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Method for screening soybean mosaic viruses based on RPA-IAC technique, RPA-IAC primer and kit

A technology of soybean mosaic virus and kit, which is applied in the direction of recombinant DNA technology, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of long biological identification cycle and cumbersome preparation of electron microscope, and reach the screening time Short, specific and sensitive effects

Inactive Publication Date: 2017-10-10
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The cycle of biological identification is long, the preparation of samples for electron microscope identification is cumbersome and requires expensive electron microscopes, and RT-PCR technology requires thermal cycle equipment

Method used

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  • Method for screening soybean mosaic viruses based on RPA-IAC technique, RPA-IAC primer and kit
  • Method for screening soybean mosaic viruses based on RPA-IAC technique, RPA-IAC primer and kit
  • Method for screening soybean mosaic viruses based on RPA-IAC technique, RPA-IAC primer and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] This embodiment provides an RPA-IAC primer pair and internal standard amplification sequence and its application.

[0049] The screening method for RPA-IAC primers is as follows: aiming at the conserved sequence of soybean mosaic virus coat protein gene (Genbank accession number U25673.1), design RPA-IAC primers for screening or assisting screening of soybean mosaic virus, and designed multiple After RPA-IAC primers, a large number of screening experiments were carried out. Based on the specificity, sensitivity and the adaptability of the primers used and the RPA-IAC amplification kit, the final screening was good in specificity, high in sensitivity and wide in application. A pair of RPA-IAC primers.

[0050] The specific sequence of this RPA-IAC primer is as follows:

[0051] Upstream primer: 5'-AAGATGAATCTTCCAATGGTTGAAGGGAAGATC-3' (SEQ ID NO: 1);

[0052] Downstream primer: 5'-CAAGCTCATATTCATCTTTAACTGCATTGTACC-3' (SEQ ID NO: 2).

[0053] Design and synthesis of int...

Embodiment 2

[0059] This embodiment provides a kit and application thereof, the kit comprising:

[0060] (1) plant total RNA extraction reagent;

[0061] (2) reverse transcription reagents;

[0062] (3) SEQ ID NO: 1, SEQ ID NO: 2 (both from Example 1), and a plasmid containing the internal standard amplification sequence shown in SEQ ID NO: 3;

[0063] (4) tube containing freeze-dried enzyme powder;

[0064] (5) rehydration buffer;

[0065] (6) Magnesium acetate solution.

[0066] 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.

[0067] 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)...

Embodiment 3

[0073] This embodiment provides a method for screening or assisting in screening soybean mosaic virus, such as screening or assisting in screening whether a plant sample to be tested is infected with soybean mosaic virus, the method uses the RPA-IAC primer of Example 1 And the plasmid containing the above-mentioned internal standard amplification sequence, or the kit of Example 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 samples to be tested according to its instructions; and the PrimeScript RT-PCR Kit with the article number RR014A from TaKaRa was used to extract the aforementioned The extracted total RNA was used as a template to reverse transcribe to obtain cDNA, and the obtained cDNA was stored at -20°C for future use.

[0077] 2. RPA-IAC amplification

[00...

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Abstract

The invention provides a primer pair and an internal standard amplification sequence. The primer pair has a nucleotide sequence represented by SEQ ID NO: 1 and a nucleotide sequence represented by SEQ ID NO: 2, and the internal standard amplification sequence has a nucleotide sequence represented by SEQ ID NO: 3. The invention further provides an application of the primer pair and the internal standard amplification sequence in the preparation of a product for screening or auxiliary screening of soybean mosaic viruses and a method for screening the soybean mosaic viruses based on an RPA-IAC technique. Experiments prove that the screening method of the soybean mosaic viruses established by combining with the internal standard amplification sequence based on an RPA-IAC primer is good in specificity, high in sensitivity, short in screening time, and wide in application range, has a good quality control effect, does not require a special instrument and has guiding significance to the screening of imported and exported relevant plants and products.

Description

technical field [0001] The invention relates to the field of biological screening, in particular to an RPA-IAC method, primers and kit for screening soybean mosaic virus. Background technique [0002] Soybean mosaic virus (SMV) is a member of the genus Potyvirus in the family Potyviridae. The source of the initial SMV invasion is mainly the infected seeds. The virus will exist in the embryo and cotyledons (the pollen carries the virus), and the virulence can last for two years or even longer. Have the virus and show symptoms. Because SMV is easy to be transmitted by contact, it may become a source of infection in the field no matter whether it is onset or not. Poison-carrying aphids are also another important factor in the natural transmission of SMV, which can cause the virus to re-transmit and have non-persistent characteristics. SMV is a widely distributed and very destructive virus worldwide. The number of pods of the affected plants decreased, the hundred-kernel wei...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/70C12Q1/68
Inventor 张永江袁俊杰相宁魏霜付伟李桂芬魏梅生
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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