Porcine reproductive and respiratory syndrome virus real-time fluorescent quantitation PCR detection kit and primer

A porcine PRRS virus, real-time fluorescence quantitative technology, applied in the determination/inspection of microorganisms, microorganisms, microorganism-based methods, etc. The detection of highly pathogenic porcine PRRS virus and porcine PRRS virus takes a long time, specificity, and sensitivity, etc., to achieve the effect of high sensitivity, short time and strong specificity

Pending Publication Date: 2019-11-15
SUZHOU XISHAN BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to solve the problem of time-consuming detection of porcine PRRS virus, complex operation, poor specificity and sensitivity, and the inability to effectively detect classic porcine PRRS virus, highly pathogenic porcine PRRS virus, and new mutations with high pathogenicity at the same time. Problems with PRRSV

Method used

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  • Porcine reproductive and respiratory syndrome virus real-time fluorescent quantitation PCR detection kit and primer
  • Porcine reproductive and respiratory syndrome virus real-time fluorescent quantitation PCR detection kit and primer
  • Porcine reproductive and respiratory syndrome virus real-time fluorescent quantitation PCR detection kit and primer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: Real-time PCR kit for detecting porcine PRRS virus

[0021] The present invention provides a kit for detecting porcine PRRS virus by real-time fluorescence quantitative PCR using SYBR Green dye method, which comprises reagents including SYBR Premix EX Taq (purchased from Bao Bioengineering (Dalian)), primers, ROX, Rnase-FreeddH 2 O. Positive reference. The following examples are all tested with CFX96 instrument without special instructions.

[0022] The primers are a pair, which are specific forward and reverse primers designed for the highly conserved region of porcine PRRS virus (including classic porcine PRRS virus, highly pathogenic porcine PRRS virus and the newly mutated strain-NADC30). Its sequences are shown in SEQ ID NO:1 and SEQ ID NO:2.

[0023] The forward primer sequence represented by SEQ ID NO: 1 is as follows:

[0024] 5’-GCCGKTTGTGCTTGCT-3’

[0025] The reverse primer sequence represented by SEQ ID NO:2 is as follows:

[0026] 5’-TGCGTGG...

Embodiment 2

[0043] Example 2: Preparation of positive standard standard curve

[0044]The positive standard of this kit is composed of plasmids containing the target fragment. The positive standard is diluted 10-fold with TE buffer. The dilution gradient is as follows: the concentrations of S1 to S10 are 1.0×10 9 copies / μL, 1.0×10 8 copies / μL, 1.0×10 7 copies / μL, 1.0×10 6 copies / μL, 1.0×10 5 copies / μL, 1.0×10 4 copies / μL, 1.0×10 3 copies / μL, 1.0×10 2 copies / μL, 1.0×10 1 copies / μL, 1.0 copies / μL. Quantitative PCR detection was carried out with the kit and method described in Example 1 using the above-mentioned fold-diluted standard as a template.

[0045] ginseng figure 1 and figure 2 , the results show that the standard concentration is between S1~S8 (1.0×10 9 copies / μL~1.0×10 2 It has a good linear relationship in the range of copies / μL), S9~S10 (1.0×10 1 copies / μL~1.0copies / μL) detected no linear relationship, and only one well of S9 showed positive amplification. in, f...

Embodiment 3

[0046] Example 3: Sensitivity and Repeatability

[0047] In order to verify the minimum detection limit and reproducibility of the detection kit, the sensitivity of porcine PRRS was tested with positive standard S4-S10 as reference and repeated three times.

[0048] ginseng image 3 , the results show that the positive standards S4~S8 are all amplified, the Ct value is ≤32, the Tm value is 86.0, and only one well of S9 has positive amplification, which does not meet the requirements, so the detection limit of this kit is 1.0 × 10 2 copies / μL.

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Abstract

The invention reveals a primer used for detecting porcine reproductive and respiratory syndrome virus, which can perform specific recognition on the porcine reproductive and respiratory syndrome virus. The invention also reveals a real-time fluorescent quantitation PCR detection kit using the primer, has the advantages of high sensitivity, strong specificity and good reproducibility, and providesa basis for timely, fast and accurate diagnosis of diseases in pig farms.

Description

technical field [0001] The present invention relates to real-time fluorescence quantitative PCR detection of virus. Background technique [0002] Porcine PRRS, also known as Porcine Reproductive and Respiratory Syndrome, is a viral swine infectious disease caused by porcine PRRS virus (porcine reproductive and respiratory syndrome virus, PRRSV). In 1992, the United States first isolated a porcine PRRS virus and named it VR2332. In 1996, China also isolated the first porcine PRRS virus strain CH-1α, which confirmed the prevalence of porcine PRRS in my country. Existence, and then many provinces and cities reported the occurrence of the disease. There are two genotypes of porcine PRRS virus, namely European type and American type, and the homology between them is about 60%. The World Organization for Animal Health has listed it as a statutory reportable animal disease, and my country has listed it as a second-class animal disease. In 2015, Zhou L et al. reported the emergenc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/6851C12Q1/701C12Q2531/113C12Q2563/107C12Q2545/113
Inventor 朱婷王立鹏时长军
Owner SUZHOU XISHAN BIOLOGICAL TECH
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