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Degenerate primers for universal reverse transcription-polymerase chain reaction (RT-PCR) detection of cowpea mosaic virus and fabavirus, detection method and application thereof

A cowpea mosaic virus, RT-PCR technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve problems such as unestablished cowpea mosaic virus genus, and achieve convenient use Effect

Inactive Publication Date: 2011-08-03
INSPECTION & QUARANTINE TECH CENT OF XIAMEN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2007, Ferrer et al established the fabavirus genus ( Fabavirus ) virus universal RT-PCR detection and identification method, but the cowpea mosaic virus genus ( Comovirus ) or a universal RT-PCR method that can be used for both virus genera

Method used

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  • Degenerate primers for universal reverse transcription-polymerase chain reaction (RT-PCR) detection of cowpea mosaic virus and fabavirus, detection method and application thereof
  • Degenerate primers for universal reverse transcription-polymerase chain reaction (RT-PCR) detection of cowpea mosaic virus and fabavirus, detection method and application thereof
  • Degenerate primers for universal reverse transcription-polymerase chain reaction (RT-PCR) detection of cowpea mosaic virus and fabavirus, detection method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] 1. Primer design and synthesis

[0017] Primers were designed according to the RNA1 genome sequences of RaMV, SqMV, RCMV, CPMV, CPSMV, BPMV, and BBWV1 viruses in Cowpea mosaic virus and Fabavirus genus, and the primer sequences were:

[0018] SEQ ID No.1: 5'-TGYGAYTAYDVHWSWTTYGATGG-3';

[0019] SEQ ID No. 2: 5'-AKYARRTTRTCATCHCCATA-3'.

[0020] 2. Extraction of total RNA

[0021] 1) Take 0.1 g of diseased leaves, add 1 ml of TRIzol reagent (Invitrogen, USA), transfer to a sterilized 1.5 mL centrifuge tube, and keep at room temperature for 5 min;

[0022] 2) Add 0.2 mL of chloroform, shake vigorously for 15 s, then keep at room temperature for 2-15 min, then centrifuge at 12000 g for 15 min at 4°C;

[0023] 3) Transfer the upper aqueous phase to a new 1.5 mL centrifuge tube, add 0.5 mL isopropanol, invert and mix well, and keep at room temperature for 15 min;

[0024] 4) Centrifuge at 12,000 g for 10 min at 4°C, and the RNA will form a precipitate on the side wall an...

Embodiment 2

[0032] Versatile Detection of Cowpea Mosaic Virus and Faba Bean Virus Generic RT-PCR Method

[0033]Cowpea mosaic virus (CPMV), cowpea heavy mosaic virus (CPSMV), bean pod mottle virus (BPMV), radish mosaic virus (RaMV), red clover mottle virus (RCMV), squash mosaic virus (SqMV), Total RNA was extracted from diseased leaves and healthy broad bean leaves of 9 isolates of 7 viruses including Broad Bean Wilt Virus No. 1 (BBWV1) according to the method in Example 1, and then amplified by RT-PCR, and the results were detected by agarose gel electrophoresis. Results A DNA fragment of about 350 bp was detected in the diseased leaves. Test results such as figure 1 shown. It shows that the established general RT-PCR method can be used for the detection of different virus species in these two virus genera, has good versatility, and can be used for the general detection of cowpea mosaic virus and faba bean virus.

Embodiment 3

[0035] The specific detection of the general RT-PCR method of cowpea mosaic virus genus and faba bean virus genus adopts the nematode-transmitted polyhedronvirus genus virus belonging to the same family of cowpea mosaic virus subfamily (respectively ArMV, TRSV, ToRSV, TBRV, PRMV, CLRV, RpRSV) diseased leaves, extract total RNA according to the method in Example 1, perform RT-PCR amplification, and perform specific detection. Test results such as figure 2 shown. It shows that the established general RT-PCR method will not cross-react with viruses of the same genus, has good specificity, and meets the detection requirements.

[0036] Sequence List

[0037] Xiamen Entry-Exit Inspection and Quarantine Bureau Inspection and Quarantine Technology Center

[0038] Degenerate primers for universal RT-PCR detection of cowpea mosaic virus and faba bean virus

[0039]

[0040] 2

[0041] Patent In version?

[0042] 1

[0043] 23

[0044] DNA

[0045] Artificial sequence...

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Abstract

The invention discloses degenerate primers for universal reverse transcription-polymerase chain reaction (RT-PCR) detection of cowpea mosaic virus and fabavirus, a detection method and application thereof. The degenerate primers consist of a positive primer and a negative primer; the nucleotide sequence of the positive primer is shown as SEQIDNo.1:5'-TGYGAYTAYDVHWSWTTYGATGG-3'; and the nucleotide sequence of the negative primer is shown as SEQIDNo.2: 5'-AKYARRTTRTCATCHCCATA-3', wherein Y=C / T, D=G / A / T, V=G / A / C, H=A / T / C, W=A / T, S=G / C, K=G / T and R=A / G. The detection method can quickly and accurately judge whether a sample contains the cowpea mosaic virus and fabavirus, variety identification is carried out according to a sequence, and guarantee for import and export safety is provided.

Description

technical field [0001] The invention relates to a degenerate primer for universal RT-PCR detection of viruses belonging to the genus Cowpea Mosaic Virus and the genus Faba Bean Virus, a detection method and application thereof. Background technique [0002] Cowpea mosaic virus ( Comovirus ) and Fabavirus ( Fabavirus ) belonging to the order Picornaviridae ( Picornavirales ), associated Vignaviridae ( Secoviridae ), Cowpea mosaic virus subfamily ( Comovirinae ) in 2 virus genera. Cowpea mosaic virus genus contains 15 kinds of viruses, the host range of a single virus is narrow, most of them are limited to a few legume plants, cowpea mosaic virus ( Cowpea mosaic virus , CPMV) is its representative species. The fabavirus genus contains 4 viruses with a wide host range, faba bean wilt virus No. 1 ( Broad bean wilt virus 1 , BBWV-1) is its representative species. These two virus genera are a class of viruses that pose an important threat to agricultural production s...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/70C12Q1/68
Inventor 林石明廖富荣黄蓬英吴媛
Owner INSPECTION & QUARANTINE TECH CENT OF XIAMEN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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