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Molecular marker method for rapidly detecting panonychus citri pyrethroid resistance

A technology of Panonychus citrus and pyrethroids, applied in the field of molecular biology, can solve the problems of increasing the time and complexity of operations, difficulty in extracting DNA from single-headed mites, etc., and achieves saving detection costs, large sample volume, and time saving Effect

Inactive Publication Date: 2015-07-01
SOUTHWEST UNIVERSITY
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Problems solved by technology

However, there is currently no rapid and efficient method for diagnosing molecular markers of pyrethroid resistance in Panonychus citrus mites
Moreover, the current relevant molecular diagnostic techniques need to extract the DNA of the sample first, and it is very difficult to extract DNA from single-headed mites, which greatly increases the time and complexity of the operation.
In addition, there is currently no detection method for the resistance-related mutation sites of the sodium ion channel gene of Panonychus citrus

Method used

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  • Molecular marker method for rapidly detecting panonychus citri pyrethroid resistance

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Embodiment Construction

[0025] The sources of reagents used in the embodiments of the present invention are as follows:

[0026] 2 × Phire animal tissue PCR buffer (Thermo Scientific company, the United States)

[0027] Phire hot start II DNA polymerase (Thermo Scientific, USA)

[0028] FastDigest Green Buffer (Thermo Scientific, USA)

[0029] BclI enzyme (Thermo Scientific company, USA)

[0030] PrimeSTAR Max premix (2×) (Takara, Japan)

[0031] The molecular marker method for rapidly diagnosing the pyrethroid resistance-related genes of Panonychus citrus mites, according to the following steps:

[0032] 1) Preparation of PCR amplification template: take Panonychus citrus as a sample, put each sample into a 200 μL PCR tube, add 3 μL ddH 2 O. Prepare a 10 μL small tip, burn the tip of the tip into a closed ball on the fire, and cool it down. Grind the Panonychus citrus in the PCR tube with the prepared small tip until there is no obvious tissue. The ground sample was used as the template for t...

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Abstract

The invention relates to a molecular marker method for rapidly detecting the pyrethroid resistance of panonychus citri. The method comprises the following steps: (1) preparing a PCR amplification template, wherein a single panonychus citri is adopted as a sample; (2) carrying out nested PCR amplification. In the first round of PCR, the sample prepared in the step 1 is adopted as an amplification template, and PCR amplification is carried out with primers A and B; and in the second round of PCR, the product of the first round of PCR is adopted as an amplification template, and PCR amplification is carried out with primers C and D; (3) carrying out enzyme digestion, wherein the product of the second round of PCR is subjected to an enzyme digestion reaction with BclI enzyme; and (4) carrying out electrophoresis, wherein the reaction liquid after enzyme digestion in the step 3 is subjected to electrophoresis with 1.5% agarose gel, and whether bands appear at 307bp and 281bp is observed. According to the method, a DNA extraction step is avoided, and single panonychus citri F1538I mutation is rapidly detected directly with nested PCR and enzyme digestion.

Description

technical field [0001] The invention belongs to the technical field of molecular biology and relates to a molecular marker, in particular to a molecular marker method for rapidly detecting the pyrethroid resistance of the citrus panonylus mites. Background technique [0002] Panonychus citrus is a worldwide harmful mite, which has a great impact on the yield and quality of citrus. It has attracted widespread attention because of its wide distribution, serious damage, and rapid development of resistance. At present, chemical control is still an important measure to control Panonychus citrus. However, improper application of chemical pesticides not only affects the safety of fruits, but also leads to serious drug resistance. Pyrethroids are a kind of insecticidal (mite) effective, low toxicity and environmentally friendly pesticides, which are widely used in pest control. However, Panonychus citrus has developed serious drug resistance to this kind of insecticide, which grea...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6848
Inventor 王进军蒋玄赵丁天波廖重宇
Owner SOUTHWEST UNIVERSITY
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