JP7813438B2 - Method for determining the possibility of contamination with type II cytolethal toxin-producing bacteria - Google Patents

Description

The present invention relates to a method for determining the possibility of contamination with type II cytolethal distending toxin-producing bacteria.

In recent years, Escherichia albertii has been attracting attention as a causative agent of food poisoning. This bacterium (hereinafter sometimes referred to as "Albertii"), like Escherichia coli O157, has a high potential to cause intestinal bleeding. Not only does it share biochemical properties with E. coli, it also possesses shared pathogenic factors, such as the eae gene, which encodes intimin, involved in adhesion to the intestinal mucosa, and the cdt gene, which is involved in cytolethal distending toxin (CDT). Therefore, it is necessary to strictly distinguish between the two.

As a method for distinguishing between the two, Patent Document 1 discloses a medium containing a sugar that is not decomposed by E. albertii and a pH indicator. By culturing using this medium, it is possible to detect E. albertii from bacteria of the genus Escherichia. However, because Shigella and Bacillus Providencia also form similar colonies in this medium, it is necessary to specifically detect E. albertii.

It is known that there are many bacteria that produce CDT other than E. coli and P. albertii. However, of the five types of cdt genes (cdtI-V) believed to be produced by E. coli, it has been shown that the cdt-I gene, cdt-III gene, cdt-IV gene, and cdt-V gene have low homology to the cdt genes possessed by P. albertii and high homology to the cdt-II gene possessed by certain strains of E. coli (Non-Patent Document 1). Based on this knowledge, Non-Patent Document 1 reveals that a PCR method (Eacdt PCR) and a nested PCR method (Nested Eacdt PCR) have been developed that can detect the cdt-II gene possessed by P. albertii, targeting the cdt-III gene, cdt-V gene, and the cdt gene of Providencia alcalifaciens (P. alcalifaciens). Furthermore, Non-Patent Document 2 discloses a primer consisting of the base sequence shown in SEQ ID NO: 69 and a primer consisting of the base sequence shown in SEQ ID NO: 70, which were used in the Eacdt PCR method.

However, the former Eacdt PCR method does not have a sufficient lower limit of detection, and the latter nested PCR method requires two-stage amplification, so new primers are needed that can detect and distinguish the cdt-II gene from the other four types of cdt genes in a single amplification stage. Furthermore, because cdt-II genes are expected to have mutations such as single nucleotide polymorphisms, a method that can detect as many cdt-II genes as possible is desirable.

Patent Publication No. 2019-024418

Noritomo Yasuda, Development and application of a detection method for Escherichia albertii discovered by re-identification of E. coli carrying the type II cytolethal distending toxin gene, Internet, <URL: https://www.osakafu-u.ac.jp/osakafu-content/uploads/sites/428/k1798.pdf> Atsushi Hinenoya et al., Diagnostic Microbiology and Infection Disease 95(2019) 119-124

The present invention was made in consideration of the above-mentioned background art, and aims to provide PCR primers that can specifically detect type II cytolethal toxin-producing bacteria using PCR that allows detection through a single amplification step rather than nested PCR.

The present invention is a method for determining the possibility of contamination with type II cytolethal toxin-producing bacteria, which includes the step of contacting a sample to be determined or DNA extracted from the sample to amplify a target gene fragment in the sample, and the step of amplifying the target gene fragment uses a specific primer set.

The present invention specifically detects the type II cytolethal distending toxin (type II CDT) gene (cdt-II gene) from various CDT-producing bacteria, enabling rapid determination of the possibility of contamination with the CDT-producing bacteria.

FIG. 1 is an explanatory diagram showing the sites where primers bind to the cdt-II gene and the amplified region. FIG. 2 is an explanatory diagram showing a part of the base sequence of the cdt-II gene and the site where the primer binds. Figure 3 shows electrophoretic images of the amplified products using various primer sets. Lane 1 shows P. albertii, lane 2 shows Providencia rustiganii, lane 3 shows Escherichia coli, lane 4 shows distilled water as a control, and lanes M1 and M2 show the markers. FIG. 4 shows representative images of melting curves obtained using various primer sets.

The method of the present invention is a method for determining the possibility of contamination with type II cytolethal toxin-producing bacteria, and includes a step of contacting a sample to be determined or DNA extracted from the sample to amplify a target gene fragment in the sample, using a specific primer set in the step of amplifying the target gene fragment.

The type II cytolethal distending toxin gene (cdt-II gene) is publicly known. According to Non-Patent Document 1, the cdt-II gene is believed to be possessed by CTEC-II (type II cytolethal distending toxin-producing E. coli), a type of E. coli. However, CTEC-II is likely to be Bacillus albertii, and if cdt-II is detected, it can be assumed that contamination with Bacillus albertii has occurred.

In the present invention, contamination with type II CDT-producing bacteria does not only mean the presence of bacteria carrying the cdt-II gene in a sample, whether live or dead, but also the presence of the target gene in a sample due to contamination by bacteria carrying the cdt-II gene or foreign matter containing the target gene. Possible contamination means that it is presumed that the sample has been contaminated with the bacteria in question for some reason. Therefore, even if contamination is determined using the method of the present invention, it cannot necessarily be concluded that live bacteria were present, and confirmation of bacterial contamination using another method is required.

The method of the present invention includes a step of contacting a target gene fragment in a sample to be evaluated or DNA extracted from the sample and amplifying the target gene fragment in the sample. This step is known as PCR (Polymerase Chain Reaction). In this step, the following primer set is used in the present invention.

The primer set used preferably has a base sequence capable of binding complementarily to the cdt-II gene. "Able to bind complementarily" is used in the sense commonly used by those skilled in the art of PCR, meaning that the bases of each primer can bind to the cdt-II gene at opposite ends. However, the primers of the present invention do not necessarily need to bind perfectly complementarily, as long as they can amplify a partial region of the cdt-II gene even if one to three bases are inserted, deleted, or substituted. The cdt-II gene is a gene that produces type II CDT, and various polymorphisms of the cdt-II gene exist, each differing by a single base. The primer set of the present invention binds complementarily to the cdt-II gene having the consensus sequence shown in Figure 2 (SEQ ID NO: 71), but can also amplify partial regions of the cdt-II gene having a base sequence different from the consensus sequence. The base sequence shown in SEQ ID NO: 71 represents 641 bases, from the 1300th base to the 1940th base from the 5' end of the consensus sequence (sense strand) of the cdt-II gene.

The primer set according to the present invention comprises one forward primer selected from the group consisting of primer group 6F consisting of primers having the base sequence shown in SEQ ID NO: 1 and each having 20 to 24 bases; primer group 7F consisting of primers having the base sequence shown in SEQ ID NO: 3 and each having 17 to 21 bases; primer group 8F consisting of primers having the base sequence shown in SEQ ID NO: 5 and each having 17 to 22 bases; and primer (1845F) having the base sequence shown in SEQ ID NO: 68 (TAATGATTCGAACGCCAAAC); primer group 6R consisting of primers having the base sequence shown in SEQ ID NO: 2 and each having 16 to 20 bases; primer group 7R consisting of primers having the base sequence shown in SEQ ID NO: 4 and each having 17 to 21 bases; The primer set is composed of a combination of primer group 8R, which consists of primers having the nucleotide sequence shown in SEQ ID NO: 6 and a reverse primer selected from the group consisting of primers having 16 to 21 bases (CTATTTCCCATCCAATAGTCT) and a primer (IchiR) having the nucleotide sequence shown in SEQ ID NO: 70, and the combinations of the forward primer and the reverse primer are primer group 6F and primer group 6R, primer group 6F and primer group 7R, primer group 6F and primer group 8R, primer group 7F and primer group 7R, primer group 8F and primer group 7R, primer group 8F and primer group 8R, primer group 6F and primer IchiR, and primer 1845F and primer group 7R.

More specifically, the forward primer can be selected from primers having the nucleotide sequences shown in SEQ ID NOs: 7 to 15 in primer group 6F, from primers having the nucleotide sequences shown in SEQ ID NOs: 27 to 36 in primer group 7F, and from primers having the nucleotide sequences shown in SEQ ID NOs: 48 to 58 in primer group 8F. Furthermore, the reverse primer can be selected from primers having the nucleotide sequences shown in SEQ ID NOs: 17 to 22, 24, and 25 in primer group 6R, from primers having the nucleotide sequences shown in SEQ ID NOs: 38 to 46 in primer group 7R, and from primers having the nucleotide sequences shown in SEQ ID NOs: 59 to 67 in primer group 8R.

In the present invention, the terms forward primer and reverse primer are used in the sense commonly used by those skilled in the art, with a forward primer referring to a primer designed to bind to the antisense strand of the cdt-II gene and a reverse primer referring to a primer designed to bind to the sense strand of the cdt-II gene.

Figures 1 and 2 show the binding positions of each primer in the cdt-II gene. The numbers in Figure 1 indicate the base positions from the 5' end of the cdt-II gene. 1 and 2 show the binding positions of representative primers in primer group 6F, primer group 7F, primer group 8F, primer group 6R, primer group 7R, and primer group 8R, respectively: primer (6F) consisting of the nucleotide sequence (TCAGATAGATGAATTAGGAAAAG) set forth in SEQ ID NO: 7; primer (7F) consisting of the nucleotide sequence (TGTGAAAACACCTGAAGAAG) set forth in SEQ ID NO: 27; primer (8F) consisting of the nucleotide sequence (GAGAGACTATTGGATGGGAAA) set forth in SEQ ID NO: 48; primer (6R) consisting of the nucleotide sequence (TTCTTCAGGTGTTTTCACA) set forth in SEQ ID NO: 17; primer (7R) consisting of the nucleotide sequence (CGTCATTTTTAGCAGGTTCC) set forth in SEQ ID NO: 38; and primer (8R) consisting of the nucleotide sequence (TGGTCTGTGTTTGGCGTTCG) set forth in SEQ ID NO: 59.

In the present invention, the combination of forward primers and reverse primers may be any of the combinations described above, but is preferably a combination of primer group 7F and primer group 7R, or a combination of primer group 8F and primer group 8R, and desirably a combination of primer group 8F and primer group 8R.

The target sample is not limited to samples in which bacteria (whether live or dead) carrying the cdt-II gene may be present, but may also be any sample in which the cdt-II gene may be present, such as vomit, feces, or food from various animals, including humans, cows, pigs, dogs, and cats. While these samples may be used directly as samples for amplification, it is preferable to use template DNA (template DNA) for amplification extracted from these samples. There are no particular limitations on the extraction and preparation of template DNA, and it can be obtained by conventional methods.

The concentration of the primers used and the amount of template used are not limited and can be adjusted as appropriate. In the present invention, in addition to the primers (primer set), buffers and DNA polymerases commonly used in PCR are also used for amplification. These buffers and DNA polymerases are also not limited, and their concentrations can be determined as appropriate by those skilled in the art.

Examples of buffers include TRIS, TRICINE, BIS-TRICINE, HEPES, MOPS, TES, TAPS, PIPES, and CAPS. The PCR reaction solution preferably contains about 1.0 to 5 mM Mg ²⁺ , and may also contain KCl.

As DNA polymerases, known polymerases, preferably those derived from thermostable bacteria, can be used. Examples of commercially available polymerases (the following are trade names) include Taq DNA polymerase and Tth DNA polymerase, which belong to Family A (Pol I type), and KOD DNA polymerase, Pfu DNA polymerase, Pwo DNA polymerase, Ultima DNA polymerase, and PrimeSTAR DNA polymerase, which belong to Family B (α type). Thermostable DNA polymerases are not limited to extracts, but may also be recombinant, or may be mutants with an amino acid sequence in which one to several amino acids are deleted, substituted, added, or inserted into the amino acid sequence of a natural polymerase. One or more types of DNA polymerases can be used.

The amplification conditions (amplification temperature, amplification cycle, holding time, etc.) in the amplification step can be set based on the common sense of those skilled in the art of PCR. The conditions described in the Examples are an example of such conditions.

The possibility of contamination is determined by the presence or absence and amount of amplification product amplified in the amplification process. Methods for determining the presence or absence and amount of amplification product include electrophoresis, which electrophoreses the amplification product in the reaction solution after the amplification process, the cycling probe method or probe method (5' nuclease method) that uses fluorescent substances, and the intercalator method. All of these determination methods are publicly known. While any of these determination methods can be used to determine the possibility of contamination, the intercalator method is preferred as it is a relatively simple method that can be used in real-time PCR.

The intercalator used in the intercalator method is not particularly limited, as long as it is a fluorescent substance that emits fluorescence when inserted between double-stranded DNA and that quenches the fluorescence when the double-stranded DNA dissociates. Examples of commercially available dyes (hereinafter, the respective product names) include, but are not limited to, ethidium bromide, cyanine dyes (e.g., TOTO, YOYO, BOBO, POPO), SYBR Green I, SYBR Green ER, SYBR Green Gold, SYBR DX, PicoGreen, LCGreen, EvaGreen, SYTOX Green, ResoLight, Acridine orange, CyQUANT GR, SYTO 9, SYTO 10, SYTO 13, SYTO 14, SYTO 82, and FUN-1. Of course, any method other than the exemplified methods may be used as long as it is capable of determining the presence or absence of an amplification product. Furthermore, melting curve analysis is performed in the intercalator method, and the conditions for melting curve analysis can be set based on the common sense of a person skilled in the art. A commercially available real-time PCR device is used for PCR amplification and melting curve analysis, and the conditions for melting curve analysis can be set in accordance with the operating manual for the real-time PCR device used.

The measurement kit of the present invention is a measurement kit for determining the possibility of contamination with the cdt-II gene, and includes at least one of the above-mentioned primer sets. This measurement kit is used to amplify the cdt-II gene present in a target sample and examine the possibility of contamination with bacteria carrying the cdt-II gene, and in addition to the primer set, may also include a buffer, DNA polymerase, a cycling probe for detecting the amplified product, an intercalator, etc.

Next, the present invention will be further explained based on the following examples, but it goes without saying that the present invention is not limited to the following examples.

The nucleotide sequences of the cdt-II gene of the 38 strains shown in Table 1 that were identified as E. albertii strains of known origin were analyzed to determine the nucleotide sequence (consensus sequence: majority sequence) of the cdt-II gene. The 38 E. albertii strains for which consensus sequences were determined include the type strain (ATCC) E. albertii (Albert19982), but not all of the other 37 strains had the same nucleotide sequence as the type strain; single nucleotide polymorphisms were found throughout the cdt-II gene, including the region of the nucleotide sequence shown in Figure 1 (bases 1300 to 1940, which were found to be relatively highly conserved). Primer sets Set 1 to Set 8 shown in Table 2 were created from the consensus sequence of this cdt-II gene, the base sequences of the cdt-I gene (NT3363-cdtI), cdt-III gene (PII4-cdtIII), cdt-IV gene (P159-cdtIV), and cdt-V gene (P336-cdtV), and the base sequences of the cdt gene (JHI-Prcdt) of Providencia rustigianii (JHI strain) and the cdt gene (AH31-Pacdt) of Providencia alcalifaciens (AH31 strain), which are considered to have relatively high homology to the cdt-II gene of P. albertii, and real-time PCR was performed. For each set in the sample name (Sample Name) in Table 2, the start position of the forward side (upper row) and the number of bases of the primer and the start position of the reverse side (lower row) are shown. For example, the forward primer of Set 6 is a primer (6F) having the base sequence shown in SEQ ID NO: 7, which is composed of 23 bases starting from the 1672nd base, and the reverse primer is a primer (6R) having the base sequence shown in SEQ ID NO: 17, which is composed of 19 bases starting from the 1816th base. The forward primer of Set 7 is a primer (7F) having the base sequence shown in SEQ ID NO: 27, which is composed of 20 bases starting from the 1798th base. The reverse primer is a primer (7R) having the base sequence shown in SEQ ID NO: 38, which is composed of 20 bases starting from the 1915th base. The forward primer of Set 8 is a primer (8F) having the base sequence shown in SEQ ID NO: 48, which is composed of 21 bases starting from the 1770th base. The reverse primer is a primer (8R) having the base sequence shown in SEQ ID NO: 59, which is composed of 20 bases starting from the 1872nd base. As a result, primer sets Set 1 to Set 5 did not show sufficient separation in the Tm values (the temperature at which 50% of double-stranded DNA is determined to dissociate into single-stranded DNA: °C) in melting curve analysis, and it was determined that it was not possible to detect P. albertii strains separately from other bacterial species based on the electrophoresis images of the amplified products (not shown).

Next, primer sets Set 6 to Set 8 described above were used to examine specificity, that is, to examine whether they could be detected and distinguished from other bacterial species. The target bacterial species were a total of 67 strains, including 39 strains of E. albertii and 28 strains of 26 species other than E. albertii, as shown in Table 3.

Template DNA was prepared from bacteria using standard methods, and a reaction solution was prepared by adding water to 1 μL of template DNA solution, 10 μL of DNA polymerase solution (Go Taq qPCR Master Mix: Promega Corporation), 7 μL each of forward primer solution (10 μM) and reverse primer solution (10 μM), for a total volume of 20 μL.

The solution was amplified under the conditions shown in Table 4, followed by melting curve analysis (hereinafter, real-time PCR was performed under the same conditions). As a result, regardless of which primer set was used, Tm values were observed for all Bacillus albertii strains, and the cdt-II gene was amplified. On the other hand, no Tm values were observed for the other 28 strains, confirming that only the cdt-II gene possessed by Bacillus albertii could be detected.

Next, real-time PCR using the Set8 primer set was compared with the Eacdt method (real-time PCR using the IchiF and IchiR primer set) described in Non-Patent Document 1. Raccoons, which are commonly infected with C. albertii, were used as subjects. Raccoons were swabbed rectally to collect samples, which were then suspended in PBS and diluted 10-fold with TSB. After centrifugation, the precipitate was decomposed with sodium hydroxide solution and neutralized with acid to prepare template DNA. The results are shown in Table 5. As a result, the positive detection rate improved from 67% with the Eacdt method to 77%. The IchiF and IchiR primer set described in Non-Patent Document 1 was designed from a highly conserved region of the cdt-II gene found in E. coli classified as CTEC-II (cdt-II gene-carrying E. coli).

Modified primers shown in Tables 6-8 were prepared based on primer sets 6-8, and E. albertii AKT5 was detected. Table 6 shows the results when modified primers based on primers from Set 6 (1672F: 6F in Figure 1 and 1816R: 6R in Figure 1) were used. Table 7 shows the results when modified primers based on primers from Set 7 (1798F: 7F in Figure 1 and 1915R: 7R in Figure 1) were used. Table 8 shows the results when modified primers based on primers from Set 8 (1770F: 8F in Figure 1 and 1872R: 8R in Figure 1) were used. The top row of each table shows the forward primer, and the bottom row shows the reverse primer. Real-time PCR was performed using a primer set combining the modified forward primer with the unmodified reverse primer in each table, and real-time PCR was performed using a primer set combining the modified reverse primer with the unmodified forward primer in each table. Numbers in parentheses in the tables indicate Tm values.

Each primer set in Sets 6 to 8 amplifies a region of approximately 250 bp at most. Therefore, detection of E. albertii (Eacdt), P. rustigainii (Prcdt), and E. coli (CTEC-III) was attempted using Sets A to I (see Figure 3 ), which consisted of combinations of forward and reverse primers from these sets. The forward and reverse primers used were primer (6F) consisting of the nucleotide sequence of SEQ ID NO: 7, primer (7F) consisting of the nucleotide sequence of SEQ ID NO: 27, primer (8F) consisting of the nucleotide sequence of SEQ ID NO: 48, primer (1845F) consisting of the nucleotide sequence of SEQ ID NO: 68, primer (6R) consisting of the nucleotide sequence of SEQ ID NO: 17, primer (7R) consisting of the nucleotide sequence of SEQ ID NO: 38, primer (8R) consisting of the nucleotide sequence of SEQ ID NO: 59, and primer (IchiR) consisting of the nucleotide sequence of SEQ ID NO: 70. Detection was not attempted with the combinations of primers (8F) and (6R) and primers (7F) and (6R) due to the narrow amplification regions. The results are shown in FIGS.
Furthermore, we attempted to detect E. albertii (Eacdt) in raccoon feces using Sets A, B, D, and I, which showed particularly good detection. The results are shown in Table 9. The strain names (Strain ID) in Table 9 indicate the names of the strains detected in the rectums of raccoons captured in the wild.

Claims (3)

1. A method for determining possible contamination with Escherichia albertii, comprising:
a step of contacting a target sample or DNA extracted from the sample with the target gene fragment to amplify the target gene fragment in the sample;
A method using the following primer set in the step of amplifying the target gene fragment:
A primer consisting of the base sequence shown in SEQ ID NO: 48;
A primer consisting of the base sequence shown in SEQ ID NO: 59;
A primer set consisting of A primer set for amplifying a partial region of the type II cytolethal distending toxin gene of Escherichia albertii, comprising:
A primer consisting of the base sequence shown in SEQ ID NO: 48;
A primer consisting of the base sequence shown in SEQ ID NO: 59;
A primer set consisting of: A measurement kit for determining the possibility of contamination with Escherichia albertii, comprising:
A measurement kit comprising the primer set according to claim 2 .