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mSNP technology based mixed sample detection method for detecting purity of carrot seeds

A detection method, carrot technology, applied in the direction of recombinant DNA technology, biochemical equipment and methods, microbial measurement/inspection, etc., can solve problems such as high quality requirements, and achieve the effect of improving detection efficiency, simple operation, and rapid detection work

Inactive Publication Date: 2021-12-07
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

AFLP has high polymorphism, and was earlier used in variety identification and purity analysis of rice and other crops, but this technology has high requirements for DNA purity and endonuclease quality

Method used

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  • mSNP technology based mixed sample detection method for detecting purity of carrot seeds
  • mSNP technology based mixed sample detection method for detecting purity of carrot seeds
  • mSNP technology based mixed sample detection method for detecting purity of carrot seeds

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Example 1: Obtaining method of specific primers

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

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

[0092] For the identified single nucleotide variation site set, screen the minimum allelic variation frequency > 0.02, the heterozygosity rate < 10%, and the deletion rate < 20%, merge the single nucleotide variation sites, and screen for single nucleotides The number of sites is located in the segment from 1 to 9, that is, mSNP (polymononucleotide polymorphism) sites. Compared with traditional SNP (single nucleotide polymorphism) sites, mSNP sites can maximize the Using the information obtained by each primer pair, that is, detecting as many SNP sites as possible without ch...

Embodiment 2

[0095] Example 2: Primer set used in the detection of carrot seed purity

[0096] The primer set used for the detection of carrot seed purity, including primer pair 1F / R~21F / R, in the primer pair 1F / R~21F / R, not only include specific primer sequences as shown in SEQIDNo.1~42, but also include general The primer sequence, the F-terminal universal primer sequence of the F primer in the primer pair 1F / R~21F / R is shown in SEQ ID No.43; the sequence of the R-terminal universal primer of the R primer in the primer pair 1F / R~21F / R is as follows shown in SEQIDNo.44;

Embodiment 3

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

[0098] After obtaining specific primers, design specific tag sequences, and then perform primer synthesis again. At this time, specific tag sequences will be added during primer synthesis. This embodiment uses 96 groups of specific tag combinations, specifically:

[0099]According to the specific tag combination, each specific primer was synthesized into 10 primers with different target tags. The primer sequences with the target tags were shown in Table 2. All F (forward primers) with the target tags were combined. and all R (reverse primers), take 10 μl of each primer, and dilute the volume to 10 ml; the concentration of each primer is 0.1 μM, in this example, a total of 96 sets of specific tag combinations are prepared to prepare primer pairs, that is, primer mixtures;

[0100] Table 2 Primer sets

[0101]

[0102] "FFF" is the F-terminal universal primer sequence, and the F-terminal universal primer sequen...

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Abstract

The invention relates to an mSNP technology based mixed sample detection method for detecting the purity of carrot seeds. The method is carried out by utilizing a primer pair 1F / R-21F / R, and the gene sequence of the primer pair 1F / R-21F / R is as shown in SEQ ID No.1-42. 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; and a mixed sample 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 a 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, in particular to a mixed sample detection method for detecting the purity of carrot seeds based on mSNP technology. Background technique [0002] Carrot is a varietal of the wild carrot species of the genus Daucus in the family Umbelliferae, a biennial herb. Carrots have high nutritional value and health care functions, and are widely grown. They have a cultivation history of about 2,000 years and are most distributed in Asia, Europe and the Americas. At present, the main types of carrots cultivated in the world are AmsterdamForcing, Nantes, Imperator, Danvers, Chantenay, Oxheart, Kuroda, Flakkee and Berlicum. Among them, Nantes and Kuroda are the most widely cultivated carrot types in the world. There are many varieties used in production in my country, and Kuroda is still the main variety. Because most of the Heitian series are conventional seeds, some are imported from abroad, some are s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6858C12N15/11
CPCC12Q1/6895C12Q1/6858C12Q2600/156C12Q2531/113C12Q2535/122C12Q2563/143C12Q2563/149C12Q2563/185
Inventor 张丛许彦芬李凝刘景艺张萌郝军会刘田龚舒高苗
Owner 石家庄博瑞迪生物技术有限公司
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