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Primer for detecting RT-PCR of Seneca Valley viruses and RT-PCR detecting method

A technology of RT-PCR and detection method, which is applied in the field of RT-PCR detection primers of Seneca Valley virus, which can solve the problems of not being able to take timely and effective prevention and control measures, not having fast and effective detection methods, and not being able to diagnose pathogens in a timely manner , to achieve good application value, fast detection speed and high sensitivity

Inactive Publication Date: 2017-08-11
WENS FOOD GRP CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, there have been reports showing that SVV infection has also appeared in China, but there is no corresponding rapid and effective detection method at present, so that once an outbreak occurs, the pathogen cannot be diagnosed in time, so that prevention and control measures cannot be taken in a timely and effective manner. bring huge losses

Method used

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  • Primer for detecting RT-PCR of Seneca Valley viruses and RT-PCR detecting method
  • Primer for detecting RT-PCR of Seneca Valley viruses and RT-PCR detecting method
  • Primer for detecting RT-PCR of Seneca Valley viruses and RT-PCR detecting method

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Experimental program
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Effect test

Embodiment 1

[0033] 1. Primer design: Since Seneca Valley virus is a newly discovered pathogenic virus, common PCR detection technology has not been established in China at that time, and the gene sequences that can be found on GenBank are also extremely limited. We will use the only Seneca virus on GenBank The gene sequences of Valley virus SVV001 strain and 11-55910-3 strain were compared, and compared with the gene sequences of various viruses of the same virus family, and the conserved region was found, and the 1336-1662 position of SVV001 strain was selected Gene fragments are used as targets, and then BLAST further confirms their conservation. A pair of primers SVVUP / SVVDN with a 327bp amplification target band were designed on the conserved sequence through the Oligo6.0 primer design software. The primer sequences are as follows:

[0034] SVVUP: 5'-GATGTATAAACCTTCTC-3' (SEQ ID NO: 1)

[0035] SVVDN: 5'-ATTGTAAGTGCCAAGAG-3' (SEQ ID NO: 2).

[0036] 2. Extraction of sample RNA: Use ...

Embodiment 2

[0041] Embodiment two specificity test

[0042] According to the RT-PCR reaction system and reaction procedure described in Example 1, the sample template RNA was replaced with foot-and-mouth disease, swine fever, pseudorabies, blue ear, and circular vaccine samples respectively, and the positive results were detected by our laboratory and identified by sequencing. The positive sample of Seneca Valley virus (i.e. named after the CH-01-2015 strain (KT321458) virus disease material RNA sample) is a positive control, and RT-PCR reaction is carried out, and the RT-PCR product is passed through 1% agarose gel Electrophoresis identification, electrophoresis results such as figure 1 As shown, the results showed that only a single band appeared at 327bp in the positive control lane, indicating that the method had good specificity.

Embodiment 3

[0043] Example 3 Sensitivity Test

[0044] According to the RT-PCR reaction system and reaction program described in embodiment one, the positive control RNA in embodiment two is used as positive control, and this positive sample RNA is pressed 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 Perform 10-fold serial dilution, use distilled water as a negative control, and carry out RT-PCR reaction. The RT-PCR product is identified by 1% agarose gel electrophoresis. The electrophoresis results are as follows: figure 2 shown. The result shows that dilution 100(10 -2 ) times the band is still obvious, diluted 1000 (10 -3 ) times, there are still inconspicuous but visible bands, indicating that the method has high sensitivity.

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Abstract

The invention relates to the technical field of detection of pig viruses, in particular to a primer for detecting RT-PCR of Seneca Valley viruses and a detecting method. Nucleotide sequences of primers are as shown in SEQ ID NO: 1 and SEQ ID NO: 2. The RT-PCR detecting method comprises the following steps: designing and synthesizing the primers; extracting a sample RNA and storing for subsequent use; taking the obtained sample RNA as a template RNA, and carrying out RT-PCR reaction by using the primers; and judging and reading a detecting result. According to the scheme, the technical means of virus detection is enriched, specificity and sensitivity are good, the detection speed is high, and the application value is also high.

Description

technical field [0001] The invention relates to the technical field of porcine virus detection, in particular to a pair of RT-PCR detection primers for Seneca Valley virus and a RT-PCR detection method. Background technique [0002] Seneca Valley virus (SVV) belongs to the genus Senecavirus of the picornaviridae family. It is a single-stranded positive-sense RNA virus with a genome length of about 7.28kb, including 5'UTR, 3'UTR and A single ORF between the two, with VPg protein covalently bound at the 5' end and a polyA tail at the 3' end. SVV was first discovered in PER.C6 cell culture in 2002. In 2007, clinical symptoms of vesicular disease appeared in pigs in a slaughterhouse in the United States. After testing and excluding various pathogens that could cause vesicles, SVV was finally identified as the cause of the disease. Original. However, no other cases have been reported since then. [0003] Until 2014-2015, Brazil reported an outbreak of vesicular disease in pig ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/686C12Q1/701C12Q2521/107C12Q2565/125
Inventor 曾喜多陈燕珊陈俊伟张冠群何晓明凌宝明温伟怡庞仕旭
Owner WENS FOOD GRP CO LTD
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