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One-step multiplex reverse transcription-polymerase chain reaction (RT-PCR) method for detecting turnip mosaic virus and special primers for method

A technology of turnip mosaic virus and primer set, which is applied in the field of biology and can solve problems such as cumbersome steps

Inactive Publication Date: 2013-09-18
BEIJING AGRO BIOTECH RES CENT
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The general RT-PCR detection method needs to be carried out in two steps: first extract the total RNA of the sample, and then perform PCR detection after reverse transcription to obtain cDNA, the steps are relatively cumbersome

Method used

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  • One-step multiplex reverse transcription-polymerase chain reaction (RT-PCR) method for detecting turnip mosaic virus and special primers for method
  • One-step multiplex reverse transcription-polymerase chain reaction (RT-PCR) method for detecting turnip mosaic virus and special primers for method
  • One-step multiplex reverse transcription-polymerase chain reaction (RT-PCR) method for detecting turnip mosaic virus and special primers for method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] The design and condition exploration of related primers in embodiment 1, one-step RT-PCR method

[0068] 1. Design of relevant primers in one-step RT-PCR method

[0069] Under natural conditions, Turnip Mosaic Virus (TuMV) is very easy to mutate through gene recombination. According to literature review, no gene recombination site was found in the TuMV-6K1 gene, the sequence was highly conserved, and the C-terminus of the TuMV-CP gene was relatively conserved.

[0070] After sequence comparison of 122 TuMV strains registered in GenBank, according to the 6K1 segment of TuMV (3526-3681 of the coding region of GenBank No. C4) Positions 9074-9450 of the coding region) Two sets of specific primers GSP1 and GSP2 were designed (see Table 1). The designed primers were tested by NCBI BLAST to ensure the uniqueness and accuracy of TuMV detection. Primers were synthesized by Shanghai Jierui Bioengineering Co., Ltd.

[0071] Table 1 is the sequence of two sets of specific prime...

Embodiment 2

[0137] Embodiment 2, one-step multiplex RT-PCR detection

[0138] In order to simplify the detection method, multiplex RT-PCR was further developed on the basis of one-step RT-PCR. Two sets of GSP primers can work in the same system without affecting each other, and the one-step multiple RT-PCR can be applied to the rapid detection of TuMV in actual production.

[0139] Carry out one-step multiplex RT-PCR amplification detection according to the reaction system and reaction program of 2 in 2 of Example 1, where the difference is that the primers are GSP1, GSP2, Oligo d(T)18Primers and Random Primer added to the system , the templates are respectively the crude extract of the sample to be tested numbered 1, the crude extract of the sample to be tested numbered 2, and the crude extract of the April slow rapeseed leaf infected with C4, and the April slow rapeseed that is not infected with any disease source as a negative control.

[0140] If the amplified product is 156bp and / o...

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Abstract

The invention discloses a one-step multiplex reverse transcription-polymerase chain reaction (RT-PCR) method for detecting turnip mosaic virus (TuMV) and special primers for the method. The invention provides a primer group for detecting the turnip mosaic virus. The primer group is a primer group C, a primer group A or a primer group B shown in the specifications, wherein the primer group C consists of the primer group A and the primer group B; the primer group A comprises a primer pair a; the primer group B comprises a primer pair b; the primer pair a consists of a DNA molecule shown as a sequence 3 in a sequence table and a DNA molecule shown as a sequence 4 in the sequence table; and the primer pair b consists of a DNA molecule shown as a sequence 5 in the sequence table and a DNA molecule shown as a sequence 6 in the sequence table. Experiments prove that: the TuMV is quickly and accurately detected by the one-step multiplex RT-PCR method.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a one-step multiple RT-PCR method for detecting turnip mosaic virus and special primers thereof. Background technique [0002] Turnip Mosaic Virus (TuMV) belongs to the family Potyviridae and the genus Potyvirus. Virus particles are curved and linear, about 720nm long and 15-20nm wide, consisting of 95% coat protein and 5% RNA composition. Viral nucleic acid is single-stranded positive-sense RNA consisting of approximately 10,000 nucleotides. TuMV has a wide range of hosts. Under artificial inoculation conditions, it can infect 43 families, 156 genera, more than 318 species of dicotyledonous plants and some monocotyledonous plants; it mainly infects cruciferous vegetable crops and ornamental species, second only to cucumber flowers. Leaf virus (CMV) is the most important virus infecting field vegetables. According to the survey, the damage of Chinese cabbage caused by TuMV causes ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 姚磊曾钢叶艳英马荣才
Owner BEIJING AGRO BIOTECH RES CENT
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