Method for synchronous detection of sweet potato feathery mottle virus (SPFMV), sweet potato virus C (SPVC), sweet potato virus G (SPVG) and sweet potato virus 2 (SPV2)

Abstract

The invention relates to method for a synchronous detection of sweet potato feathery mottle virus (SPFMV), sweet potato virus C (SPVC), sweet potato virus G (SPVG) and sweet potato virus 2 (SPV2), and belongs to the field of plant protection. The method comprises the following steps of respectively designing and synthesizing specific forward primers and a universal reverse primer of SPFMV, SPVC, SPVG and SPV2, extracting total RNA of tissue of a diseased plant through a cetyltrimethyl ammonium bromide (CTAB) method, and carrying out one step quadruplex reverse transcription-polymerase chain reaction (RT-PCR) magnification process to realize a synchronous detection of SPFMV, SPVC, SPVG and SPV2. Primers designed by the method have strong singularities and SPFMV, SPVC, SPVG and SPV2 share areverse primer thus an interaction of primers is reduced. In the invention, a CTAB method is utilized for extracting RNA from tissue of an RNA virus infected plant, thus a quality of RNA is guaranteed and a detection cost is reduced effectively; and the inverse transcription and the multiple PCR are completed in one step thus a detection time is saved. The method has the advantages of rapid detection speed, high efficiency, strong singularity, high sensitivity and low cost and can realize a synchronous detection of four kinds of sweet potato virus thus has wide application prospects.

Description

Technical field: [0001] The invention relates to a method for synchronously detecting SPFMV, SPVC, SPVG and SPV2, which belongs to the field of plant protection. Background technique: [0002] Sweet potato is the seventh largest food crop in the world and the fifth largest food crop in developing countries after rice, wheat, corn and cassava, with an annual output of 127 million tons. China is the world's largest sweet potato producer, with an annual output accounting for 82.7%. Sweet potatoes mainly rely on seedlings and tubers for asexual reproduction, which can easily cause the spread and accumulation of viruses. Virus disease is one of the most restrictive factors for sweet potato production after the sweet potato weevil. The annual loss of sweet potato due to virus infection is as high as 30%-50%. The annual loss of sweet potato virus disease in China is as high as 4 billion yuan. . According to reports, there are more than 20 kinds of viruses infecting sweet potato,...

AI Technical Summary

Problems solved by technology

The ELISA method has a short detection cycle and high sensitivity, and can detect large-scale samples at one time. However, because SPFMV, SPVC, SPVG, and SPV2 are very closely related in phylogenetic evolution, they have some serological cross-reactions, which often result in the leakage of some viruses. false positive

Molecular biology technology, especially RT-PCR technology is not only simple to operate, but also has higher specificity and sensitivity than ELISA method. However, ordinary RT-PCR method can only detect one virus at a time. Then it is necessary to carry out a separate detection for each virus, which requires a lot of work and is not efficient

In addition, before the usual RT-PCR detection, it is necessary to use expensive RNA extraction reagents to extract total RNA from diseased tissues, resulting in high detection costs

In many existing multiple RT-PCR detection technologies for plant viruses, each virus requires a pair of detection primers, which increases the probability of interaction between primers, often easily leads to false negatives and non-specificity, and affects the accuracy of detection results.

[0004] The patent application No. 200810049503.2 discloses a multiplex RT-PCR detection method capable of simultaneously detecting three sweet potato viruses of sweet potato latent virus (SPLV), SPVG and SPFMV. Recently, the primers designed by this method cannot distinguish between SPFMV and SPVC, and the primer SEQ6 can combine with the genomic RNA of SPVG and the genomic RNA of SPV2

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