JP4658816B2 - Primer for detection of peanut and detection method thereof - Google Patents

Description

  The present invention targets a peanut genus that may cause allergies, and even when a single plant of the peanut genus is contained in a food raw material or product, the amount of the peanut genus is detected with high sensitivity. The present invention relates to a new primer and a detection method thereof.

About the primer for the detection of the peanut genus and its detection method, the applicant of this application has already developed and applied for a patent (patent document 1).
In the detection method, any one of the three types of primer pairs is used. That is, the first is a pair of primers made in the peanut ITS-1 sequence and the primer made in the 5.8S rRNA gene sequence, the second is the primer and ITS-2 sequence made in the 5.8S rRNA gene sequence The pair of primers prepared in the third is the pair of the primer prepared in the ITS-1 sequence and the primer prepared in the ITS-2 sequence.
For the first pair, the touchdown PCR method is used in the detection method, which complicates the PCR temperature conditions, and in particular examines samples containing peanut plants at low concentrations near the detection limit. In some cases, different test results may be given due to the influence of subtle differences in temperature characteristics among PCR devices.
Although the second pair and the third pair do not employ the touchdown PCR method, some related plants belonging to the same leguminous family as the peanut genus may be erroneously detected. In addition, the PCR target amplification product is also relatively long, and in the case of the second pair, it is 253 to 259 bp, and in the case of the third pair, it is 384 to 390 bp. There is a concern that the detection sensitivity will be lower than the method with a short PCR target amplification product.

JP 2003-199599 A

In carrying out the PCR method for detecting plants belonging to the genus Peanut, the object is to not use the touchdown PCR method and thereby solve the above-mentioned problems caused by the touchdown PCR method.
Another object of the present invention is to detect plants of the genus Peanut with high sensitivity even in processed products.
It is another object of the present invention to prevent some related plants belonging to the same leguminous family as the peanut genus from being erroneously detected.

As a result of extensive studies by the present inventors for the above object, it was found that the object can be effectively achieved by using a primer having a specific base sequence, and the present invention has been completed. That is, the present invention provides a primer for detecting a peanut genus comprising a base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2.
The present invention also provides a primer pair for detecting a peanut genus in which a primer comprising the base sequence represented by SEQ ID NO: 1 and a primer comprising the base sequence represented by SEQ ID NO: 2 are combined.
Furthermore, the present invention provides a step of obtaining an amplification product containing at least a part of the ITS-1 sequence of the peanut genus using the primer pair, and a step of determining the presence of the peanut genus using the presence of the amplification product as an index; A method for detecting a peanut genus comprising:

  According to the peanut genus detection primer and its detection method of the present invention, the PCR temperature condition resulting from the touchdown PCR method becomes complicated by not using the touchdown PCR method in performing the PCR method. When inspecting a sample containing a peanut genus plant at a low concentration near the limit, it is possible to reduce the possibility of giving different test results due to the subtle differences in temperature characteristics of each PCR device. In addition, by shortening the PCR target amplification product to 85 to 87 bp, it is possible to detect plants of the peanut genus with high sensitivity even in processed products. Further, by changing to the primer of the present invention, there is an advantage that some related plants belonging to the same leguminous family as the peanut genus can be prevented from being erroneously detected.

In the present invention, first, a primer for detecting peanuts was designed. The designed primer is a primer designed in the ITS-1 region of peanut genus and capable of hybridizing under stringent conditions with a nucleic acid molecule having a base sequence in the ITS-1 sequence of peanut genus, As a specific primer that satisfies the requirements, it is necessary that the oligonucleotide has a property that the 3 ′ end hybridizes to the base in the ITS-1 sequence of the peanut genus when hybridized with the molecule. The oligonucleotide represented by the following SEQ ID NO: 1 or SEQ ID NO: 2 was found.
CAAAACCCCGGCGCGGAAA (SEQ ID NO: 1)
GTCGCCCCGACCGGATGC (SEQ ID NO: 2)

“Hybridize under stringent conditions” means that two DNA fragments hybridize with each other under standard hybridization conditions as described by Sambrook J et al. (Expression of cloned genes in E. coli (Molecular Cloning: A laboratory manual (1989)) Cold Spring harbor Laboratory Press, New York, USA, 9. 47-9.62 and 11.45-11.61). More specifically, for example, hybridization and washing (for example, about 2.0 × SSC, 50 ° C.) are performed based on the Tm value obtained by the following formula.
Tm = 81.5 + 16.6 (log10 [Na +]) + 0.41 (fraction G + C)-(600 / N)
Moreover, the genus as used in this specification means the thing containing all the peanuts contained in a genus, or the thing containing some species selected from the peanuts contained in a genus.

Next, PCR is performed using one of the primers as a sense primer and the other as an antisense primer, and the presence of a PCR target amplification product containing at least a part of the ITS-1 sequence of the peanut genus is used as an indicator to determine the presence of the peanut genus. To detect. Specifically, using a primer pair for detecting a peanut genus in which a primer composed of the base sequence represented by SEQ ID NO: 1 and a primer composed of the base sequence represented by SEQ ID NO: 2 are combined, the ITS- An amplification product including at least a part of one sequence is obtained, and the presence of the peanut genus is determined using the presence of the amplification product as an index.
The PCR is described in, for example, Saiki RK, et al., Science, 230: 1350-1354 (1985), separate volume of plant cell engineering, plant PCR experiment protocol, supervised by Isao Shimamoto and Takuji Sasaki (1995), etc. Based on the usual methods, conditions such as denaturation, annealing, extension step and temperature, enzyme (DNA polymerase) type and concentration, dNTP concentration, primer concentration, magnesium chloride concentration, template DNA amount, etc. Change and select the best one. However, a general PCR method using conditions in which the annealing temperature is constant over all cycles can be adopted, and there is no need to adopt a so-called touchdown PCR method.

  After the PCR, contamination of the peanut genus is detected using as an index the presence of a PCR target amplification product having a size of 85 to 87 bp. In this detection, whether or not a PCR amplification product of a target size (85 to 87 bp) exists in the reaction solution after PCR, that is, a target containing at least a part of the ITS-1 sequence of the peanut genus If there is a PCR amplification product of the size, it means that a plant belonging to the genus Peanut is mixed in the sample to be examined, and conversely, the peanut is absent even if the PCR amplification product is not present or present. If there is no target-sized PCR amplification product containing at least a portion of the genus ITS-1 sequence, then the peanut plant is not contaminated in the sample to be examined. And confirmation of whether the PCR target amplification product of 85-87 bp size exists, the reaction solution after PCR of DNA extracted from the sample to be inspected such as food materials and products, for example, by electrophoresis, Whether or not a peanut genus is present in the sample to be examined is detected depending on whether or not a single band appears in the amplification product size of 85 to 87 bp. In the present invention, this detection can be performed with high sensitivity, for example, at a level of 2 ppm.

Example 1
(Sample preparation)
The seeds were ultrasonically washed in 1% SDS solution, then ultrasonically washed in distilled water, and air-dried at 50 ° C. About 0.3 g of seeds and one zirconia bead of φ7 mm diameter (manufactured by Nikkato Co., Ltd.) were placed in a 2 ml tube (manufactured by eppendorf), and finely pulverized with Retsch MM 300 (manufactured by QIAGEN). After adding 1 ml of buffer G2 (QIAGEN), 10 μl Proteinase K (20 mg / ml) (QIAGEN) and 1 μl RNase A (100 mg / ml) (QIAGEN) to the tube after grinding And kept at 50 ° C. for 1 hour. Thereafter, the mixture was centrifuged at about 3,000 × g for 10 minutes to obtain the supernatant. The obtained supernatant was subjected to Genomic-tip 20 / G (QIAGEN) equilibrated with 1 ml of buffer QBT (QIAGEN) in advance to adsorb the DNA to the tip. Thereafter, the tip was washed with 4 ml of buffer QC (QIAGEN), and the DNA was eluted with 1 ml of buffer QF (QIAGEN) preheated to 50 ° C. After adding 4 volumes of buffer NT2 (MACHEREY-NAGEL) to the eluate and mixing, apply 650 μl at a time to two NucleoSpin Extract Columns (MACHEREY-NAGEL) at approximately 6,000 × g for 1 minute. The DNA was adsorbed on the column by centrifugation. This was repeated until the entire liquid volume was processed. Then, add 600 μl of buffer NT3 (manufactured by MACHEREY-NAGEL) to the column, centrifuge at about 6,000 × g for 1 minute to wash the column, add 600 μl of buffer NT3 again, and centrifuge at maximum speed for 1 minute. The buffer NT3 remaining in the Column was completely removed. Finally, 100 µl of Buffer NE (manufactured by MACHEREY-NAGEL) preheated to 70 ° C is added to the Column, left at room temperature for 1 minute, and then centrifuged at maximum speed for 1 minute to elute the DNA from the Column. The precipitate recovered by isopropanol precipitation was dissolved in 50 μl of sterilized ultrapure water. The DNA concentration in the solution was measured, and appropriately diluted with sterile ultrapure water was used as a PCR template DNA sample. The template DNA used was a DNA solution measured with a spectrophotometer, a spectrum of 220 to 350 nm was measured, and a maximum value was observed at 260 nm.

(PCR reaction conditions)
The PCR reaction conditions are as shown in Table 1. For Taq polymerase, HotStarTaq PCR Master Mix manufactured by QIAGEN was used.

(detection)
The obtained PCR reaction solution was confirmed by subjecting to ethidium bromide-containing 3% agarose gel electrophoresis.
The PCR thermal cycler usually uses GeneAmp PCR System 9600 (Applied Biosystems), but it is confirmed that GeneAmp PCR System 2400 (Applied Biosystems) and DNA Engine (MJ Research) can also be used. is doing.
The detection result by the electrophoresis is shown in FIG.
As is clear from the results of FIG. 1, for peanuts, the target amplification product is clearly present at a size of about 86 bp, while for plants other than peanuts such as wheat and corn, the size is about 86 bp. It can be seen that there is no target amplification product. This showed that the primer pair specifically detects peanuts. In addition, the presence of a target amplification product of about 86 bp in size was confirmed from 100 fg (2 ppm weight / weight) of peanut DNA added to 50 ng salmon sperm DNA, indicating that peanut can be detected with a sensitivity of 2 ppm level.

落花生以外のいずれの配列からも、標的増幅産物は得られないことが予測された。 (Example 2)
PCR simulations using Amplify 1.0 (Bill Engels) were conducted for ITS-1 areas of peanut-related species, specific raw materials other than peanuts, legumes, major food ingredients, and nuts. The results are shown in Table 2.

It was predicted that no target amplification product could be obtained from any sequence other than peanut.

  By utilizing the primer of the present invention and the PCR method using the primer, it can be greatly useful for detecting whether or not a trace amount of peanut is mixed in food or food raw materials.

It is a detection result when the obtained PCR reaction solution is subjected to ethidium bromide-containing 3% agarose gel electrophoresis. Example 1

Claims (2)

A primer pair for detecting a peanut genus, which is designed in the ITS-1 region of the peanut genus and combines a primer consisting of the base sequence represented by SEQ ID NO: 1 and a primer consisting of the base sequence represented by SEQ ID NO: 2. A step of obtaining an amplification product comprising at least a part of the ITS-1 sequence of the peanut genus using the primer pair according to claim 1, and the presence of the peanut genus is determined using the presence of the amplification product of 85 to 87 bp in size as an index A method for detecting a peanut genus comprising a step.

Images