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Real-time fluorescent quantitative PCR (polymerase chain reaction) detection method and kit for resistance of plutella xylostella to Bt insecticidal proteins

A technology of insecticidal protein and diamondback moth, applied in the biological field, can solve the problems of high material requirements, low sensitivity, and poor repeatability, and achieve the effect of less material requirements, high sensitivity and accuracy, and short detection cycle

Inactive Publication Date: 2012-11-07
INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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  • Abstract
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AI Technical Summary

Problems solved by technology

[0007] The present invention aims at the problems of low sensitivity, long period, poor repeatability and high material requirements in the existing Plutella xylostella Bt insecticidal protein resistance detection technology. Establish a fast and accurate real-time fluorescence quantitative PCR molecular detection method and a fast, simple, accurate and efficient detection kit for detecting Bt resistance of diamondback moth, providing an effective tool for effective detection of Bt resistance in diamondback moth

Method used

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  • Real-time fluorescent quantitative PCR (polymerase chain reaction) detection method and kit for resistance of plutella xylostella to Bt insecticidal proteins
  • Real-time fluorescent quantitative PCR (polymerase chain reaction) detection method and kit for resistance of plutella xylostella to Bt insecticidal proteins
  • Real-time fluorescent quantitative PCR (polymerase chain reaction) detection method and kit for resistance of plutella xylostella to Bt insecticidal proteins

Examples

Experimental program
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Effect test

Embodiment 1

[0050] Detection of midgut cDNA samples of 4th instar larvae of DBM1Ac-S and DBM1Ac-R by real-time fluorescence quantitative PCR technology and establishment of the detection kit and its application method.

[0051] 1. Taking the conserved fragment region of the alkaline phosphatase gene in the membrane-bound form on the brush border membrane vesicles of the diamondback moth midgut as the target sequence (sequence 1 in the sequence listing), according to the principle of real-time fluorescent quantitative PCR primer design, design specific The sequences of fluorescent quantitative primers are as follows:

[0052] Forward primer (qALP-F): 5'-GCACACACCATGACCGTAGCAG-3' (sequence 2 in the sequence listing);

[0053] Reverse primer (qALP-R): 5'-GGCTCTTCGTGACATCG-3' (sequence 3 in the sequence listing);

[0054] The specific primer amplifies the fragment sequence as figure 1 As shown, its amplification specificity is as figure 2 (In which, 1: midgut cDNA sample of 4th instar Plu...

Embodiment 2

[0089] Through the other two Bt-resistant diamondback moth populations T 2 -R and SH-R verify the usability of the kit and detection method in Example 1, and the specific experimental process is as shown in Example 1.

[0090] The final fluorescent quantitative PCR reaction test results are as follows: Figure 6 Shown in, wherein, A: negative control sample of the midgut cDNA of the 4th instar larvae of the sensitive Plutella xylostella population DBM1Ac-S to Bt insecticidal protein in embodiment 1; B: produce resistance to Bt Cry1Ac insecticidal protein in embodiment 1 positive control sample of midgut cDNA of 4th instar larvae of Plutella xylostella population DBM1Ac-R; C: cDNA test sample of midgut cDNA of 4th instar larvae of Plutella xylostella population T2-R resistant to Bt Cry1Ac insecticidal protein; D: A test sample of midgut cDNA from the 4th instar larvae of Plutella xylostella population SH-R resistant to Btk.

[0091] Under specific wavelength conditions, accor...

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Abstract

The invention discloses real-time fluorescent quantitative PCR (polymerase chain reaction) detection method and kit for resistance of plutella xylostella to Bt insecticidal protein. The real-time fluorescent quantitative PCR detection kit for detecting the resistance of plutella xylostella to bacillus thuringiensis Bt insecticidal proteins is characterized by containing the following primer sequences: a forward primer: 5'-GCACACACCATGACCGTAGCAG-3', and a reverse primer: 5'-GGCTCTTCGTGACATCG-3'. The real-time fluorescent quantitative PCR detection method for the resistance of plutella xylostella to Bt insecticidal proteins disclosed by the invention has the characteristics of small manual labor and sample amount, large detection amount, high flexibility, strong specificity, rapidness and convenience and accuracy and reliability, thereby being capable of effectively detecting the resistance of plutella xylostella to Bt insecticidal proteins and suitable for early rapid diagnosis of resistance of plutella xylostella to Bt insecticidal protein; and at the same time, the real-time fluorescent quantitative PCR detection method and kit can be used for real-time monitoring and early warning and forecasting of resistance of plutella xylostella to Bt insecticidal protein, so that the application prospect is wide.

Description

technical field [0001] The invention relates to the field of biology, in particular to a real-time fluorescent quantitative PCR detection method and a kit thereof for the resistance of diamondback moth to Bt insecticidal protein. Background technique [0002] Diamondback moth Plutella xylostella (L.), also known as diamondback moth, box moth, belongs to Lepidoptera (Lepidoptera) Plutellidae (Plutellidae), is a kind of important vegetable pests of worldwide damage, with more than 40 kinds of hosts. has become a major obstacle to the production of cruciferous vegetables worldwide. In my country, since the 1970s, diamondback moth has gradually become the main pest of cruciferous vegetables in the middle and lower reaches of the Yangtze River, and its distribution has continued to expand northward (Zhao Jianzhou et al., 1994). The harm of this insect in Northwest China has also become increasingly serious. Plutella xylostella mainly damages leaves with larvae in the whole growt...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N21/64
Inventor 张友军郭兆将吴青君王少丽徐宝云谢文朱勋雷妍圆陈得峰符伟杨家强
Owner INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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