisms. On the other hand, no DNA fragmentation was observed when the mtDNA primer set was used, making it clear that primers targeting mtDNA can minimize the effects of enzymes and microorganisms. This is thought to be because the extent of the effects of the insect's own enzymes and microorganisms differs between the 18SrDNA present in the genomic DNA and the mitochondrial DNA.

From the results of Figures 10 and 12, the ΔCt values of the long chain amplification products of the 18SrDNA primer set and the mtDNA primer set were calculated. In this way, the time of death of the insect can also be estimated by comparing the Ct values of the PCR designed on the genomic DNA with the Ct values of the PCR designed on the mitochondrial DNA.

(Determining the time of death of Smoky Brown Cockroaches)
The Smoky Brown cockroach was obtained from a commercially available test insect, and the one that died from freezing was used in the experiment. A part of the untreated Smoky Brown cockroach was crushed with Bio Masher II (manufactured by Nippi). DNA was extracted from the crushed insect body using Blood & Tissue Kit (manufactured by QUIAGEN). Next, one Smoky Brown cockroach was placed in a 50 mL centrifuge tube, lightly covered, and placed in a thermostatic chamber (LU-112T, manufactured by ESPEC) at 25 ° C., and was taken out after storing for 1, 2, 3, 4, 5, 6, 7, and 10 days. A part of the Smoky Brown cockroach stored at 25 ° C. for a certain period of time was also crushed with Bio Masher, and DNA was extracted using Blood & Tissue Kit. Then, the extracted DNA was used as a template DNA to perform real-time PCR.

Real-time PCR was performed using a 7900HT Fast Real Time PCR System (Applied Biosystems) under the following conditions: 10 minutes at 95°C, followed by 45 cycles of 30 seconds at 95°C, 10 seconds at 58°C, and 2 minutes at 72°C. From the results, the ΔΔCt value was calculated at an arbitrarily set threshold of 0.256.

The results are shown in Figures 13-1 and 13-2. When Smoky Brown Cockroaches were stored at 25°C for a certain period of time after death, the ΔΔCt value tended to increase over time compared to untreated specimens. From these results, it can be determined that for Smoky Brown Cockroaches, if the ΔΔCt value is 1.0 or less, it has been within 3 days since death.

(Determination of time of death by electrophoresis)
The Smoky Brown cockroach was obtained from a commercially available test insect, and was used in the experiment after it died from freezing. A part was cut from the Smoky Brown cockroach immediately after death, crushed with Bio Masher II, and DNA was extracted with Blood & Tissue Kit. The Smoky Brown cockroach was also stored at 25°C for 7 days, and DNA was extracted in the same manner. Furthermore, the Smoky Brown cockroach was placed in a 50 mL centrifuge tube, and curry was added so that the entire Smoky Brown cockroach was immersed, and the tube was also stored at 25°C for 3 days. After storage, the Smoky Brown cockroach was taken out and the curry sauce components attached thereto were removed with sterilized water. DNA was extracted from the Smoky Brown cockroach stored at 25°C for a certain period of time using Blood & Tissue Kit.

1% agarose was dissolved and ethidium bromide (BIORAD) was added to 0.5 μg/mL to prepare an agarose gel for electrophoresis. DNA from a Smoky Brown cockroach immediately after death, DNA from a Smoky Brown cockroach stored at 25°C for 7 days, and DNA from a Smoky Brown cockroach stored in curry at 25°C for 3 days were electrophoresed. Electrophoresis was performed at 100 V using Mupid-2 (Mupid). A 200 bp DNA ladder (TAKARA) was used as a molecular weight marker. After electrophoresis, analysis was performed using a FluorImager 595 (GE Healthcare), and a large difference was found in the electrophoretic images of DNA from the sample immediately after death and DNA from the sample stored at 25°C for a certain period after the insect's death (Figure 14). In this way, by extracting DNA from an unknown sample and performing electrophoresis, it is possible to determine whether a person died immediately after death or if a certain period of time has passed since death.

(Determining the heat treatment of mealworm larvae)
Mealworm larvae were obtained from commercially available pet food, and those that died from freezing were used in the experiment. Unheated mealworm larvae were ground with BioMasher II (manufactured by Nippi Co., Ltd.). Nucleic acid was extracted from the grounded bodies using ISOHAIR (manufactured by Nippon Gene Co., Ltd.). At this time, RNase treatment was not performed, and the nucleic acid sample may contain not only DNA but also RNA. Next, the mealworm larvae were placed in water, sealed, and subjected to a heat treatment at 121°C for 20 minutes. After heating, the bodies were removed and the moisture was removed. The bodies were ground with BioMasher II, and nucleic acid was extracted with ISOHAIR. At this time, RNase treatment was not performed, and the nucleic acid sample may contain not only DNA but also RNA.

Real-time PCR was performed using a 7900HT Fast Real Time PCR System (Applied Biosystems). The thermal cycle consisted of 10 minutes at 95°C, followed by 45 cycles of 30 seconds at 95°C, 10 seconds at 58°C, and 2 minutes at 72°C. Primers and probes used were E100 Primer & Probe Mix (amplification length approximately 100 base pairs) and E350 Primer & Probe Mix (amplification length approximately 350 base pairs) included in the FRED Assay Kit for Eukaryotic DNA (manufactured by Nippon Gene).

The results are shown in Figure 15. When mealworm larvae were not heated, the short chain amplification curve by E100 Primer & Probe Mix and the long chain amplification curve by E350 Primer & Probe Mix had substantially the same Ct value at an arbitrarily set threshold of 0.256 (ΔΔCt value = 1.5). On the other hand, when mealworm larvae were heated, the efficiency of generating long chain amplification products decreased, and the difference in Ct value with the short chain amplification product at the set threshold of 0.256 increased (ΔΔCt value = 13.5). From these results, it was confirmed that it is possible to determine whether an unknown sample has undergone a heating process equivalent to retort sterilization by extracting nucleic acid from an unknown sample, performing real-time PCR, and comparing the amplification curves of the short chain amplification products and the long chain amplification products.

1% agarose was dissolved in Tris-acetate EDTA Buffer (Nippon Gene Co., Ltd.) and ethidium bromide (Nippon Gene Co., Ltd.) was added to a concentration of 0.5 μg/mL to prepare an agarose gel for electrophoresis. Nucleic acids derived from unheated mealworm larvae samples and those heated at 121°C for 20 minutes were electrophoresed. Electrophoresis was performed at 100V for approximately 30 minutes using a Mupid-2 plus (Mupid Co., Ltd.). 1 kb DNA ladder (New England Biolabs) was used as a molecular weight marker. After electrophoresis, the electrophoretic images were analyzed using a UV imaging device Printgraph (ATTO Co., Ltd.), and a large difference was found between the electrophoretic images of the unheated sample and the heated sample (Figure 16). In this way, by extracting nucleic acids from an unknown sample and performing electrophoresis, it is possible to determine whether the sample has undergone a heating process similar to that of retort sterilization.

(Determining the time of death of mealworm larvae)
The bodies of mealworm larvae that had died from freezing were ground with a BioMasher II (manufactured by Nippi Co., Ltd.). Nucleic acid was extracted from the ground bodies using an ISOHAIR (manufactured by Nippon Gene Co., Ltd.). At this time, RNase treatment was not performed, and the nucleic acid sample may contain not only DNA but also RNA. Furthermore, mealworm larvae were placed in plastic tubes, sealed, and left at 25°C for 2 days or 5 days. After treatment, the mealworm larvae were removed and ground with a BioMasher II, and nucleic acid was extracted with an ISOHAIR. At this time, RNase treatment was not performed, and the nucleic acid sample may contain not only DNA but also RNA.

Real-time PCR was performed using a 7900HT Fast Real Time PCR System. The thermal cycle consisted of 10 minutes at 95°C, followed by 45 cycles of 30 seconds at 95°C, 10 seconds at 58°C, and 2 minutes at 72°C. Primers and probes used were E100 Primer & Probe Mix (amplification length approximately 100 base pairs) and E350 Primer & Probe Mix (amplification length approximately 350 base pairs) included in the FRED Assay Kit for Eukaryotic DNA.

The results are shown in Figure 17. When mealworm larvae died immediately after their death, the short chain amplification curves using E100 Primer & Probe Mix and the long chain amplification curves using E350 Primer & Probe Mix had substantially the same Ct value at an arbitrarily set threshold of 0.256 (ΔΔCt value = 1.5). On the other hand, when mealworm larvae were heated, the efficiency of producing long chain amplification products decreased, and the difference in Ct value with the short chain amplification product at the set threshold of 0.256 increased (ΔΔCt value = 4.1 (after 2 days), 4.8 (after 5 days)). In this way, the time of death of the sample can be evaluated by extracting nucleic acid from an unknown sample, performing real-time PCR, and comparing the amplification curves of the short chain amplification products and the long chain amplification products.

1% agarose was dissolved in Tris-acetate EDTA Buffer (Nippon Gene Co., Ltd.) and ethidium bromide (Nippon Gene Co., Ltd.) was added to a concentration of 0.5 μg/mL to prepare an agarose gel for electrophoresis. Nucleic acids from mealworm larvae samples immediately after death, samples two days after death, and samples five days after death were analyzed by electrophoresis. Electrophoresis was performed at 100 V for approximately 30 minutes using a Mupid-2 plus (Mupid Co., Ltd.). 1 kb DNA ladder (New England Biolabs) was used as a molecular weight marker. After electrophoresis, the electrophoretic images were analyzed using a UV imaging device, Printgraph (manufactured by ATTO Corporation), and a large difference was found in the electrophoretic images of the unheated sample and the sample where the post-death period had been long (Figure 18). In this way, the time of death of a sample can be evaluated by extracting nucleic acid from an unknown sample and performing electrophoresis.

(Electrophoretic analysis of nucleic acids fragmented by heating at or below 100° C.)
Unheated German cockroach bodies were ground with BioMasher II (manufactured by Nippi Co., Ltd.). Nucleic acid was extracted using ISOHAIR (manufactured by Nippon Gene Co., Ltd.). At this time, RNase treatment was not performed, and the nucleic acid sample may contain not only DNA but also RNA. In addition, German cockroaches were placed in water, sealed, and subjected to heat treatment at 80°C for 30 minutes or 100°C for 30 minutes. After heating, the German cockroach was removed and the water was removed. The heated insect bodies were ground with BioMasher II, and nucleic acid was extracted with ISOHAIR. At this time, RNase treatment was not performed, and the nucleic acid sample may contain not only DNA but also RNA.

1% agarose was dissolved in Tris-acetate EDTA Buffer (Nippon Gene Co., Ltd.) and ethidium bromide (Nippon Gene Co., Ltd.) was added to a concentration of 0.5 μg/mL to prepare an agarose gel for electrophoresis. Nucleic acids derived from unheated samples of the German cockroach, samples treated at 80°C, and samples treated at 100°C were analyzed by electrophoresis. Electrophoresis was performed at 100 V for approximately 30 minutes using a Mupid-2 plus (Mupid Co., Ltd.). A 1 kb DNA ladder (New England Biolabs Co., Ltd.) was used as a molecular weight marker. After electrophoresis, the electrophoretic images were analyzed using a UV imaging device Printgraph (ATTO Co., Ltd.), and significant differences were found in the electrophoretic images of each sample (Figure 18). In this way, the degree of heating of an unknown sample can also be evaluated by extracting nucleic acid from the sample and performing electrophoresis. With real-time PCR, it was difficult to evaluate nucleic acid fragmentation caused by heating below 95°C, but electrophoresis makes it possible to perform the evaluation.

Next, real-time PCR was performed using each nucleic acid sample with a 7900HT Fast Real Time PCR System. The thermal cycle consisted of holding at 95°C for 10 minutes, followed by 45 cycles of holding at 95°C for 30 seconds, holding at 58°C for 10 seconds, and holding at 72°C for 2 minutes. Primers and probes used were E100 Primer & Probe Mix (amplification length approximately 100 base pairs) and E350 Primer & Probe Mix (amplification length approximately 350 base pairs) included in the FRED Assay Kit for Eukaryotic DNA.

The results are shown in Figure 20. Compared with nucleic acids derived from unheated samples and samples treated at 80°C from the German cockroach, nucleic acids derived from samples treated at 100°C showed a large decrease in the efficiency of long-chain amplification products at an arbitrarily set threshold of 0.256, and a large difference in Ct value with short-chain amplification products (ΔΔCt value = -0.4, 0.6, and 2.0 for unheated samples, 80°C-treated samples, and 100°C-treated samples, respectively).

These results confirm that the fragmentation of nucleic acids caused by heating to 100°C can also be evaluated using electrophoresis and real-time PCR.

Claims (16)

A method for determining when a dead organism was mixed into food, comprising:
evaluating the degree of fragmentation of nucleic acid derived from the carcass of the organism, and evaluating, based on the degree of fragmentation, whether or not the organism was subjected to nucleic acid degradation during the food production process;
The degree of fragmentation of the nucleic acid is evaluated by generating at least two types of amplification products, each having a different sequence length, by PCR using at least two different primer pairs and using nucleic acid derived from the corpse of the organism as a template nucleic acid under polymerase chain reaction conditions, and comparing the value of the difference in production efficiency of the at least two types of amplification products with a reference value which is the value of the difference in production efficiency of at least two types of amplification products generated by the polymerase chain reaction using nucleic acid extracted from the corpse of an organism that is the same as or thought to be the same as the organism, which has been subjected to the decomposition action or has not been subjected to the decomposition action, as a template nucleic acid. The method according to claim 1, wherein the decomposition is a heat treatment during the manufacturing process of a food product. The method according to claim 2, wherein the heat treatment is carried out at 100°C or higher. The method according to any one of claims 1 to 3, wherein the organism is an insect. The degree of fragmentation of the nucleic acid is evaluated based on the production efficiency of each of the at least two types of amplification products, and the evaluation is calculated according to the following formula:
ΔCt (sample) = Ct _{target A (sample)} - Ct _{target B (sample)} ... formula (4)
[Indicating the value obtained by subtracting the Ct value of target B (Ct _{target B (sample)} ) from the Ct value of target A (Ct _{target A (sample)} ) among the at least two types of amplification products]
The method according to claim 1, wherein the method is carried out based on the ΔCt value obtained by The method of claim 5, wherein target A produces long amplification products and target B produces short amplification products, or target A produces short amplification products and target B produces long amplification products. The method according to claim 5, wherein target A is designed on genomic DNA and target B is designed on mitochondrial DNA, or target A is designed on mitochondrial DNA and target B is designed on genomic DNA. The degree of fragmentation of the nucleic acid is evaluated based on the production efficiency of each of the at least two types of amplification products, and the evaluation is calculated according to the following formula:
ΔΔCt=ΔCt(sample)−ΔCt(NFC) Equation (1)
[wherein ΔCt(sample) is expressed by the following formula:
ΔCt(sample)=Ct _{long chain(sample)} −Ct _{short chain(sample)} Formula (2)
and represents a value obtained by subtracting the Ct value of the short chain amplification product (Ct _{short chain (sample)} ) from the Ct value of the long chain amplification product (Ct _{long chain (sample)} ) of the at least two types of amplification products,
ΔCt(NFC) is calculated using the following formula:
ΔCt(NFC)= _{Ctlong chain(NFC)} −Ctshort _chain(NFC) ...Equation (3)
and indicates the value obtained by subtracting the Ct value of the short amplification product (Ct short (NFC) ) from the Ct value of the long amplification product (Ct _{long (NFC)} ) of at least two types of amplification products generated under the same conditions as the polymerase chain reaction from unfragmented DNA ( _{NFC: No Fragmentation Control} ) corresponding to the template nucleic acid.
The method according to claim 1, wherein the method is based on the ΔΔCt value obtained by The method of claim 8, wherein the template nucleic acid comprises at least a portion of a nucleic acid encoding 18S rRNA or mitochondrial DNA. The method according to any one of claims 1 to 9, wherein the organism is a Smoky-breasted cockroach, the plurality of different primer pairs include primers comprising the following sequence or a mutant thereof, and the mutant is a base sequence that hybridizes under stringent conditions to a base sequence complementary to the following sequence and has 90% or more identity with the following sequence :
Forward primer 5'-ACTAGTCGCATCCGGTATCCTC-3' (SEQ ID NO:1);
Short strand amplification reverse primer 5'-CTCAATCTCGTGCGGCTAGA-3' (SEQ ID NO:2); and long strand amplification reverse primer 5'-AAGGGCAGGGACGTAATCAA-3' (SEQ ID NO:3). The method according to any one of claims 1 to 9, wherein the organism is a German cockroach, and the plurality of different primer pairs include primers comprising the following sequence or a mutant thereof, the mutant being a base sequence that hybridizes under stringent conditions to a base sequence complementary to the following sequence and has 90% or more identity with the following sequence :
Forward primer 5'-TAGTCGCATCCGGCATCCTT-3' (SEQ ID NO: 4);
Short strand amplification reverse primer 5'-CTCAATCTCGTGCGGCTAGG-3' (SEQ ID NO:5); and long strand amplification reverse primer 5'-AAGGGCAGGGACGTAATCAC-3' (SEQ ID NO:6). The method according to any one of claims 1 to 9, wherein the organism is a Smoky-breasted cockroach, the plurality of different primer pairs include a primer comprising the following sequence or a mutant thereof, and the mutant comprises a base sequence that hybridizes under stringent conditions to a base sequence complementary to the following sequence and has 90% or more identity with the following sequence :
Forward primer 5'-GAACATCATTGAGAATATTAATTCGTGCT-3' (SEQ ID NO: 9);
Short strand amplification reverse primer 5'-GAAAGCATGTGCAGTTACAATCAC-3' (SEQ ID NO: 10); and long strand amplification reverse primer 5'-ATGGTGGGTATAACTGTTCAACCTGTA-3' (SEQ ID NO: 11). The method according to any one of claims 1 to 9, further comprising a step of identifying the species of the organism based on the presence or absence of an amplification product generated from the template nucleic acid. A primer pair for 18S rDNA of the Smoky Brown Cockroach for use in the method according to any one of claims 1 to 9, comprising a primer comprising the following sequence or a mutant thereof, the mutant being a base sequence that hybridizes under stringent conditions to a base sequence complementary to the following sequence and has 90% or more identity with the following sequence :
Forward primer 5′-ACTAGTCGCATCCGGTATCCTC-3′ (SEQ ID NO:1);
The short amplification reverse primer was 5'-CTCAATCTCGTGCGGCTAGA-3' (SEQ ID NO:2); and the long amplification reverse primer was 5'-AAGGGCAGGGACGTAATCAA-3' (SEQ ID NO:3). A primer pair for 18S rDNA of the German cockroach for use in the method according to any one of claims 1 to 9, comprising a primer or a mutant thereof comprising the following sequence, wherein the mutant is a base sequence that hybridizes under stringent conditions to a base sequence complementary to the following sequence and has 90% or more identity with the following sequence :
Forward primer 5′-TAGTCGCATCCGGCATCCTT-3′ (SEQ ID NO:4);
The short amplification reverse primer was 5'-CTCAATCTCGTGCGGCTAGG-3' (SEQ ID NO:5); and the long amplification reverse primer was 5'-AAGGGCAGGGACGTAATCAC-3' (SEQ ID NO:6). A primer pair for mitochondrial DNA of the Smoky Brown cockroach for use in the method according to any one of claims 1 to 9, comprising a primer comprising the following sequence or a mutant thereof, the mutant being a base sequence that hybridizes under stringent conditions to a base sequence complementary to the following sequence and has 90% or more identity with the following sequence :
Forward primer 5′-GAACATCATTGAGAATATTAATTCGTGCT-3′ (SEQ ID NO:9);
The short amplification reverse primer was 5'-GAAAGCATGTGCAGTTACAATCAC-3' (SEQ ID NO: 10); and the long amplification reverse primer was 5'-ATGGTGGGTATAACTGTTCAACCTGTA-3' (SEQ ID NO: 11).