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Dual real-time fluorescence RT-PCR (reverse transcription-polymerase chain reaction) kit for detecting and identifying wild strain and vaccine strain of CSFV (classical swine fever virus) in swine umbilical cord blood and application of dual real-time fluorescence RT-PCR kit

A technology of RT-PCR and swine fever virus, applied in the field of dual real-time fluorescent PCR kits, can solve the problems of inability to effectively distinguish wild strain infection and vaccination, less kits, and long operation time, and achieve direct and effective diagnosis , reliable technical support, and highly specific effects

Inactive Publication Date: 2017-02-22
HUNAN XINNANFANG CULTURE SERVICE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still few commercial kits independently developed in China
At the same time, the ordinary PCR detection method has the defects of prone to false positives, long operation time, nested amplification, and high technical requirements for technical operation.
[0005] The above diagnostic methods for swine fever have their own shortcomings in terms of sensitivity, specificity and timeliness, and these methods cannot effectively distinguish between wild strain infection and vaccination.

Method used

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  • Dual real-time fluorescence RT-PCR (reverse transcription-polymerase chain reaction) kit for detecting and identifying wild strain and vaccine strain of CSFV (classical swine fever virus) in swine umbilical cord blood and application of dual real-time fluorescence RT-PCR kit
  • Dual real-time fluorescence RT-PCR (reverse transcription-polymerase chain reaction) kit for detecting and identifying wild strain and vaccine strain of CSFV (classical swine fever virus) in swine umbilical cord blood and application of dual real-time fluorescence RT-PCR kit
  • Dual real-time fluorescence RT-PCR (reverse transcription-polymerase chain reaction) kit for detecting and identifying wild strain and vaccine strain of CSFV (classical swine fever virus) in swine umbilical cord blood and application of dual real-time fluorescence RT-PCR kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071]Example 1 The composition of the dual real-time fluorescent RT-PCR kit for detection and identification of swine fever virus wild strain and vaccine strain in pig umbilical cord blood

[0072] (1) 2× fluorescent RT-PCR reaction solution (containing enzyme): the raw material was purchased from Nanjing VAZYME Company; the reaction solution contains UNG enzyme system, which can effectively solve the phenomenon of amplification pollution during the amplification process Pollution ability, etc.;

[0073] (2) RT-PCR primer sets CSFV-F and CSFV-R: synthesized by Shanghai Sangon Bioengineering Co., Ltd., prepared with DEPC water to a concentration of 10 μM.

[0074] Upstream amplification primer CSFV-F: 5'-GCCATGCCCATAGTAGGA-3', which is the sequence of SEQ ID NO:1;

[0075] Downstream amplification primer CSFV-R: 5'-CTACTGACGACTGYCCTGTA-3', which is the sequence of SEQ ID NO:2, wherein Y=C / T;

[0076] (3) Specific fluorescent probes CSFV-LV and CSFV-Wt: synthesized by BGI Cor...

Embodiment 2

[0089] Example 2 The method of using the dual real-time fluorescent RT-PCR kit for detecting and distinguishing the wild strain of classical swine fever virus and the vaccine strain in pig umbilical cord blood

[0090] The method for using the kit of the present invention specifically includes the following steps: (1) sample collection; (2) sample processing; (3) RNA extraction; (4) double real-time fluorescent RT-PCR: utilizing the specific primers and probes designed by the present invention Perform double real-time fluorescent RT-PCR detection; (5) judge the result.

[0091] (1) Sample collection

[0092] 1. Take a clean penicillin bottle and cork, wash it, boil and sterilize it for 30 minutes, dry it and collect it for later use;

[0093] 2. When the piglets are born, squeeze the "cord blood" of all the piglets delivered by each sow into a clean penicillin bottle, 3-5 drops per piglet, and squeeze the "cord blood" into the penicillin bottle bottle, sealed;

[0094] Prec...

Embodiment 3

[0119] Example 3 Verification of the dual real-time fluorescent RT-PCR kit for detection and identification of swine fever virus field strains and vaccine strains in pig umbilical cord blood

[0120] 1. Validation of amplification efficiency

[0121] Perform a 10-fold serial dilution of the positive control clone plasmid pEASY-5UTR2 to make its copy number: 10 8 -10 1 copies / μl, each gradient was repeated three times for real-time fluorescent RT-PCR, and a standard curve was made according to the amplification results.

[0122] figure 1 It is the gradient amplification standard curve (FAM channel) of 10-fold dilution of the positive control of the present invention, wherein the parameters of the standard curve are as follows: slope: -3.56, intercept: 39.78, correlation coefficient: 0.998, amplification efficiency: 0.91.

[0123] figure 2 In order to invent a 10-fold diluted gradient amplification standard curve (VIC channel) for the positive control, the parameters of the...

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Abstract

The invention discloses a dual real-time fluorescence RT-PCR (reverse transcription-polymerase chain reaction) kit for detecting and identifying a wild strain and a vaccine strain of a CSFV (classical swine fever virus) in swine umbilical cord blood and an application of the dual real-time fluorescence RT-PCR kit. The kit comprises a pair of primers and two fluorescent probes, wherein the sequences of the pair of primers are shown as SEQ ID NO.1 and SEQ ID NO.2, and the sequences of the two fluorescent probes are shown as SEQ ID NO.3 and SEQ ID NO.4. The kit has the advantages of high specificity, sensitivity and accuracy and excellent repeatability, meanwhile, the wild strain and the vaccine strain of the CSFV can be identified and detected by detecting the same sample once, and the problem of genetic crossover of the CSFV with the bovine viral diarrhea virus and the border disease virus which belong to the same genus can be solved. The kit is applicable to fast identification and detection of the wild strain and the vaccine strain of the CSFV in scientific research and clinical detection, can be used for precisely evaluating and diagnosing CSFV carrying and expelling conditions of sows and latent infection conditions of piglets and can be further used for evaluating effects of CSFV vaccines.

Description

technical field [0001] The invention relates to the technical field of molecular diagnosis of animal pathogens, in particular to a dual real-time fluorescent PCR kit for detecting and distinguishing wild strains and vaccine strains of classical swine fever virus in pig umbilical cord blood and its application. Background technique [0002] Classical swine fever is a highly contagious and acute infectious disease of pigs caused by classical swine fever virus (CSFV). Sexual spotting, multiple hemorrhage, necrosis and infarction in internal organs, contagious and high fatality rate. In recent years, great changes have taken place in the prevalence and incidence characteristics of CSF in my country. Mild CSF or atypical CSF caused by low-virulence CSF wild strains or persistent infection are more common. The pathological features are also different from typical swine fever, which brings great troubles to veterinary clinical diagnosis. [0003] As a class of infectious disease, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 喻正军李增强石建廖娟红
Owner HUNAN XINNANFANG CULTURE SERVICE CO LTD
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