Mixed sample detection method for detecting purity of Chinese cabbage seeds based on mSNP technology

A detection method, Chinese cabbage technology, applied in the direction of recombinant DNA technology, biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of cumbersome operation steps, high cost, complicated operation, etc., to improve detection efficiency, The effect of low cost and simple operation

Inactive Publication Date: 2021-11-16
石家庄博瑞迪生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Molecular markers can be divided into three categories: one is the marking technology based on enzyme digestion, such as RFLP, which has stable polymorphism but complicated operation, high cost and less polymorphism; the other is the marking technology based on PCR reaction. , such as RAPD, SSR, SCAR, etc., such markers are low in cost, easy to operate but poor in stability; the third is a combination of enzyme digestion and PCR technology, such as AFLP, which has the advantages of both RFLP and RAPD, and has a higher Reliability and high efficiency, less DNA consumption, but cumbersome operation steps, high cost

Method used

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  • Mixed sample detection method for detecting purity of Chinese cabbage seeds based on mSNP technology
  • Mixed sample detection method for detecting purity of Chinese cabbage seeds based on mSNP technology
  • Mixed sample detection method for detecting purity of Chinese cabbage seeds based on mSNP technology

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Effect test

Embodiment 1

[0089] Embodiment 1: Obtaining method of specific primer

[0090] The method for obtaining specific primers of the present invention is specifically:

[0091] Using the whole genome resequencing data of Chinese cabbage, use BWA-mem (http: / / bio-bwa.sourceforge.net / ) to post to the reference genome of Chinese cabbage, and use GATK (https: / / software.broadinstitute.org / gatk / ) for single nucleotide variant identification.

[0092] For the set of identified single nucleotide variation sites, screen the minimum allelic variation frequency > 0.02, heterozygosity rate < 15%, and deletion rate < 20%, combine the single nucleotide variation sites, and screen the single nucleotide The number of sites is located in the segment of 2-10, that is, the mSNP (polynucleotide polymorphism) site. Compared with the traditional SNP (single nucleotide polymorphism) site, the mSNP site can maximize the Using the information obtained by each primer pair, that is, as many SNP sites as possible can be...

Embodiment 2

[0095] Embodiment 2: Chinese cabbage seed purity detection primer set used

[0096] The primer set used for the detection of the purity of Chinese cabbage seeds includes the primer pair 1F / R~22F / R. In the primer pair 1F / R~22F / R, it not only includes the specific primer sequence shown in SEQIDNo.1~44, but also includes Universal primer sequence, the F-terminal universal primer sequence of the F primer in the primer pair 1F / R~22F / R is shown in SEQIDNo.45; the sequence of the R-terminal universal primer of the R primer in the primer pair 1F / R~22F / R As shown in SEQ ID No.46;

Embodiment 3

[0097] Embodiment 3: the obtaining method of primer mixture:

[0098] After obtaining specific primers, design specific tag sequences, and then re-synthesize primers. At this time, specific tag sequences will be added during primer synthesis. In this example, 96 sets of specific tag combinations are used, specifically:

[0099]According to the combination of specific tags, each specific primer was synthesized into 10 primers with different target tags. The sequence form of the primers with the target tags is shown in Table 2. And all R (reverse primers), take 10 μl of each primer, and dilute to 10ml; the concentration of each primer is 0.1 μM, and this embodiment prepares a total of 96 sets of primer pairs containing specific label combinations, that is, the primer mixture;

[0100] Table 2 Primer Set

[0101]

[0102]

[0103] "FF" is the F-terminal universal primer sequence, and the F-terminal universal primer sequence is AACGACATGGCTACGATCCGACTT, as shown in SEQ ID N...

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Abstract

The invention relates to a mixed sample detection method for detecting the purity of Chinese cabbage seeds based on an mSNP technology, which is carried out by utilizing a primer pair 1F / R-22F / R, and the gene sequence of the primer pair 1F / R-22F / R is as shown in SEQ ID No.1-44; according to the invention, an mSNP technology is adopted, so that more SNP variations can be detected under the condition that amplicons are not changed; a sample mixing method is adopted for detection, the sequencing cost is reduced while the amplification workload is reduced, the detection speed is also increased, and detection of 1440 seeds can be completed within one day at most. According to the method, the single nucleotide polymorphism is directly read by using a sequencing technology, and the purity result is directly interpreted through a program, so that the result is visual and reliable, and the influence of subjective factors during the seed development period and the result interpretation is avoided.

Description

technical field [0001] The invention belongs to the field of seed purity detection, and in particular relates to a mixed-sample detection method for detecting the purity of Chinese cabbage seeds based on mSNP technology. Background technique [0002] Chinese cabbage (Brissica campestris L.ssp.pekinesis), also known as head cabbage, yellow sprouts, and Chinese cabbage, belongs to the Brassica family Brassica. Chinese cabbage originated in my country, has a cultivation history of more than 5,000 years, is rich in germplasm resources, and has various ecological types. It is one of the main traditional vegetables in my country. In the 1970s, the use of heterosis played a dominant role in the breeding of new varieties of Chinese cabbage, and achieved fruitful results. In order to breed excellent new varieties in a short period of time, breeders will select a small number of high-quality backbone parents, so that the genetic basis of the varieties will continue to narrow. Variet...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6869C12N15/11
CPCC12Q1/6895C12Q1/6869C12Q2600/156C12Q2600/13C12Q2531/113C12Q2523/308
Inventor 刘景艺许彦芬高苗李凝张萌郝军会刘田龚舒张丛
Owner 石家庄博瑞迪生物技术有限公司
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