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

A technology of RT-PCR and pepper, which is applied in the field of crop virus detection technology, can solve problems such as time-consuming, high cost, and environmental pollution, and achieve the effects of reducing reagents and consumables, improving detection efficiency, and increasing virus types

Inactive Publication Date: 2016-07-13
HUNAN AGRICULTURAL UNIV
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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...

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

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Embodiment 1 4

[0039] The establishment and optimization of embodiment 1 quadruple RT-PCR detection system

[0040] Primer design:

[0041] Among them, TMV, CMV, PVY and BBWV-2 are the main viruses in pepper production in my country. Since there are no reports on multiple RT-PCR detection for these four viruses, in order to ensure the successful establishment of the detection system, we compared and analyzed these four viruses. According to the requirements of multiplex PCR for primers and the conserved sequences of each virus, 2 sets of 8 pairs of primers were designed with Primer 5.0, and a preliminary RT-PCR test was carried out on the 2 sets of primers, and the better working ones were screened out. Group 1, on this basis, redesigned and tested the primers of BBWV2, and finally determined four pairs of primers. The lengths of the four pairs of primers differ by at least 100bp, and there is no or little interaction between the four pairs of primers.

[0042] Primers were synthesized by Sha...

Embodiment 2 4

[0060] Embodiment 2 quadruple RT-PCR detection field capsicum plant sample

[0061] 43 samples of pepper plants with yellowing and mosaic symptoms were investigated in Changsha, the main pepper producing area in Hunan, and brought back to the laboratory with soil, and the young leaves were selected, ground with liquid nitrogen, and RNA was extracted. Repeat RT-PCR to detect whether there is CMV, TMV, PVY and BBWV-2 infection, the test results are shown in Figure 4 , and the results showed that quadruple RT-PCR could well detect the virus in pepper samples from the main producing areas of Hunan. Among the 43 samples, CMV, TMV, BBWV-2 and PVY were detected in 35, 16, 10 and 1 samples, and the detection rates of CMV, TMV, BBWV-2 and PVY were 81.4%, 37.21%, 23.26% and 2.33%, 43 samples were infected by a single virus or a combination of multiple viruses, indicating that the occurrence of CMV in peppers in our sampling area is the most serious, and the occurrence of PVY is the li...

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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...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2537/143
Inventor 胡新喜汪沛熊兴耀
Owner HUNAN AGRICULTURAL UNIV
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