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Method for fast detecting botryis cinerea's resistance to QoI type bactericides and primer composition

A technology for Botrytis cinerea and detection primers, applied in the biological field, can solve the problems of expensive thermal cycler, time-consuming, low accuracy, etc., achieve the effect of getting rid of the dependence of thermal cycler, saving instruments and equipment, and increasing application value

Active Publication Date: 2015-12-09
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] Purpose of the invention: In view of the time-consuming, laborious, high cost, low accuracy and other shortcomings in the existing Botrytis cinerea resistance identification method for QoI fungicides and PCR detection technology requires expensive thermal cycle equipment and cumbersome electrophoresis operation process, Unable to meet the needs of rapid detection

Method used

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  • Method for fast detecting botryis cinerea's resistance to QoI type bactericides and primer composition
  • Method for fast detecting botryis cinerea's resistance to QoI type bactericides and primer composition
  • Method for fast detecting botryis cinerea's resistance to QoI type bactericides and primer composition

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Embodiment 1

[0042] The specific experiment of embodiment 1LAMP reaction primer composition

[0043] In order to verify the specificity of the LAMP reaction primer composition, LAMP primers were designed based on the mutation of the 143rd amino acid codon GGT (Gly) → GCT (Ala) of the Botrytis cinerea cytb gene (AB262969.1), and the mutation position The point is located at the 3' end of the forward internal primer FIP, and any or any two bases are carried out for mismatch mutation between the upstream and downstream of the mutation site. A total of 7 sets of LAMP primers are designed, which are sensitive to Botrytis cinerea. The bacterial strain and the DNA of Botrytis cinerea resistant genotype G143A to QoI fungicides were used as templates for LAMP experiments, and a LAMP primer composition capable of specifically detecting the resistance of Botrytis cinerea to QoI fungicides was optimized.

[0044] The specific sequences of each primer are as follows:

[0045] FIP: 5'-CCAATTCATGGTACAGC...

Embodiment 2

[0050] Embodiment 2 LAMP reaction system and detection kit system optimization

[0051] In order to save the identification cost and ensure the stability and reliability of the identification method, BstDNA polymerase (8U / μL) (0.8-4.0U), Mg 2+ (25mM) concentration (0.6-2.0μL), primer FIP / BIP (40μM) and F3 / B3 (10μM) concentration (0.1-0.5μL), betaine (8M) concentration (0.4-2.0μL), HNB (2.5mM ) concentration (0.4-1.2μL) was optimized, and the best system (1mL detection solution) of each component in the kit was determined to be: 1mL detection solution, the detection solution comprising: 1.2μM forward internal primer FIP, 1.2 μM reverse inner primer BIP, 0.3 μM forward outer primer F3, 0.3 μM reverse outer primer B3, 60 μL 10×ThermoPolBuffer, 1.5 mM MgCl 2 , 1.0mMdNTPs, 0.2M betaine, 1.5mM hydroxybromophenol blue (HNB), 160UB stDNA polymerase, prepared by adding sterile ultrapure water, 100 packs, transported at low temperature, stored at -20°C, valid for 1 year.

[0052] Wher...

Embodiment 3

[0057] Embodiment 3 LAMP reaction parameter optimization

[0058] In order to obtain the most suitable reaction temperature and time and ensure the high efficiency of the detection method, when the time is set to 60min, the reaction temperature is optimized at 52-68°C; when the temperature is set at 60°C, the reaction time is optimized at 15-120min; After observing the color change and the gel electrophoresis ladder strip, it is concluded that the reaction temperature is 59-61°C ( figure 1 , figure 2 ) and the reaction time is 45 ~ 120min ( image 3 , Figure 4 ) can achieve LAMP amplification.

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Abstract

The invention discloses a method for fast detecting botryis cinerea's resistance to QoI type bactericides and a primer composition. The method is based on the loop-mediated isothermal amplification technology. The LAMP detecting primer composition comprises a forward inner primer FIP, a backward inner primer BIP, a forward outer primer F3 and a backward outer primer B3. The method has the advantages that the method is simple and convenient, good in practicality, high in sensitivity, high in specificity and high in accuracy, isothermal amplification is realized, a new technical platform is provided to the detection of the botryis cinerea's resistance to the QoI type bactericides, the QoI type bactericide resistance groups of botryis cinerea can be monitored, and the development trend of the resistance groups can be learned about timely; the method has important practical significance to manage crop grey mold resistance, reduce pesticide environmental pollution and lower agricultural production cost.

Description

technical field [0001] The invention belongs to the field of biological technology, and specifically relates to a method and a primer composition for detecting Botrytis cinerea to QoI fungicide-resistant genotype G143A strain based on ring-mediated constant temperature amplification technology; it can be used for Botrytis cinerea to kill QoI fungicides The dynamic monitoring and resistance risk assessment of the development of pesticide-resistant populations can provide scientific guidance for the early warning of crop resistance, resistance management and rational use of botrytis cinerea. Background technique [0002] Botrytis cinerea, caused by Botrytis cinerea, is an important worldwide disease. The disease has a wide range of hosts and can infect more than 235 kinds of plants. It occurs all over my country. It not only infects fruits, but also infects stems, leaves, and flowers. It can also cause fruit rot on greenhouse vegetables and cause yield losses of 20 -30%, local...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/645
CPCC12Q1/6844C12Q1/6895C12Q2531/119C12Q2565/125C12Q2563/173
Inventor 段亚冰周明国陈长军杨莹王建新张晓柯
Owner NANJING AGRICULTURAL UNIVERSITY
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