Quadruple RT-PCR method simultaneously detecting multiple kinds of pepper viruses and application of method

Abstract

The invention discloses a quadruple RT-PCR method simultaneously detecting multiple kinds of pepper viruses and an application of the method.The method comprises the steps that firstly, tender pepper blades with mosaic symptoms in a filed are selected, saved on ice and brought back to a laboratory, and RNA is extracted through a Trizol method; secondly, the RNA obtained in the first step serves as a template, 6-base Random Primers serve as reverse primers, and by means of inverse transcription, cDNA is obtained; thirdly, the cDNA obtained in the second step serves as a template, and PCR amplification is conducted on the tender pepper blades through four pairs of primers; fourthly, PCR products are subjected to gel electrophoresis, a gel-imaging system takes pictures, and the virus infection situation is judged according to the size of a target fragment and positive control.According to the detection method, one RT-PCR is used for simultaneously detecting main pepper viruses including a cucumber mosaic virus (CMV), a tobacco mosaic virus (TMV), a potato virus Y (PVY) and a broad bean wild virus-2 (BBWV-2).The kinds of detected virus are increased, the detection efficiency is improved, and the detection cost is low.

Description

technical field [0001] The invention belongs to the technical field of crop virus detection, and relates to a method for simultaneously detecting a variety of pepper viruses by quadruple RT-PCR and its application, more specifically, adopting the quadruple RT-PCR method to simultaneously detect cucumber mosaic virus, the main virus of pepper (CMV), tobacco mosaic virus (TMV), potato virus Y (PVY) and broad bean wilt virus-2 (BBWV-2). Background technique [0002] Capsicum belongs to the Solanaceae Capsicum genus. It is an important vegetable crop and condiment widely planted in Central and South America, Europe, Asia and other regions. It can be eaten fresh or processed into various pepper products. It has rich nutritional value and deep Loved by people, the vitamin C content of capsicum ranks first in vegetables, and the red pigment in capsicum is still a natural food coloring agent widely used by people at present. In recent years, the demand and sales of peppers have bee...

AI Technical Summary

Problems solved by technology

Therefore, the RT-PCR detection of capsicum virus at home and abroad mostly stays in single and double RT-PCR. [9,10] , the RT-PCR detection method of triple and above is rarely reported, although Huang Ya has established a five-fold RT-PCR detection system for pepper PMMoV, ChiVMV, PVY, TSWV and TuMV [11] , but does not contain the most common CMV, TMV and BBWV-2 in pepper production in my country. Therefore, using the existing RT-PCR detection method to detect TMV, CMV, PVY and BBWV-2 viruses requires multiple reactions to complete , time-consuming, laborious, waste of reagent kit consumables, high cost, and also cause environmental pollution

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