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Molecule detection method for cowpea severe mosaic virus

A molecular detection and leaf virus technology, applied in the biological field, can solve the problems of insufficient speed, sensitivity, and accuracy, and achieve high sensitivity, good specificity, and good repeatability

Inactive Publication Date: 2010-09-01
ANIMAL AND PLANT & FOOD DETECTION CENTER JIANGSU ENTRY EXIT INSPECTION AND QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, quarantine ports mostly use the traditional DAS-ELISA (double-antibody sandwich enzyme-linked immunosorbent assay) technology to detect CPSMV, which is not fast, sensitive and accurate enough.

Method used

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  • Molecule detection method for cowpea severe mosaic virus
  • Molecule detection method for cowpea severe mosaic virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1 utilizes the inventive method to detect cowpea heavy mosaic virus

[0019] The specific detection method is as follows:

[0020] 1. Preparation of sample virus crude extract

[0021] 1.1 Selection and sowing of infected CPSMV seeds: according to the symptoms and characteristics of cowpea infection after the virus, select seeds with small grains and flattened grains, and wash three times with sterile water after surface disinfection with 3% (m / v) sodium hypochlorite for 10 min. , sow the seeds in sterilized soil, and the pots and utensils used are all sterilized.

[0022] 1.2 Selection of infected plants and preparation of virus crude extracts: after the emergence of cowpea seedlings and the flattening of the first pair of true leaves, prepare a sample from a single plant showing suspicious symptoms; for asymptomatic plants, take 1 leaf per plant, A sample was prepared from the leaves of each plant. Each sample contains 0.1 g of fresh leaf tissue, to whic...

Embodiment 2

[0054] Embodiment 2 utilizes the inventive method to detect the specificity of cowpea heavy mosaic virus

[0055] 1. Viral RNA extraction for specificity verification

[0056] Select cowpea mosaic virus (CPMV) and bean pod mottle virus (BPMV) of the same genus as cowpea heavy mosaic virus (comovirus genus Comovirus), another nematode-borne polyhedron virus of the same family (comoviridae) Genus (Nepovirus) Tobacco Ringspot Virus (TRSV), and Black-eyed Cowpea Mosaic Virus (BlCMV), another important seed-transmitted virus on cowpea, Potatovirus Y, were used for RT-Real time PCR of Cowpea Heavy Mosaic Virus (CPSMV) Verification of specificity. The above-mentioned tested viruses were all from the American Type Culture Collection (ATCC). The negative control was the leaves of healthy cowpea.

[0057] Add 1 mL of sample extraction buffer to the leaves (0.1 g) of plants infected with each virus and grind, centrifuge for 10 min at a centrifugal force of 10,000 g, and the supernatan...

Embodiment 3

[0081] Embodiment 3 utilizes the inventive method to detect the sensitivity of cowpea heavy mosaic virus IC-RT-Real time PCR

[0082] 1. Preparation of Cowpea Heavy Mosaic Virus Sample Crude Extract

[0083] Weigh 100mg of cowpea heavy mosaic virus diseased leaf tissue, add 1mL of sample extraction buffer to grind, centrifuge at 10000g for 10min, the supernatant is the virus crude extract, and the concentration of the supernatant is 100mg / mL virus crude extract. Then use the sample extraction buffer to dilute step by step with a 10-fold gradient to a crude virus extract with a concentration of 10 mg / mL, 1 mg / mL, 100 μg / mL, 10 μg / mL, 10 ng / mL, and 1 ng / mL.

[0084] 2. Preparation of Immunocapture PCR Tubes

[0085] Take the CPSMV coating antibody, dilute it to the working concentration with coating buffer, pipette 25 μL into the PCR tube, and place it at room temperature for 4 hours or incubate overnight in a refrigerator at 4°C. Wash 3 times with washing buffer (PBST), add 2...

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Abstract

The invention relates to a molecule detection method for cowpea severe mosaic virus, which belongs to the technical field of biology. The method comprises the following steps of: obtaining coarse virus extract; preparing an immune capturing PCR tube; releasing cowpea severe mosaic virus RNA in the immune capturing PCR tube and taking the cowpea severe mosaic virus RNA as a template, and performing reverse transcription reaction by using CPSMVR of a sequence 5'-GGCTTCTGCAGGTGTTCCAA-3' as a primer to obtain a reverse transcription product; and performing real-time fluorescent PCR reaction by using the reverse transcription product as a template and using a forward primer 5'-GGTCAATCCCGGCATTATTG-3', a reverse primer CPSMVR and a Taq Man probe CPSMVPro 5'FAM-TGTAGCACAATCAGGGCAAACACAGCA-TAMRA 3', wherein when the initial cycle number is less than or equal to 35, a sample is infected with the cowpea severe mosaic virus.

Description

technical field [0001] The invention relates to a detection method of cowpea heavy mosaic virus, in particular to an immunocapture-reverse transcription-real-time fluorescent PCR molecular detection method (or IC-RT-Real time PCR method), which belongs to the field of biotechnology. Background technique [0002] Cowpea severe mosaic virus (CPSMV) is a definitive species of the genus Comovirus in the family Comoviridae. The natural host of the virus is leguminous plants, which can seriously infect various crops such as cowpea and soybean. After the virus infects cowpea, it can cause mosaic and mottled leaves, and in severe cases, the whole plant can die; the infection rate of cowpea in the field can reach 100%, resulting in a loss of yield of up to 50%. [0003] At present, CPSMV is mainly distributed in the Americas, such as the United States, Brazil, Peru, Venezuela, Trinidad, Puerto Rico, Costa Rica, and Suriname. There are no reports of occurrence and harm in China. [...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
Inventor 李彬吴翠萍粟寒李艳华吴晶陈贯源陈青安榆林
Owner ANIMAL AND PLANT & FOOD DETECTION CENTER JIANGSU ENTRY EXIT INSPECTION AND QUARANTINE BUREAU
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