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Fluorogenic quantitative PCR kit for detecting swine transmissible gastroenteritis virus

A technology for infectious and gastroenteritis, applied in the field of fluorescent quantitative PCR kits, can solve the problems of long time consumption and low sensitivity, and achieve the effects of short time consumption, high sensitivity and rapid detection

Inactive Publication Date: 2015-05-13
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Traditional detection methods, such as virus isolation and identification and serum examination, often take too long
The patent application with publication number 103740860A discloses a method for detecting porcine transmissible gastroenteritis by PCR. The minimum detection limit of porcine transmissible gastroenteritis that can be detected is 35 fg / μL, that is, 875 copies / μL, and the sensitivity is low

Method used

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  • Fluorogenic quantitative PCR kit for detecting swine transmissible gastroenteritis virus
  • Fluorogenic quantitative PCR kit for detecting swine transmissible gastroenteritis virus
  • Fluorogenic quantitative PCR kit for detecting swine transmissible gastroenteritis virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] The preparation of embodiment 1 kit of the present invention

[0039] 1. Materials and instruments

[0040] The same experimental materials and instruments as mentioned above.

[0041] 2. Experimental method

[0042] 2.1 Fluorescence quantitative PCR primer design

[0043] According to the full sequence of the TGEV SC-H strain isolated in our laboratory, bioinformatics software was used to analyze the full sequence of TGEV, and the N gene of the TGEV SC-H strain was selected as the target sequence to design 3 pairs of fluorescent quantitative primers (Table 1). And sent to Shanghai Sangon Bioengineering Co., Ltd. for synthesis.

[0044] Table 1 Fluorescent quantitative RCR primers

[0045]

[0046] 2.2 Extraction of viral RNA and synthesis of cDNA

[0047] In the ultra-clean workbench, take out a small piece of TGEV attenuated vaccine, dilute it with 500 μL ultrapure water, and extract RNA. According to the instruction of TIANGEN Total RNA Extraction kit.

[0...

Embodiment 2

[0126] Embodiment 2 specificity experiment

[0127] 1. Test method

[0128] Separately extract porcine transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine rotavirus (PoRV), Japanese encephalitis B (JEV), swine fever virus (CSFV), pig reproductive With the gene of respiratory syndrome virus (PRRSV), the second pair of primers (F2 and R2F: GCCAAGCATTACCCACAAC; R: TGGACGAGCATAGGCATTA) were used for fluorescent quantitative PCR, and DEPC-treated water was used as a blank control. To verify the specificity of the gene chip and kit of the present invention.

[0129] The fluorescent quantitative PCR reaction system is as follows, with a total volume of 20 μL:

[0130]

[0131] Reaction conditions: initial template denaturation at 95°C, 1min; denaturation at 95°C, 20sec; annealing at 58°C, 30sec; extension at 68°C, 30sec; 35 cycles.

[0132] 2. Results

[0133] Experimental results such as Figure 5 Shown, adopt the method of the present...

Embodiment 4

[0135] Embodiment 4 sensitivity test

[0136] 1. Test method

[0137] With concentration from 6.37×10 5 , 6.37×10 4 , 6.37×10 3 , 6.37×10 2 , 6.37×10 1 , 6.37×10 0 The copy / μL standard of porcine transmissible gastroenteritis virus (TGEV) plasmid was used as a template, and the second pair of primers (F2 and R2F: GCCAAGCATTACCCACAAC; R: TGGACGAGCATAGGCATTA) were used for fluorescent quantitative PCR to obtain the lowest template that can be detected copy number, while using DEPC-treated water as a blank control.

[0138] The fluorescent quantitative PCR reaction system is as follows, with a total volume of 20 μL:

[0139]

[0140] Reaction conditions: initial template denaturation at 95°C, 1min; denaturation at 95°C, 20sec; annealing at 58°C, 30sec; extension at 68°C, 30sec; 35 cycles.

[0141] 2. Results

[0142] The result is as Image 6 as shown,

[0143] With concentration from 6.37×10 5 , 6.37×10 4 , 6.37×10 3 , 6.37×10 2 , 6.37×10 1 , 6.37×10 0 The co...

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Abstract

The invention discloses a fluorogenic quntitative PCR kit for detecting swine transmissible gastroenteritis virus. The fluorogenic quantitative PCR kit comprises primer pairs showed in SEQ ID NO: 1-2 for amplifying swine transmissible gastroenteritis virus genes. According to the kit disclosed by the invention, the swine transmissible gastroenteritis virus can be detected accurately and effectively, and the kit has the advantages of strong specificity, high sensitivity, short consumed time, high detecting speed and good application prospect.

Description

technical field [0001] The invention relates to a fluorescent quantitative PCR kit for detecting porcine transmissible gastroenteritis virus. Background technique [0002] Porcine transmissible gastroenteritis (TGEV) is a highly contagious intestinal disease that can affect pigs of different ages and breeds, especially for suckling piglets, often leading to disease, morbidity and mortality in the whole litter High, the severity of the disease varies with age, and the younger the age, the higher the mortality rate. Piglets under the age of two weeks have vomiting, severe watery diarrhea, sometimes in the form of jets, and the pigsty where the diarrhea occurs has a foul smell, and the whole body of the pig is polluted by feces, and the feces contain undigested curd flakes and appear milky or off-white; In the later stage of the disease, the feces of pigs become viscous, and the pigs become thin and lose weight; they generally die within 3-7 days, and the mortality rate of pig...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/701
Inventor 黄小波文心田李蕊彤曹三杰文翼平伍锐李亚青邓丽卿盈张雨迪陈杰
Owner SICHUAN AGRI UNIV
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