Method for detecting bean pod mottle virus based on RPA technology, RPA primer and kit

A mottle virus, bean pod technology, applied in microorganism-based methods, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problems of reducing soybean quality, agricultural production loss, soybean yield loss, etc. Easy-to-judge, wide-ranging effects

Inactive Publication Date: 2017-05-31
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Depending on the time of virus infection, the virus can cause 3% to 52% yield loss and reduce soybean quality. If mixed with soybean mosaic virus infection,

Method used

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  • Method for detecting bean pod mottle virus based on RPA technology, RPA primer and kit
  • Method for detecting bean pod mottle virus based on RPA technology, RPA primer and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] This embodiment provides an RPA primer and its application.

[0053]The screening method for RPA primers is as follows: Aiming at the conserved sequence of the bean pod mottle virus genome (Genbank accession number M62738.1), design RPA primers for detecting or assisting the detection of bean pod mottle virus, design multiple RPA primers and conduct a large number of screening In the experiment, a pair of RPA primers with good specificity, high sensitivity and wide application range were finally screened out based on the specificity, sensitivity and the adaptability of the primers used to the RPA amplification kit.

[0054] The specific sequence of this RPA primer is as follows:

[0055] Upstream primer: 5'-TATTTGTATGCTATGGTGCATGATAGTTCAGTGTC-3' (SEQ ID NO: 1);

[0056] Downstream primer: 5'-TGTTCTCACTGTTGCCATTTGATTCCAAAC-3' (SEQ ID NO: 2).

[0057] The above RPA primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0058] The above RPA primers were u...

Embodiment 2

[0061] This embodiment provides a kit and application thereof, the kit comprising:

[0062] (1) plant total RNA extraction reagent;

[0063] (2) reverse transcription reagents;

[0064] (3) upstream primer and downstream primer (from embodiment 1);

[0065] (4) tube containing freeze-dried enzyme powder;

[0066] (5) rehydration buffer;

[0067] (6) Magnesium acetate solution.

[0068] The plant total RNA extraction reagent is the reagent from Tiangen Biotechnology Co., Ltd. Plant Total RNA Extraction Kit (Cat. No.: DP432); the reverse transcription reagent is from TaKaRa Company PrimeScript RT-PCR Kit (Cat. No.: RR014A) reagent.

[0069] The above-mentioned tubes containing lyophilized enzyme powder, rehydration buffer (Rehydration Buffer) and magnesium acetate solution (280mmol / L) are all derived from RPA amplification kit TwistAmp Basic kits (TwistDX company, product number: TABAS03KIT).

[0070] Experiments have proved that the combined use of the above-mentioned pla...

Embodiment 3

[0073] This embodiment provides a method for detecting or assisting in the detection of bean pod mottle virus, such as detecting or assisting in detecting whether a plant sample to be tested is infected with bean pod mottle virus, or detecting or assisting in detecting whether the virus to be tested is or is a candidate for bean pod mottle virus Virus, the method uses the RPA primer of embodiment 1 and the test kit of embodiment 2.

[0074] Above-mentioned method comprises the steps:

[0075] 1. RNA extraction and cDNA synthesis

[0076] The plant total RNA extraction kit with the article number DP432 from Tiangen Biotechnology Co., Ltd. was used to extract the total RNA of the plant sample or the virus to be tested according to its instructions; and the PrimeScriptRT-PCR Kit with the article number RR014A from TaKaRa was used to extract Instructions The total RNA extracted above was used as a template to reverse transcribe to obtain cDNA, and the obtained cDNA was stored at ...

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Abstract

The invention provides a primer pair, wherein one primer comprises the nucleotide sequence shown as the SEQ ID NO: 1, the other primer comprises the nucleotide sequence shown as the SEQ ID NO: 2. The invention also provides the application of the primer pair to detection or aided detection on bean pod mottle virus, or to detection or aided detection on bean pod mottle virus products and a method for detecting the bean pod mottle virus based on a RPA technology. Experiments show that the method for detecting the bean pod mottle virus based on the RPA technology is good in specificity and high in sensitivity, the detection time is short, a special instrument is not needed, the applicable range is wide, and guiding significance is realized in import and export plant and product testing.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to an RPA method, primers and a kit for detecting bean pod mottle virus. Background technique [0002] Bean pod mottle virus is an important virus that harms soybeans and other leguminous plants. At present, there is no distribution of this virus in my country. It mainly occurs in soybean-producing areas such as the United States, Canada, Brazil, Peru, and Ecuador. The natural hosts include soybeans, cowpea, Common bean, the experimental host has 20 genera and 25 species of plants, which can be spread by sap inoculation, beetle dispersal and long-distance seed dispersal. [0003] Depending on the time of virus infection, the virus can cause 3% to 52% yield loss and reduce soybean quality. If mixed with soybean mosaic virus infection, it can cause up to 85% soybean yield loss. Once colonized, it will be difficult to eradicate and will cause great losses to my country's agricultural...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/94
CPCC12Q1/701C12Q1/6844C12Q2521/507C12Q2522/101C12Q2531/119
Inventor 张永江相宁魏霜付伟刘建华李桂芬魏梅生
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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