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Kit and method for detecting reversely transcribed PCR molecule of cowpea severe mosaic virus

A technology for molecular detection and leaf virus, which is applied in the direction of biochemical equipment and methods, and microbial measurement/inspection, can solve the problems of not being fast enough, sensitive, accurate, and difficult to be suitable for quarantine ports, etc., and achieve good specificity and good application prospects , Highly repeatable effect

Inactive Publication Date: 2012-07-18
ANIMAL AND PLANT & FOOD DETECTION CENTER JIANGSU ENTRY EXIT INSPECTION AND QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0006] At present, CPSMV is mostly detected by DAS-ELISA kit (i.e., double-antibody sandwich enzyme-linked immunosorbent assay), but this method is not fast, sensitive, and accurate enough to be suitable for use at quarantine ports

Method used

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  • Kit and method for detecting reversely transcribed PCR molecule of cowpea severe mosaic virus
  • Kit and method for detecting reversely transcribed PCR molecule of cowpea severe mosaic virus
  • Kit and method for detecting reversely transcribed PCR molecule of cowpea severe mosaic virus

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Effect test

Embodiment 1

[0022] Embodiment 1 cowpea heavy mosaic virus reverse transcription PCR molecular detection kit

[0023] The composition of cowpea heavy mosaic virus reverse transcription PCR molecular detection kit of the present invention is as follows:

[0024] dNTP mix (10mmol / L), RNase-free ddH 2 O, 5×RT Buffer, RNase inhibitor (40U / μL), RTase (200U / μL), 10×PCR Buffer (without Mg 2+ ), MgCl 2 (25mM), dNTP mixture (2.5mM), CPSMVr (10μM), CPSMVf (20μM), CPSMVr (20μM), Taq DNA polymerase (5U / μL), ddH 2 O and positive control.

[0025] Among them, the design process of CPSMVf and CPSMVr is as follows: Search through GeneBank (http: / / www.ncbi.nlm.nih.gov), design a pair of oligonucleotides according to the conserved sequence in the CPSMV coat protein coding gene (accession numbers: NC003544, M83309) Nucleotide primers, using CPSMV (American Type Culture Collection standard strain number PV-273) as a template to amplify the product by reverse transcription PCR (RT-PCR) and then sequence, a...

Embodiment 2

[0029]Embodiment 2 utilizes kit of the present invention to detect susceptible cowpea leaf and seed samples

[0030] 1. extract the RNA of sample and described cowpea heavy mosaic virus freeze-dried powder respectively

[0031] The RNA of the sample and the lyophilized powder of cowpea heavy mosaic virus were extracted by Trizol method.

[0032] Using CPSMV standard strain (American Culture Collection No. PV-273) as material, inoculate susceptible cowpea variety (Blackeye Early Ramshorn), after the onset, collect leaves and diseased seeds, and use the seeds and leaves of the diseased plants as samples.

[0033] Preparation of seed samples: according to the symptoms and characteristics of cowpea infection after the virus, select seeds with small grains and flat grains, and after surface disinfection with 3% (m / v) sodium hypochlorite for 10 minutes, wash three times with sterile water, and select seed embryos , add liquid nitrogen, fully grind into powder, and make samples.

...

Embodiment 3

[0066] Embodiment 3 utilizes kit of the present invention to detect the specificity of reverse transcription PCR of cowpea heavy mosaic virus

[0067] 1. Specificity verification material and viral RNA extraction

[0068] 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), Black-eyed Cowpea Mosaic Virus (BlCMV) and Soybean Mosaic Virus (SMV) of another important seed-borne virus Potatovirus Y on cowpea, carry out cowpea heavy mosaic virus ( CPSMV) RT PCR specificity verification. The virus sources above are from the American Type Culture Collection (ATCC). Viral RNA was extracted using the Trizol method.

[0069] 2. Reverse transcription reaction

[0070] Prepare the following mixes in PCR tubes:

[0071] CPSMVr (10 μM): 1 μL

[0072] dNTP mix (10mmol / L): 1μL

...

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Abstract

The invention provides a kit and a method for detecting the reversely transcribed PCR molecule of a cowpea severe mosaic virus, belonging to the technical field of biology. The kit comprises primers CPSMVf and CPSMVr, wherein the CPSMVf is 5'-ACACARGTNMGNCCNGATAC-3', R=A or G, N=I and M=A or C; and the CPSMVr is 5'-GCTACNGGACTRCTRCTCA-3', N=I and R=A or G. The detection steps are that: the RNA ofa sample is extracted and reverse transcription reaction is conducted by taking the CPSMVr as the primer; and PCR reaction is conducted by taking the reverse transcription product as a template and by taking CPSMVf and CPSMVr as the primers, electrophoresis is conducted and the sample is infected by CPSMV if a specific amplified band exists at 356bp of the RNA of the sample. The method has the advantages of rapidness, simple and convenient operation, good specificity, high repetitiveness and the like.

Description

technical field [0001] The invention relates to a kit for detecting Cowpea severe mosaic virus (CPSMV) and an application thereof, belonging to the field of biotechnology. Background technique [0002] Cowpea heavy mosaic virus (CPSMV) is an established species belonging to the genus Comovirus of 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. [0004] CPSMV can be transmitted over long distances through seeds, wherein the seed transmission rate ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 李彬粟寒吴翠萍李艳华陈贯源郑斯竹杨静安榆林
Owner ANIMAL AND PLANT & FOOD DETECTION CENTER JIANGSU ENTRY EXIT INSPECTION AND QUARANTINE BUREAU
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