Multiple RT-PCR (reverse transcription-polymerase chain reaction) detection method for SPVD (sweet potato virus disease)

A technology of RT-PCR and detection method, which is applied in the field of bioengineering to achieve the effect of strengthening early warning and control

Active Publication Date: 2011-06-29
INST OF PLANT PROTECTION HENAN ACAD OF AGRI SCI
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  • Abstract
  • Description
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Problems solved by technology

[0004] The technical problem to be solved in the present invention: overcome the shortcoming of existing sweet potato virus detection, provide a kind of detection whether dangerous sweet potato virus disease SPVD exists in a PCR reaction And the method of existence type provides a simple, efficient and low-cost method for the specific detection of sweet potato virus

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  • Multiple RT-PCR (reverse transcription-polymerase chain reaction) detection method for SPVD (sweet potato virus disease)
  • Multiple RT-PCR (reverse transcription-polymerase chain reaction) detection method for SPVD (sweet potato virus disease)
  • Multiple RT-PCR (reverse transcription-polymerase chain reaction) detection method for SPVD (sweet potato virus disease)

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Embodiment 1

[0048] Embodiment 1: the multiplex RT-PCR detection method of sweet potato virus disease SPVD

[0049] (1) Materials and methods

[0050]1. Design and synthesis of primers:

[0051] According to the genome sequences of SPCSV and SPFMV viruses, the following primers were designed, and the primers were synthesized by TaKaRa Company. The primer sequences are shown in Table 1 below.

[0052]

[0053] Note: where Y=C / T, D=G / A / T, R=A / G, M=A / C.

[0054] 2. Virus material

[0055] Sweet potato stems with typical SPVD symptoms were collected in Guangdong, Sichuan, Jiangsu and other places and planted in insect-proof greenhouses. Serological methods and single PCR methods were used to detect SPCSV and SPFMV to determine the SPVD virus, and confirmed by nucleotide sequencing. For the strain type of SPFMV, several strains of SPVD susceptible sweet potato plants were finally selected for future use.

[0056] 3. Enzyme and reagent (box)

[0057] UNIQ-10 column total RNA extraction ...

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Abstract

The invention relates to a multiple RT-PCR (reverse transcription-polymerase chain reaction) detection method for hazardous SPVD (sweet potato virus disease), which comprises the following steps: respectively synthesizing an SPCSV (sweet potato chlorotic stunt virus) primer, an SPFMV (sweet potato feathery mottle virus) primer and an SPFMV-CH GPV forward primer; extracting the total RNA (ribonucleic acid) of sweet potato leaves infected with SPVD as a PCR template, and carrying out reverse transcription; placing the primers in a PCR reaction system, and carrying out PCR amplification by using the reverse transcription product as a template, wherein the PCR reaction process is as follows: pre-denaturing at 94 DEG C for 3 minutes, denaturing at 94 DEG C for 30 seconds, annealing at 54 DEG C for 30 seconds, extending at 72 DEG C for 40 seconds, and extending at 72 DEG C for 10 minutes after 30 cycles; and finally, detecting the amplification product by agarose gel electrophoresis. The method provided by the invention can detect whether SPVD is present or not in one-step PCR reaction, can effectively differentiate the type of SPFMV strains, has high detection efficiency and can warn the hazard level of SPVD in time, thereby providing reliable evidence for the control and prevention of the disease.

Description

technical field [0001] The invention relates to RT-PCR detection of sweet potato virus, belongs to the technical field of bioengineering, in particular to a multiple RT-PCR detection method of dangerous sweet potato virus disease SPVD. Background technique: [0002] Sweet potato virus disease (Sweet potato virus diseases, SPVD) is caused by sweet potato chlorotic dwarf virus ( Sweet potato chlorotic stunt virus , SPCSV) and sweet potato feather mottle virus ( Sweet potato feathery mottle virus , SPFMV) virus disease caused by symbiotic infection of sweet potato. SPVD was first discovered in Africa in the 1970s, and is currently mainly distributed in some countries in Africa and South America. Sweet potatoes infected with SPVD showed symptoms such as plant dwarfing, leaf chlorosis, distortion, and bright veins. The disease has a great impact on the yield of sweet potato. In Africa, when the disease is severe, it can cause a yield reduction of more than 80%, or even no h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 张振臣张德胜乔奇秦艳红田雨婷王永江
Owner INST OF PLANT PROTECTION HENAN ACAD OF AGRI SCI
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