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Multiplex detection method based on suspension chip system and aiming at drug-resisting related loci of botrytis cinerea

A botrytis cinerea and suspension chip technology, applied in the field of molecular biology, can solve the problems of short life cycle, rapid disease spread, large spore production, etc., and achieve the effect of good repeatability, good specificity and high sensitivity

Inactive Publication Date: 2016-11-16
BEIJING UNIV OF TECH
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Problems solved by technology

Due to the short life cycle of Botrytis cinerea, large spore production, rapid disease spread, and the long-term and widespread use of a single type of fungicide and unreasonable spraying techniques, Botrytis cinerea has generally produced certain conventional fungicides. drug resistance, and even multi-resistance
[0003] The reported molecular detection methods of Botrytis cinerea resistance loci can only detect one fungicide, so these molecular detection methods are not enough to meet the needs of agricultural production, and there is an urgent need for a fast, simple, high-throughput The detection method realizes the simultaneous detection of multiple fungicide resistance mutations

Method used

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  • Multiplex detection method based on suspension chip system and aiming at drug-resisting related loci of botrytis cinerea
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  • Multiplex detection method based on suspension chip system and aiming at drug-resisting related loci of botrytis cinerea

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Embodiment Construction

[0012] Application of suspension chip technology to a strain of BenA-E198(GAG), BenA-198A(GCG), SdhB-H272(CAC), SdhB-272Y(TAC), BcOS1-I365( High-throughput detection of eight genotypes of ATC), BcOS1-365S (AGC), erg27-F412 (TTC), and erg27-412S (TCC).

[0013] In the first step, use the genomic DNA of the strain or sterile water without a template as a template, and use four pairs of primers to simultaneously amplify BenA, SdhB, BcOS1, and erg27 including drug-resistant mutation sites in the same reaction system For the four gene fragments, the sample and the blank control are duplicated. The 25μl reaction system is shown in Table 1:

[0014] Table 1 Multiplex PCR reaction system

[0015]

[0016] PCR reaction program: 94°C for 5min; 35 cycles of 94°C for 30sec, 54°C for 30sec, and 72°C for 25sec; 72°C for 10min; store at 4°C until use.

[0017] In the second step, ExoSAP-IT processes the quadruple PCR products to remove redundant primers and deoxynucleotides (dNTPs). T...

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Abstract

The invention provides a multiplex detection method based on a suspension chip system and aiming at drug-resisting related loci of botrytis cinerea, relates to the field of molecular biology, and in particular relates to a multiplex detection technology adopting the suspension chip system, being in combining with an ASPEPCR method, and aiming at grey mould drug-resisting loci related to drug resistance of four fungicides. The invention discloses the multiplex detection method based on the suspension chip system and aiming at drug-resisting mutation loci of botrytis cinerea. According to the multiplex detection method, the purpose of simultaneously detecting genotypes relevant to drug-resisting types of the four fungicides is realized through five sequential steps including multiple PCR, EXO / SAP treatment on the PCR product, multiple ASPR PCR treatment, hybridization of ASPR PCR products with biotin labels and magnetic beads, and Bio-Plex 200 system detection. The multiplex detection method has the advantages of high flux, high sensitivity, good specificity, good repeatability, small number of needed samples, and the like.

Description

technical field [0001] The present invention relates to the field of molecular biology, in particular to a suspension chip system combined with Allele Specific Primer Extension (Allele Specific Primer Extension, ASPE) PCR method to target Botrytis cinerea resistance related to four kinds of fungicide resistance Point multiple detection technology. Background technique [0002] Gray mold is an important worldwide plant fungal disease caused by Botrytis cinerea Pers. The pathogen has a wide range of hosts and harms a variety of vegetables, economic crops, food crops, and ornamental plants. Botrytis cinerea has been listed as one of the top ten fungal diseases in the world. Due to the lack of disease-resistant varieties at present, the prevention and control of Botrytis cinerea mainly adopts the method of applying a large amount of chemical fertilizers and pesticides, which causes many problems such as environmental pollution, soil damage, and frequent occurrence of drug-resi...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q2535/125C12Q2537/143C12Q2563/107
Inventor 马雪梅张鑫吕宝北赵鹏翔谢飞苏泽华
Owner BEIJING UNIV OF TECH
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