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Multi-PCR (Polymerase Chain Reaction) method for synchronously detecting four chilli viruses

A simultaneous detection and RT-PCR technology, applied in the field of crop disease diagnosis and molecular biology, can solve problems such as difficulty in disease identification, aggravation of disease symptoms, and weakening of host resistance.

Inactive Publication Date: 2017-05-31
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in nature, it is very common for the same pepper plant to be infected by two or more viruses at the same time (composite infection). Compound infection can not only weaken the resistance of the host, aggravate

Method used

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  • Multi-PCR (Polymerase Chain Reaction) method for synchronously detecting four chilli viruses
  • Multi-PCR (Polymerase Chain Reaction) method for synchronously detecting four chilli viruses
  • Multi-PCR (Polymerase Chain Reaction) method for synchronously detecting four chilli viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1. Extraction of Capsicum Leaf Tissue Total RNA

[0045] Total RNA was extracted from healthy and co-infected pepper plants with BBWV2, PVY, ChiVMV and CMV using TRIzol reagent from Invitrogen, USA. The operation steps are as follows: 1) After taking fresh or frozen pepper leaves and adding liquid nitrogen to fully grind, quickly transfer 100 mg dry powder tissue to a 1.5 ml sterile centrifuge tube that has been frozen in liquid nitrogen and treated with DEPC; 2) Add 1 mL TRIzol reagent , mix well, and let stand at room temperature for 5 minutes; 3) add 0.2mL chloroform, shake vigorously by hand for 15 seconds, and let stand at room temperature for 2-3 minutes; centrifuge at 12000g / 4°C for 15 minutes; Isopropanol, mix well, place on ice for 10 minutes; centrifuge at 12000g / 4°C for 15 minutes, remove the supernatant; 5) add 1 mL of pre-cooled 75% ethanol (prepared with sterile DEPC water) to the precipitate for washing, 7500g / 4°C Centrifuge for 5 minutes, repeat...

Embodiment 2

[0046] Embodiment 2. The design of four kinds of virus-specific primer pairs

[0047] According to the nucleotide sequences of four viruses such as BBWV2, PVY, ChiVMV and CMV published by NCBI, VectorNTI software was used to design specific primer pairs for each virus. The product does not form a secondary structure, the GC content is between 40%-60%, the bases are not complementary among the four primers, and the primers are synthesized by Shanghai Bioengineering Co., Ltd. (see Table 1 for the conditions of the four primers).

[0048] Table 1. Specific primers in the multiplex PCR detection system of BBWV2, PVY, ChiVMV and CMV four viruses

[0049]

Embodiment 3

[0050] Example 3. The specific detection of four pairs of primers

[0051] The four pairs of primers designed in Example 2 can detect four viruses, BBWV2, PVY, ChiVMV and CMV, respectively. The genus Fabavirus to which BBWV2 belongs includes 5 viruses. Except BBWV2 infecting peppers, the other four viruses are Broad bean wilt virus 1 (BBWV-1), wild sesame light mosaic Lamium mild mosaic virus (LMMV), Cucurbit mild mosaic virus (CMMV) and Gentian mosaic virus (GMV), none of them infect peppers. Similarly, the other three species belonging to the same genus Cucumovirus as CMV - Peanut stunt virus (PSV), tomato infertility virus (Tomato aspermy virus, TAV) and Shepenis light mottled Virus (Gayfeather mildmottle virus, GMMV), also does not infect pepper. However, PVY and ChiVMV both belong to the genus Potyvirus (Potyvirus), and the primers of the two did not amplify the band of the other party, indicating that the primers of the two are unique.

[0052] In order to ensure the ...

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Abstract

The invention belongs to the technical field of crop disease diagnosis and molecular biology and discloses a multi-PCR (Polymerase Chain Reaction) method for synchronously detecting four chilli viruses. A PCR system contains four pairs of specific primers (SEQ ID NO: 1 to NO: 8); In one PCR system, the four viruses are synchronously amplified to obtain 1406bp, 1202bp, 600bp and 376bp specific bands; a multi-RT-PCR reaction system capable of synchronously detecting BBWV2 (Broad Bean Wilt Virus 2), PVY (Potato Virus Y), ChiVMV (Chili Veinal Mottle Virus) and CMV (Cucumber Mosaic Virus) is established and optimized. A detection result shows that the optimized multi-RT-PCR reaction system can be used for rapidly, efficiently and accurately detecting field samples. The multi-PCR method has very important meanings on predication of field occurrence condition and disease epidemics of four common virus diseases including PVY and the like on chilies.

Description

technical field [0001] The invention belongs to the technical fields of crop disease diagnosis and molecular biology, and in particular relates to a system of reverse transcription polymerase chain reaction (RT-PCR) and its application, which can be used for synchronous detection of four RNAs infecting peppers Viruses, to achieve the purpose of accurate, rapid and efficient detection. Background technique [0002] Capsicum (Capsicum spp.) is a plant of the genus Capsicum in the family Solanaceae. It has become a favorite vegetable and condiment because of its unique fruit flavor and rich nutrition. It is also used for the extraction of food pigments, medicines and industrial components. It is widely planted in Asia, Africa, Oceania and other regions. With the increase of pepper demand and trade volume, there is a trend of further expansion. my country's pepper planting area is 1.33 million hm 2 , with a total economic output value of 70 billion yuan, ranking first in the w...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/94
CPCC12Q1/701C12Q2600/16
Inventor 李莉冯耿王锡锋刘文文
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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