RT-PCR kit for identification and diagnosis of porcine epidemic diarrhea virus and application of RT-PCR kit

A porcine epidemic diarrhea and RT-PCR technology, which is applied in the field of porcine epidemic diarrhea virus RT-PCR differential diagnosis kits, can solve the problems of inability to realize the differential diagnosis of natural infection and vaccine immune strains, and achieves high sensitivity and specificity. strong effect

Active Publication Date: 2014-11-19
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The present invention aims to solve the technical problem that the current method for diagnosing PEDV infection cannot realize the differential diagnosis of natural infection and vaccine immune strains, and provides a porcine epidemic diarrhea virus RT-PCR differential diagnosis kit, which has strong specifi

Method used

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  • RT-PCR kit for identification and diagnosis of porcine epidemic diarrhea virus and application of RT-PCR kit
  • RT-PCR kit for identification and diagnosis of porcine epidemic diarrhea virus and application of RT-PCR kit
  • RT-PCR kit for identification and diagnosis of porcine epidemic diarrhea virus and application of RT-PCR kit

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0033] Example 1 Detection of piglet diarrhea samples and analysis of ORF3 gene sequence of epidemic strain

[0034] (1) Primer design

[0035] According to PEDV ZJCZ-4 (GenBank sequence number: JX524137), a pair of primers ORF3-P1 / ORF3-P2 were designed and synthesized to amplify the full ORF3 gene. With reference to the ORF3 gene sequence of PEDV CV777 (GenBank sequence number: AF353511), ZJCZ-4 and vaccine strain DR13 (GenBank sequence number: EU054930), a pair of identification primers ORF3-JD1 / ORF3-JD2 was designed and synthesized, and the identification primers were located at ORF3 The conserved regions at both ends of the deletion region (245nt~294nt), whose amplification range covers the entire deletion region. The above two pairs of primers are shown in Table 1. All primers were synthesized by Shanghai Yingjun Biotechnology Co., Ltd.

[0036] Table 1 Amplification of PEDV ORF3 gene primers and identification primers

[0037]

[0038] (2) RNA extraction and reverse transcript...

Example Embodiment

[0046] Example 2 Optimization of RT-PCR reaction conditions

[0047] (1) Differential diagnosis RT-PCR amplification and determination of the best Tm value

[0048] The cDNAs of HLJ-1 strain and ZJCZ4 strain were amplified by PCR with identification primer ORF3-JD1 / JD2. The total volume of the PCR reaction is 20μL, containing 2μL of 10×PCR buffer, 1.5μL of dNTP (2.5mmol / L), 0.5μL of upstream and downstream primers, 0.5μL of rTaq DNA polymerase, 2μL of cDNA, and add water to 20μL. The PCR program is: 95°C pre-denaturation 5min; 94°C denaturation 1min, annealing for 30s, 72°C extension for 30s, a total of 35 cycles; finally 72°C extension for 10min. Among them, there are 5 gradients of PCR annealing temperature, namely: 45℃, 48℃, 50℃, 52℃, 55℃. The obtained PCR products were observed by 1% agarose gel electrophoresis.

[0049] (2) Determination of the optimal primer concentration for RT-PCR

[0050] On the basis of the above reaction conditions, the upstream and downstream identifica...

Example Embodiment

[0053] Example 3 RT-PCR specific identification test

[0054] Extract the RNA of TGEV, PoRV, PRRSV (HuN4 strain), CSFV and PEDV (HLJ-1 strain and ZJCZ4 strain) stored in our laboratory, and use ORF3-JD1 / JD2 primers to verify the specificity of RT-PCR amplification .

[0055] The results showed that only ZJCZ4 and HLJ-1 strains can amplify specific bands of different sizes, about 300 bp and 250 bp in size, respectively, and no specific bands appeared with other viral nucleic acids as templates (see Figure 5 ). Figure 5 Among them, M: Maker DL2000; 1: TGEV; 2: PoRV; 3: PRRSV (HuN4 strain); 4: CSFV; 5: HLJ-1 strain; 6: ZJCZ4 strain; 7: negative control.

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Abstract

The invention discloses a RT-PCR kit for identification and diagnosis of a porcine epidemic diarrhea virus (PEDV). The kit comprises a pair of primers designed for deleted regions of nucleotide sequences at positions of 245-294 of an ORF3 gene of an attenuated vaccine strain of the PEDV, wherein the forward and reverse primers of the pair of primers are respectively located in conserved regions at both ends of the deleted regions of the ORF3 gene. The RT-PCR kit for identification and diagnosis of the PEDV based on the ORF3 gene disclosed by the invention has the characteristics of strong specificity and high sensitivity. By virtue of the RT-PCR kit, whether the PEDV is present in diarrhea samples of a piglet suffering from diarrhea or not can be rapidly detected and a naturally infected wild strain can be also rapidly and accurately distinguished from the attenuated vaccine strain. The RT-PCR kit is of important significance for rapid diagnosis, prevention and control of early stage of porcine epidemic diarrhea.

Description

technical field [0001] The invention relates to the technical field of porcine epidemic diarrhea virus infection detection, in particular to a porcine epidemic diarrhea virus RT-PCR differential diagnosis kit and application thereof. Background technique [0002] Porcine epidemic diarrhea (PED) has been reported in some European and Asian countries with developed pig farming industry, and it is a serious infectious disease. In the past two years in some pig farms in my country, piglet diarrhea epidemics characterized by watery diarrhea, vomiting and high mortality in unweaned piglets have broken out again. The disease mainly occurs in piglets within 7 days of age. The mortality rate of piglets is high, and it is easy to show repeated attacks in the same pig farm. It once caused a serious situation in some pig farms that there were no piglets in staged farrowing rooms, which brought significant economic losses to the pig industry. The positive detection rate of porcine epidem...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/686C12Q1/70C12Q2531/113
Inventor 童光志周艳君吴玉璐
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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