RT-qPCR detection method and primers and TaqMan probe for detecting swine transmissible gastroenteritis virus N gene

An infectious and gastroenteritis technology, applied in the field of primers for N gene RT-qPCR of porcine infectious gastroenteritis virus, can solve the problems of low specificity, low general value of primers, low conservation, etc. High general value, high stability effect

Inactive Publication Date: 2015-06-03
GUANGDONG WENS DAHUANONG BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The two methods are aimed at the S gene of TGEV. The S protein gene has strong antigenicity and low conservation. The primers designed for the S gene have low general value, and the two methods use SYBR Green with relatively low specificity. Ⅰ dye probe

Method used

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  • RT-qPCR detection method and primers and TaqMan probe for detecting swine transmissible gastroenteritis virus N gene
  • RT-qPCR detection method and primers and TaqMan probe for detecting swine transmissible gastroenteritis virus N gene
  • RT-qPCR detection method and primers and TaqMan probe for detecting swine transmissible gastroenteritis virus N gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1: Design and synthesis of primers and TaqMan probes

[0066] There are seven open reading frames (ORFs) in the TGEV genome, among which the N gene encodes the nucleocapsid protein, which is highly conserved. Taking the N gene of the TGEV PY-D strain isolated and preserved in our laboratory as the target gene, using molecular biology software to compare and analyze it with the gene sequence published on GenBank, design the upstream primer TGEV-P1, downstream primer TGEV-P2 and The sequence of the probe TGEV-Probe is shown in Table 1,

[0067] Table 1 Primers and Probes

[0068] serial number

[0069] In a further scheme, the fluorescent reporter group labeled at the 5' end of the probe is FAM, the fluorescent quencher group labeled at the 3' end is TAMRA, and the probe is named TGEV-Probe.

Embodiment 2

[0070] Embodiment 2: the cultivation of TGEV

[0071] After the PK-15 cells were subcultured until they grew into a single layer, normal PK-15 cells such as figure 1 As indicated, the freeze-dried TGEV PY-D strain preserved in this experiment was diluted with DMEM and inoculated on PK-15 cells at 37°C in 5% CO 2 After 1 hour of adsorption in the incubator, discard the virus solution, add DMEM maintenance solution, and place it in the incubator again. After culturing for 45-55 hours, scrape the cells to harvest the virus solution after obvious lesions appear, and the cultured PK-15 cells are as follows: figure 2 shown.

Embodiment 3

[0072] Embodiment 3: the preparation of positive standard plasmid

[0073] 1) Preparation of cDNA

[0074] Viral RNA was extracted by AxyPrep Body Fluid Viral DNA / RNA Miniprep Kit (AXYGEN), or viral RNA was extracted by methods known in the prior art, and the extracted RNA was reverse-transcribed by a reverse transcription kit to prepare cDNA.

[0075] The RNA / DNA extraction kit and gel recovery kit can use AxyPrep Body Fluid Viral DNA / RNA Miniprep Kit and Gel Extraxtion Kit products from AXYGEN Company respectively.

[0076] 2) Target fragment PCR amplification

[0077] Using the cDNA prepared in step 1) of this embodiment as a template, and using primers TGEV-P1 and primer TGEV-P2 as specific primers, PCR amplification of the positive standard plasmid was carried out, and the PCR amplification conditions were as follows:

[0078] Use 25 μL reaction system: l0×Buffer 2.5 μL; primer TGEV-P1 1 μL; primer TGEV-P2 1 μL; dNTP 2.0 μL, Taq enzyme 0.15 μL, ddH 2 O 17.35 μL, cDNA...

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Abstract

The invention provides an RT-qPCR detection method, primers and TaqMan probe for detecting a swine transmissible gastroenteritis virus N gene. Particularly, the invention provides a set of RT-qPCR detection method for detecting the swine transmissible gastroenteritis virus gene and a forward primer shown in SEQ ID No.1 in a sequence table, a reverse primer shown in SEQ ID No.2 in the sequence table, and a TaqMan probe sequence shown in SEQ ID No.3 in the sequence table. The invention provides an RT-qPCR detection method for detecting the swine transmissible gastroenteritis virus N gene on the basis of the primers and the TaqMan probe. The detection method has excellent detection sensitivity, and in the case of 1*10<5>copies/microliter to 1*10<1>copies/microliter, Ct values and fluorescence values show typical amplification curves. At the same time, the detection method has good repeatability, strong specificity and convenience in operation.

Description

technical field [0001] The invention relates to primers for RT-qPCR of porcine transmissible gastroenteritis virus N gene, TaqMan probes and applications thereof. Background technique [0002] Porcine transmissible gastroenteritis (TGE) is a highly contagious intestinal disease caused by porcine transmissible gastroenteritis virus (TGEV), clinically characterized by vomiting, severe diarrhea and dehydration in sick pigs. Pigs of different breeds and ages can be infected with the disease, especially piglets and weaned piglets within two weeks of age are the most susceptible, and the fatality rate can be as high as 100%. The disease is a worldwide disease that brings huge economic losses to animal husbandry production. Early detection and diagnosis are of great significance to the prevention and treatment of this disease. [0003] At present, the routine detection methods for detecting TGEV mainly include the following categories: [0004] 1. Etiological detection, such as v...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/686C12Q1/701C12Q2561/101C12Q2531/113
Inventor 刘好朋贺东生陈瑞爱王阿明吴凤鸣胡京京裴仉福李琳
Owner GUANGDONG WENS DAHUANONG BIOTECH
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