Porcine epidemic diarrhea virus G1/G2 type RT-PCR identification primer set and diagnostic kit thereof

A porcine epidemic diarrhea, RT-PCR technology, applied in the field of porcine epidemic diarrhea virus detection, can solve the problems of long time-consuming, long waiting time, and endangering pig production

Inactive Publication Date: 2019-05-31
GUANGXI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Studies have shown that PEDV infection can cause diarrhea in pigs, seriously endangering pig production, and it is difficult to distinguish PEDV G1 and G2 strains based on clinical symptoms and epidemiology, and laboratory testing techniques must be used for differential diagnosis
Among them, gene cloning, sequence analysis and other operations are more complicated and the waiting time is longer; ELISA pathogen rapid detection kits and conventional RT-PCR methods are widely used, but the accuracy of serological methods is low, and the commonly used single PCR method takes a long time

Method used

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  • Porcine epidemic diarrhea virus G1/G2 type RT-PCR identification primer set and diagnostic kit thereof
  • Porcine epidemic diarrhea virus G1/G2 type RT-PCR identification primer set and diagnostic kit thereof
  • Porcine epidemic diarrhea virus G1/G2 type RT-PCR identification primer set and diagnostic kit thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1, porcine epidemic diarrhea virus G1 / G2 type RT-PCR diagnostic kit assembly

[0023] The kit contains the following reagents: PCR standard substance, positive control substance, negative control substance, primers; the primers include two pairs of specific primers, namely primers PEDV-BF and PEDV-BR, primers PEDV-F and PEDV-R, respectively It has the base sequence of SEQ.ID.NO.1 to SEQ.ID.NO.4 in the sequence listing.

[0024] Table 1 The duplex PCR primers

[0025]

[0026] PCR standard products include PEDV G1 strain standard and PEDV G2 strain standard; positive control contains positive plasmid mixture of PEDV G1 strain and PEDV G2 strain (the concentration of plasmid mixture is about 1.937×10 9 copies / μL); the negative control was sterilized deionized water.

[0027] The PEDV G1 type strain standard product and the PEDV G2 type virus strain standard product respectively have the base sequences of SEQ.ID.NO.5 to SEQ.ID.NO.6 in the sequence table.

...

Embodiment 2

[0037] Embodiment 2, the present invention detects double RT-PCR specific experiment

[0038] Step 1: Primer Synthesis

[0039] Two pairs of specific primer sequences (see Table 1) of the virus designed in the present invention were synthesized by Shanghai Jieli Biotechnology Co., Ltd., and the synthetic amount was 1 OD of primers per tube.

[0040] Step 2: Dual RT-PCR Rapid Detection Kit Specific Detection

[0041] According to the double PCR reaction system, Green Taq Mix (Vazyme) 12.5 μL, upstream primer mix 0.5 μL, downstream primer mix 0.5 μL, ddH 2 O 8.5 μL, add 3 μL positive sample cDNA template respectively 1: mixed virus of PEDV G1 and G2 strains; 2: PEDV G2 strain; 3: PEDV G1 strain; 4: porcine transmissible gastroenteritis virus; 5: porcine rotavirus group A; 6: porcine circovirus type 2; 7: swine fever virus; 8: swine influenza virus; 9: porcine reproductive and respiratory syndrome virus; 10: porcine pseudorabies virus; 11: porcine Astrovirus; 12: Negative cont...

Embodiment 3

[0043] Embodiment 3, the present invention detects double RT-PCR sensitivity experiment

[0044] Step 1: Primer Synthesis

[0045] With the first step in embodiment 2.

[0046] Step 2: Dual RT-PCR Rapid Detection Kit Sensitivity Detection

[0047] Double PCR sensitivity detection reaction system: Green Taq Mix (Vazyme) 12.5 μL, upstream primer mix 0.5 μL, downstream primer mix 0.5 μL, ddH 2 O 8.5 μL, respectively add 3 μL 10-fold diluted PEDV G1 type strain, PEDV G2 type strain positive plasmid mixture (1.937 × 10 8 copies / μl~1.937×10 -1 copies / μl) template. The reaction was carried out in a life ProFlex PCR amplification instrument. The reaction program was as follows: pre-denaturation at 94°C for 5 min, followed by 34 cycles of denaturation at 94°C for 40 s, annealing at 55°C for 40 s, extension at 72°C for 50 s, and finally 10 min at 72°C. Take 10 μL of the PCR product and detect it by 1% agarose gel electrophoresis.

[0048] see results figure 2 , the experimental ...

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Abstract

The invention discloses a porcine epidemic diarrhea virus G1 / G2 type RT-PCR identification primer set. The porcine epidemic diarrhea virus G1 / G2 type RT-PCR identification primer set comprises two pairs of specific primers of PEDV-BF and PEDV-BR and PEDV-F and PEDV-R respectively which have the base sequences shown in SEQ. ID. NO. 1-SEQ. ID. NO.4 in the sequence listing. Accordingly, the inventionalso provides a corresponding diagnostic kit. The porcine epidemic diarrhea virus G1 / G2 type RT-PCR identification primer set has the advantages that infection types of PEDV G1and G2 can be identified in the same reaction tube, thereby providing a quick and simple tool for accurate detection of swinery PEDV, laying a solid foundation for the clinical rapid identification of toxic strains of the PEDV G1and G2 types and the laboratorial epidemiology research, helping the pig industry accurately judge the causes of diseases and timely formulate a therapeutic schedule, and reducing the economic losses; experimental studies show that the porcine epidemic diarrhea virus G1 / G2 type RT-PCR identification primer set has good specificity, high sensitivity, good repeatability, short consumed time and low cost.

Description

technical field [0001] The invention belongs to the technical field of porcine epidemic diarrhea virus detection, in particular to a porcine epidemic diarrhea virus G1 / G2 type RT-PCR identification primer set and a diagnostic kit thereof. Background technique [0002] In recent years, with the intensive and large-scale development of pig farming, the occurrence of diarrhea in pig herds has become more and more serious. Porcine epidemic diarrhea virus (PEDV) is the main pathogen of viral enteropathy in suckling piglets. After long-term mutation, it has been divided into G1-type strains and G2-type strains. G1-type strain vaccines are less effective against G2-type strains Therefore, timely and accurate diagnosis of PEDV infection with G1-type strains or G2-type strains is of great significance to the pig industry. [0003] Porcine epidemic diarrhea (PED) is an acute intestinal infectious disease of pigs caused by porcine epidemic diarrhea virus (PEDV). The main clinical mani...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
Inventor 欧阳康王若木孙连静钟莲黄伟坚韦祖樟陈樱
Owner GUANGXI UNIV
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