Primer group for nest type RT-PCR detection of seneca valley virus (SVV), kit and application

A technology of RT-PCR and a kit, which is applied in the field of primer sets for porcine Seneca virus nested RT-PCR detection, and can solve the problem of nested RT-PCR detection methods and nested RT-PCR detection reagents. box and other problems, to achieve the effect of fast detection time, short detection time and high sensitivity

Active Publication Date: 2019-09-13
派生特(福州)生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But so far, there is no nested RT-PCR detection method specially used for SVV detection, let alone a nested RT-PCR detection kit specially used for this virus detection

Method used

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  • Primer group for nest type RT-PCR detection of seneca valley virus (SVV), kit and application
  • Primer group for nest type RT-PCR detection of seneca valley virus (SVV), kit and application
  • Primer group for nest type RT-PCR detection of seneca valley virus (SVV), kit and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] The configuration of embodiment 1 porcine Seneca virus nested RT-PCR detection kit (10 times detection amount)

[0043] 1) RT Buffer: 10×, 1 tube (30 μL);

[0044] 2) RNase inhibitor: 40U / μL, 1 tube (20μL);

[0045] 3) Reverse transcriptase: 200U / μL, 1 tube (20μL);

[0046] 4) MgCl 2 : 25mmol / L, 1 tube (60μL);

[0047] 5) DEPC water: 1 tube (1mL);

[0048] 6) PCRBuffer: 10×, 1 tube (30 μL);

[0049] 7) External upstream primer: 10 μmol / L, 1 tube (30 μL);

[0050] 8) Outer downstream primer: 10 μmol / L, 1 tube (30 μL);

[0051] 9) Inner upstream primer: 10 μmol / L, 1 tube (30 μL);

[0052] 10) Inner downstream primer: 10 μmol / L, 1 tube (30 μL);

[0053] 11) Taq DNA polymerase: 2.5U / μL, 1 tube (10μL);

[0054] 12) dNTPs: 10mmol / L, 1 tube (20μL);

[0055] 13) Positive control sample of porcine Seneca virus, 1 tube (5 μL);

[0056] 14) Negative control sample without porcine Seneca virus, 1 tube (50 μL).

Embodiment 2

[0057] The detection method of embodiment 2 porcine Seneca virus nested RT-PCR detection kit

[0058] The detection method of above-mentioned porcine Seneca virus nested type RT-PCR detection kit comprises the following steps:

[0059] 1) Reverse transcription reaction: Add 2 μL of total RNA of the sample to be tested, 0.5 μL of the outer downstream primer with a concentration of 10 μmol / L and 8.5 μL of DEPC water with a concentration of 25 mmol / L MgCl in the PCR tube 2 4 μL; 2 μL of 10×RT buffer, 2 μL of 10 mmol / L dNTPs, 0.5 μL of reverse transcriptase at a concentration of 200 U / μL, 0.5 μL of RNase inhibitor at a concentration of 40 U / μL, incubate the reaction at 25°C for 10 min, and then Reverse transcription at 42°C for 15 minutes, and extension at 70°C for 15 minutes to synthesize cDNA.

[0060] 2) The first round of RT-PCR reaction: Add 4 μL of cDNA of the sample to be tested in the PCR tube, add 0.5 μL of Taq DNA polymerase at a concentration of 2.5 U / μL, 0.5 μL of dNT...

Embodiment 3

[0063] Example 3: Specificity determination of porcine Seneca virus nested RT-PCR detection kit

[0064] 1) Extraction of RNA: samples carrying porcine seneca virus (SVV), foot-and-mouth disease virus (FMDV), porcine reproductive disorder and respiratory syndrome virus (PRRSV), and swine fever virus (CSFV) were used as materials, and each Add 560 μL Carrier RNA working solution to 140 μL sample, mix well, incubate at room temperature for 15 minutes, shake gently during the mixing period; add 560 μL absolute ethanol, mix well, incubate at room temperature for 5 minutes, transfer all the liquid in the tube to the adsorption column and centrifuge at 8000 rpm for 1 minute and discard Add 500μL buffer GD, centrifuge at 8000rpm for 1min, discard the waste solution, add 500μL eluent RW, centrifuge at 8000rpm for 1min, discard the waste solution, add 500μL eluent RW again, centrifuge at 8000rpm for 1min, discard the waste solution, and put the adsorption column back In the collection ...

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Abstract

The invention provides a primer group for nest type RT-PCR detection of seneca valley virus (SVV), a kit and application and belongs to the technical field of molecular biological detection of viruses. Two pairs of specific primers are designed according to an SVV gene sequence, first round of RT-PCR amplification reaction is performed with cDNA of extracted RNA after reverse transcription as a template, second round of RT-PCR amplification reaction is performed with a first round of RT-PCR amplification product as a template, the amplification product is detected by agarose gel electrophoresis, if specific target fragments with size of 523bp exist, the detected virus is determined as the SVV. The primer group detects SVV by the nest type RT-PCR technology, has the advantages of high specificity, high sensitivity and short detection time, can meet requirements of enterprises for production research and development and agricultural production and provides a new thought for monitoring and control of the SVV.

Description

technical field [0001] The invention relates to the technical field of virus molecular biology detection, in particular to a primer set, kit and application for porcine Seneca virus nested RT-PCR detection. Background technique [0002] Porcine Seneca virus (Seneca Valley virus, SVV) is a member of the genus Senecavirus of the Picornaviridae family, and was first discovered on embryonic retinal cells (PER.c6). The SVV virus is icosahedral, with a diameter of 30nm and a genome of about 7.2kb, encoding 4 structural proteins (VP1-4) and 7 non-structural proteins (2A-C, 3A-D). SVV has a narrow host range and mainly infects pigs. After the virus infects pigs, it can produce a variety of symptoms, typical symptoms include nasal mirror, mouth and hoof blisters or ulcers, etc., which endanger the normal growth of pigs and affect the economic benefits of farms. Swine SVV outbreaks have been confirmed in many countries around the world. [0003] At present, the establishment of rap...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6848C12R1/93
CPCC12Q1/70C12Q1/6848C12Q2549/113C12Q2521/107C12Q2531/113
Inventor 赖隆永刘小龙谭礼宁叶盛聪邱灵姗刘楚楚陈盛勇徐磊刘友霖
Owner 派生特(福州)生物科技有限公司
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