Fluorescence quantitative RT-PCR primers and probe and method for detecting APPV (atypical porcine pestivirus) as well as application

A type of atypical pestivirus, fluorescent quantitative technology, applied in biochemical equipment and methods, microbe measurement/inspection, DNA/RNA fragments, etc., can solve the problems of large gene sequence differences, rapid gene sequence variation, and inapplicability, etc. Achieve the effect of less operation steps, saving time and avoiding false positives

Active Publication Date: 2018-10-02
SOUTHWEST UNIVERSITY FOR NATIONALITIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, according to research data, the gene sequence of the virus mutates rapidly, and the gene sequence differences between different strains are large. The detection methods established abroad are not applicable in China, and the common PCR and fluorescence detection kits in China are easy to detect. The problem of false positives occurs, so the accuracy of the test results needs to be studied

Method used

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  • Fluorescence quantitative RT-PCR primers and probe and method for detecting APPV (atypical porcine pestivirus) as well as application
  • Fluorescence quantitative RT-PCR primers and probe and method for detecting APPV (atypical porcine pestivirus) as well as application
  • Fluorescence quantitative RT-PCR primers and probe and method for detecting APPV (atypical porcine pestivirus) as well as application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Primer Design

[0025] According to the domestic and foreign strain sequences of the APPV gene registered by NCBI (KU041637, KU041638, KU041639, KU194229, KX929062, KX929063, KX929069, LT594521), the specificity of the 5' end gene of APPV was designed using Beacon Designer7.7 software Primers, all primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd.

[0026] Upstream primer APPV F: YGAGTAGTACACCCAAAG (wherein Y represents T or C), SEQ ID NO: 1;

[0027] Downstream primer APPV R: CACCACCGATTTCTCTTTT, SEQ ID NO: 2;

[0028] Probe sequence APPV Probe: CYGAGCCTCAGTAGACCCT (where Y represents T or C) SEQ ID NO:3.

Embodiment 2

[0029] Embodiment 2 detects the establishment of the fluorescent quantitative RT-PCR method of porcine atypical fever virus:

[0030] The method includes the following steps:

[0031] (1) RNA extraction of samples to be tested:

[0032] The blood samples to be tested were centrifuged at 8000g for 5min, and the upper serum was taken for each use; the tissue and organ samples should be mixed with sterilized double distilled water at a mass volume ratio of 1:5, centrifuged at 8000g for 5min after grinding, and the supernatant was taken for later use.

[0033] (2) The extracted RNA was added to a reverse transcription kit purchased from Bao Bio Engineering (Dalian) Co., Ltd. for reverse transcription to obtain a cDNA template:

[0034] The reverse transcription step is as follows: mix 4 μL of RNA template, 4 μL of No. 1 5×PrimeScript Buffer, 1 μL of No. 2 PrimeScriptRT Enzyme Mix, 2 μL of No. 4 Random 6mers and 9 μL of No. 5 RNase Free dH2O, and put them into a PCR machine for re...

Embodiment 3

[0037] The determination of optimization, sensitivity, stability and specificity of embodiment 3 annealing temperature, primer concentration and probe concentration

[0038] (1) Optimization of annealing temperature: the annealing temperature in the APPV F / R primer reaction conditions is set at 48°C-54°C, observe the amplification peak pattern of the fluorescent quantitative PCR instrument, and select the one with the smallest CT value as the optimal annealing temperature, like figure 1 As shown, the optimum annealing temperature of the present invention is 54°C.

[0039] (2) Optimization of primer concentration: 20 μL reaction system for PCR, 0.2 μL to 1 μL for upstream and downstream primers, 0.5 μL for probe. Select the primer concentration with the smallest CT value as the optimal primer concentration, such as figure 2 As shown, the optimal primer concentration is 0.5 μL.

[0040] (3) Optimization of probe concentration: 20 μL reaction system for PCR, 0.8 μL of upstrea...

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Abstract

The invention discloses fluorescence quantitative RT-PCR primers and probe and method for detecting APPV (atypical porcine pestivirus) as well as an application. The sequences of the primers and probeare shown in SEQ ID NO:1-3. The APPV can be rapidly, specifically and sensitively detected, and the lowest detection concentration is 5 copies/mu L. The used operation method is simple, a reaction result is easy to judge, the sensitivity and detection rate are high, the false positive phenomenon can be avoided, and the primers and probe are applicable to disease monitoring, scene emergency and detection of clinical samples and are suitable for popularization and application on a large scale.

Description

technical field [0001] The invention belongs to the technical field of virus detection, and in particular relates to a fluorescent quantitative RT-PCR primer, a probe, a method and an application for detecting porcine SARS virus. Background technique [0002] Atypical porcine pestivirus (APPV) is a new type of pestivirus newly discovered in recent years. It is closely related to classical swine fever virus (CSFV), bovine viral diarrhea virus (BVDV) Pestivirus (Border disease virus, BDV) and the like belong to the Pestivirus (Pestivirus) Flaviviridae (Flaviviridae). The onset is characterized by bilateral spasm and contraction of skeletal muscles within a few hours after birth, resulting in the inability to suck milk. In severe cases, it can damage the brain and spinal cord of piglets and lead to death of piglets. Clinically, the symptoms vary. Some piglets have consistent and rhythmic tremors all over the body, unable to stand, and the tremors are reduced or stopped after l...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11
CPCC12Q1/6851C12Q1/701C12Q2531/113C12Q2563/107
Inventor 张斌周可磊岳华汤承王远微
Owner SOUTHWEST UNIVERSITY FOR NATIONALITIES
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