Real-time fluorescent quantitative PCR detection method and application of sweet potato chlorotic dwarf virus West African strain
A chlorotic dwarf virus, real-time fluorescence quantitative technology, applied in the direction of microbial measurement/inspection, biochemical equipment and methods, etc., to achieve high practical value, good repeatability, and strengthen the effect of early monitoring and early warning
Active Publication Date: 2016-04-06
INST OF PLANT PROTECTION HENAN ACAD OF AGRI SCI
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At present, there is no research report on the real-time fluorescent quantitative PCR detection method of SPCSV. How to use real-time fluorescent quantitative PCR technology to establish an efficient and sensitive detection technology for SPCSV and strengthen the early monitoring and early warning of SPCSV is of great significance to the prevention and control of SPVD.
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Embodiment 1
[0045] Embodiment 1: Real-time fluorescent quantitative PCR detection method of sweet potato chlorotic dwarf virus West African strain
[0046] 1 Materials and methods
[0047] 1.1 Plasmid and sweet potato virus materials
[0048] Four genes containing coat protein (CP) genes of sweetpotatofeatherymottlevirus (Sweetpotatofeatherymottlevirus, SPFMV), sweetpotatolatentvirus (SPLV), sweetpotatovirus G (SweetpotatovirusG, SPVG), and sweetpotatoveinmosaicvirus (SPVMV), respectively Four kinds of recombinant plasmids are used for the specificity test of fluorescent quantitative PCR, and these four kinds of recombinant plasmids are all constructed and preserved by our laboratory;
[0049] Sweet potato stems with typical SPVD symptoms were collected in Zhejiang, Sichuan, Jiangsu and other places, and planted in insect-proof greenhouses for nitrocellulose membrane enzyme-linked immunosorbent assay (NCM-ELISA), conventional RT-PCR and real-time fluorescence quantification Comparison o...
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The present invention relates to a real-time fluorescent quantitative PCR detection method and application of sweet potato chlorotic dwarf virus West African strain. The method uses the nucleotide sequence of sweet potato chlorotic dwarf virus West African strain SPCSV-WA as a template to design and amplify SPCSV -WA? cp Gene-specific primers and specific CSV probes to extract total RNA from susceptible sweet potato leaves; use CSV-DL2 primers to reverse transcribe the first strand of cDNA, and then use CSV-CP-P2 primers to reverse transcribe the second cDNA Chain, using CSV-CP-P1 and reverse primer CSV-CP-P2 for PCR amplification reaction; with SPCSV-WA ? cp The recombinant plasmid of the gene was used to draw a standard curve for the positive standard plasmid. By optimizing the reaction system and reaction conditions, a real-time fluorescent quantitative PCR detection method for SPCSV-WA was established. The results show that this method can only detect the target virus, the slope and correlation coefficient of the standard curve are -3.239 and 1, respectively, and the amplification efficiency is 103.568%; the lowest positive plasmid can be detected at about 3.31 copies / μL, and the sensitivity is higher than that of conventional PCR. 1000 times higher. The real-time fluorescent quantitative PCR method of the invention can be used for the detection of sweet potato samples in the field, and provides technical means for early warning and epidemiological research of SPCSV.
Description
technical field [0001] The invention relates to a virus detection method in the technical field of bioengineering, in particular to a real-time fluorescent quantitative PCR detection method and application of sweet potato chlorotic dwarf virus West African strain. Background technique [0002] Sweetpotatochloroticstuntvirus (Sweetpotatochloroticstuntvirus, SPCSV) is a Closteroviridae (Closteroviridae) Trichovirus (Crinivirus) virus, is one of the main viruses infecting sweet potato. The SPCSV genome is a double-component single-stranded positive-sense RNA with a size of about 17kb. According to the serological relationship and nucleotide sequence, SPCSV can be divided into East African (EA) and West African (WA) strains. Currently, the West African strains are mainly infecting sweet potatoes in my country. SPCSV and sweetpotato feathery mottle virus (Sweetpotatofeatherymottlevirus, SPFMV) co-infect sweetpotato, which can cause the occurrence of sweetpotatovirus disease (SPV...
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IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/6851C12Q1/70C12Q2561/113C12Q2563/107C12Q2545/114C12Q2531/113
Inventor 张振臣王丽张德胜王爽乔奇秦艳红田雨婷王永江
Owner INST OF PLANT PROTECTION HENAN ACAD OF AGRI SCI