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Multiple RT-PCR method for fast detecting four kinds of pepper viruses

An RT-PCR, pepper technology, applied in the directions of microorganism-based methods, biochemical equipment and methods, and the determination/inspection of microorganisms, can solve problems such as reports of multiple RT-PCR detection methods that have not yet been seen, and achieve rapid and accurate detection. The effect of testing requirements, reducing the number of operations, and easy operation

Active Publication Date: 2016-07-20
XINJIANG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, CMV, PMMoV, BBWV and TMV have established single-plex RT-PCR molecular detection techniques, but there are no reports on multiplex RT-PCR detection methods for these four important pepper viruses

Method used

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  • Multiple RT-PCR method for fast detecting four kinds of pepper viruses
  • Multiple RT-PCR method for fast detecting four kinds of pepper viruses
  • Multiple RT-PCR method for fast detecting four kinds of pepper viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1. Design and synthesis of primers

[0052] According to the conserved fragments of the coat protein (CP) gene sequence of CMV, PMMoV, and TMV isolates reported in the literature and GenBank and the conserved fragment of the nucleotide sequence of the non-coding region of the BBWV 5' end part of the genome, Primer5.0 primer design software was used The specific primers for these 4 viruses were designed respectively. Since multiplex PCR requires simultaneous amplification of multiple target fragments at the same annealing temperature, soft DNAMAN is used to calculate the Tm value of the primers, screen a large number of designed primers, and select primers with relatively consistent Tm values ​​to ensure that the primers in the same annealing temperature Four pathogens, CMV, PMMoV, BBWV and TMV, were detected at the same temperature. The detailed virus-specific primer sequences are shown in Table 1, and all primers were synthesized by Shanghai Sangon Bioengineer...

Embodiment 2

[0055] Embodiment 2. The establishment of multiple RT-PCR reaction system

[0056] 1. Extraction of plant total RNA

[0057] About 0.1 μg of fresh or frozen pepper samples to be tested were ground into powder in liquid nitrogen, and the total RNA of the samples was extracted using a plant total RNA extraction kit. Extract RNA product and add DEPC-ddH 2 dissolved in O and stored at -70°C for later use.

[0058] 2. Synthesis of cDNA

[0059] Using the extracted total RNA of the pepper sample to be tested as a template, the cDNA template was prepared by reverse transcription. The reverse transcription system is 20 μL including: RNA template 2 μL, 2.5 mmol / L dNTPs 2 μL, 10 μmol / L random primer 2 μL, 10×RTmix 2 μL, QuantReverseTranscriptase 2 μL, RNase-freeddH 2 O10 μL. Reaction conditions: 37°C, 60min.

[0060] 3. Establish and optimize the multiplex RT-PCR reaction system and reaction procedures

[0061] (1) Hybrid template

[0062] Use the recombinant plasmids PGM-CMV, P...

Embodiment 3

[0080] Example 3. Sensitivity detection of multiple RT-PCR reactions

[0081] In order to determine the detection sensitivity of multiplex RT-PCR, the total RNA (90?ng / μL) of plants co-infected by four viruses containing CMV, PMMoV, BBWV and TMV was diluted to 10 1 ~10 7 Six gradients to obtain dilutions with concentrations of 9.0ng / μL, 0.9ng / μL, 90pg / μL, 9.0pg / μL, 0.9pg / μL, 90fg / μL, and 9fg / μL, and use the dilutions as templates According to the reaction system and procedure in Step 2 of Example 2, cDNA was obtained by reverse transcription, and multiple PCR reactions were performed according to the optimal reaction system and the optimal reaction procedure.

[0082] Take 5?μL of PCR products and electrophoresis in agarose 1×TAE buffer system with a concentration of 15?g / L. After EB staining, use a gel imaging system to observe and record the results with photography.

[0083] The experimental results are attached Image 6 As indicated, the RNA of the four virus co-infecti...

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Abstract

The invention relates to a multiple RT-PCR method for fast detecting four kinds of pepper viruses, and belongs to the field of virus detection.According to the method, specific primer pairs, namely, CMV-F / R, PMMoV-F / R, BBWV-F / R and TMV-F / R are designed and synthesized aiming at the nucleotide conserved sequences of the four kinds of viruses, multiple RT-PCR system optimization is carried out on the primer concentration, Mg2+ concentration, Taq DNA polymerase concentration, dNTPs concentration, annealing temperature and other aspects influencing multiple RT-PCR amplification, and the detecting method capable of detecting field samples compositely infected by the viruses of CMV, PMMoV, BBWV and TMV in pepper tissue at the same time is built.According to the multiple RT-PCR detecting method, the pepper poisoned condition can be detected fast, accurately, conveniently and economically so that effective prevention measures can be taken as early as possible, and very important significance is achieved on control of diseases in the field, reduction of economic losses, screening of disease resistance and predication of disease epidemiology.

Description

technical field [0001] The invention relates to the technical field of plant virus detection, in particular to a multiple RT-PCR method for rapidly detecting four pepper viruses. Background technique [0002] Capsicum (Capsicumannuum L.), which belongs to the genus Capsicum in the family Solanaceae, is an important vegetable crop. It can not only be eaten fresh, but also be processed into various pepper products. Due to the good economic and social benefits of pepper planting, pepper has become the second largest vegetable crop in my country with an annual cultivated area of ​​about 1.33 million hm 2 . However, due to the impact of various diseases, the yield per unit area of ​​pepper in my country is relatively low. Capsicum virus disease is the primary disease in pepper production in my country. In recent years, viral diseases have become more and more serious in pepper growing areas across the country, which has become an important factor affecting the yield and qualit...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/94
Inventor 罗明韩剑张强李克梅赵冰梅张祥林
Owner XINJIANG AGRI UNIV
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