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Seneca valley virus real-time fluorescence quantification PCR detection primer and kit

A technology for real-time fluorescence quantification and detection of primers, which is used in the determination/inspection of microorganisms, microorganisms, and microorganism-based methods, etc. It can solve the problems of indistinguishability, difficulty in diagnosis, and complicated operation methods, and achieve high accuracy and repeatability. , the effect of efficient identification

Active Publication Date: 2016-09-07
SOUTH CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

The clinical symptoms caused by SVV are very similar to some other porcine vesicular diseases, and it is difficult to distinguish them clinically and histopathologically, which makes the diagnosis of the disease difficult, and its differential diagnosis usually requires the help of laboratories Diagnostic technology, the traditional detection method is time-consuming and laborious, and the operation method is cumbersome, which is not conducive to the timely diagnosis and treatment of diseases

Method used

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  • Seneca valley virus real-time fluorescence quantification PCR detection primer and kit
  • Seneca valley virus real-time fluorescence quantification PCR detection primer and kit
  • Seneca valley virus real-time fluorescence quantification PCR detection primer and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Preparation of standard positive template

[0023] 1. Extraction of total viral RNA

[0024] According to the Invitrogen company TRIZOL LS Reagent RNA extraction kit instruction manual. Add 250 µL of the aliquoted virus supernatant from cell propagation and 750 µL TRIZOL to a 1.5 mL Leppendorf tube, mix thoroughly, and place at room temperature for 10 min; add 200 µL of chloroform, shake vigorously for 15 sec, and let stand at room temperature for 5 min, 4 Centrifuge at 12,000 rpm for 15 min at ℃; transfer the supernatant to a new 1.5 mL eppendorf tube, add 500 µL of isoamyl alcohol, mix well, place at room temperature for 10 min, and centrifuge at 12,000 rpm for 10 min at 4°C; discard the supernatant , precipitated with 1000 µL of ice-cold 70% ethanol, mixed gently, washed once, and centrifuged at 12,000 rpm for 10 min at 4°C; discarded the supernatant and air-dried; dissolved RNA with 20 µL of DEPC-treated triple distilled water, Store at -80°C or use dire...

Embodiment 2

[0039] Example 2 Design and Screening of Primers and TaqMan Probes

[0040] Specific primers and TaqMan probes were designed according to the 3C conserved fragments in the SVV gene sequence, and were synthesized by Huada Company. The labeled quencher is TAMRA.

[0041]

[0042] The primers and probes in Table 1 were used to perform PCR on SVV, and it was found that the other 3 pairs of primers had non-specific amplification or primer dimers, which did not meet the experimental requirements, so only primer 1 and primer 2 can be used as specific Amplify primers for detection of SVV.

Embodiment 3

[0043] Example 3 Fluorescent quantitative PCR reaction condition optimization and standard curve making

[0044] To optimize primer concentration, probe concentration and annealing temperature conditions, Real-Time PCR 20 μL reaction system is Premix Ex Taq (Probe qPCR) (2×) 10 μL, each specific upstream and downstream primers (10 μM) 0.4 μL, ROXReference Dye II (50×) 0.2 μL, fluorescent probe (10 μM) 0.8 μL, DNA template 2.0 μL, ddH 2 O 6.2 μL. The optimal reaction conditions were: 95°C for 30 s, 95°C for 5 s, 60°C for 34 s, 40 cycles.

[0045] Determination of OD of standard recombinant positive plasmid 260nm and OD 280nm value and its ratio, calculate the plasmid concentration, and convert it into copy number, serially dilute it into 7 gradients with double distilled water 10 times (10 8 ~10 3 copies / μL). Using this as a template, a 20 μL reaction system was established, and amplified on an ABI 7500 fluorescent quantitative PCR instrument according to the optimized PC...

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Abstract

The invention belongs to the technical field of biological detection, and discloses a Seneca valley virus real-time fluorescence quantification PCR detection primer and a kit. Firstly, the Seneca valley virus real-time fluorescence quantification PCR detection primer and a probe are obtained through design and screening, the sequences of an upstream primer, a downstream primer and the probe are shown as SEQ ID NO: 1-3; the primer and the probe are used to specifically amplify a Seneca valley virus, real-time fluorescence quantification monitors the condition of combination of a double-stranded DNA fluorescent dye with a PCR amplification product in the PCR process in real time, then fluorescent data are acquired, and the SVV (Seneca Valley Virus) is identified according to a CT value; the SVV is identified after the method is used to carry out PCR amplification, the accuracy is high, the specificity and the repeatability are good, the SVV can be identified accurately, rapidly and efficiently, and the popularization and the application in clinical practice benefits are facilitated.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a real-time fluorescent quantitative PCR detection primer and a kit for Seneca Valley virus. Background technique [0002] Seneca Valley virus (Seneca Valley virus SVV) is a single-stranded positive-sense RNA virus and a typical representative of the genus Senecavirus in the family Picornaviridae. It was initially considered to be a contaminant in PER.C6 cell cultures, and was subsequently isolated in pigs in the United States and Canada. In 2007, clinical signs of blistering disease appeared in pigs in a slaughterhouse in the United States, and about 80% of them Pigs appear lame, and PCR detection of porcine foot-and-mouth disease, porcine vesicular disease, vesicular stomatitis and vesicular rash are all negative, but SVV is positive, which is considered to be primary vesicular disease. The recent infection and outbreak of swine senega valley virus (SVV) in pigs i...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 马静云赵晓亚陈桂华伍绮文
Owner SOUTH CHINA AGRI UNIV
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