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Fluorescence quantitative PCR kit for detecting porcine epidemic diarrhea virus and use thereof

A porcine epidemic diarrhea and fluorescence quantitative technology, which is applied in the field of molecular biology, can solve the problems of inability to meet clinical diagnosis and insufficient sensitivity, and achieve the effects of effective detection, good repeatability and good application prospects.

Inactive Publication Date: 2016-12-14
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the sensitivity of the RT-PCR detection kit prepared in the existing literature is still not high enough to meet the needs of clinical diagnosis.

Method used

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  • Fluorescence quantitative PCR kit for detecting porcine epidemic diarrhea virus and use thereof
  • Fluorescence quantitative PCR kit for detecting porcine epidemic diarrhea virus and use thereof
  • Fluorescence quantitative PCR kit for detecting porcine epidemic diarrhea virus and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1 Preparation of fluorescent quantitative PCR kit of the present invention

[0047] 1. Experimental method

[0048] 1.1 Primer design

[0049] According to the analysis of the sequences of the virulent strain (AF353511) and the attenuated strain (EU054930), the ORF3 gene was selected as the target sequence to design and synthesize a pair of specific fluorescent quantitative primers (see Table 1), and the primers amplified the virulent strain and the attenuated strain The obtained fragments have different sizes, among which a 238bp fragment is obtained by amplifying the strong virus strain, and a 187bp fragment is obtained by amplifying the weak virus strain. Therefore, it can also be used for differential diagnosis of strong and weak PEDV strains.

[0050] Among them, ORF3-F / ORF3-R were used to construct standard plasmids; F1 / R1 were used as RT-PCR detection primers.

[0051] Table 1 Fluorescence quantitative primers

[0052]

[0053] 1.2 Establishment ...

experiment example 1

[0088] Experimental Example 1 Specificity Test

[0089] 1. Experimental method

[0090] Porcine transmissible gastroenteritis virus (TGEV), Japanese encephalitis virus (JEV), porcine rotavirus (PoRV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus (PCV), The total RNA of porcine parvovirus (PPV) and porcine pseudorabies virus (PRV) was reverse-transcribed into cDNA, and carried out together with porcine epidemic diarrhea virus (PEDV) for EG-qRT-PCR, and a negative control (template for negative control) was set up at the same time Sterilized ddH was used 2 O)

[0091] The fluorescence quantitative reaction system is the same as in Table 2, and the reaction program is: 95°C for 30s; 95°C for 5s, 57°C for 10s, 40 cycles; finally a melting curve is generated.

[0092] The specificity of the kits and methods of the invention is evaluated.

[0093] 2. Experimental results

[0094] See Figure 9-10 .

[0095] Depend on Figure 9-10 It can be ...

experiment example 2

[0097] Experimental Example 2 Sensitivity Test

[0098] 1. Experimental method

[0099] to 10 -3 ~10 6 copies / μL standard plasmid as a template (initial plasmid concentrations of strong and weak strains were 4.79×10 11 copies / μL, 5.17×10 11 copies / μL, diluted from initial concentration to 10 -3 copies / μL, choose 10 -3 ~10 6 copies / μL for sensitivity experiment), EG-qRT-PCR detection was carried out, and its sensitivity was evaluated.

[0100] The EG-qRT-PCR reaction system is as follows:

[0101]

[0102] Reaction program: 95°C for 30s; 95°C for 5s, 57°C for 10s, 40 cycles; finally generate a melting curve.

[0103] 2. Experimental results

[0104] At a concentration of 4.79×10 -3 copies / μL~4.79×10 6 copies / μL, 5.17×10 -3 copies / μL~5.17×10 6 Copies / μL plasmid standards were used as templates for EG-qRT-PCR, and the lower limit of detection of strong toxicity was 4.79×10 0 copies / μL( Figure 11 ). The lower limit of detection of attenuated virus is 5.17×10 0...

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Abstract

The invention provides a fluorescence quantitative PCR kit for detecting a porcine epidemic diarrhea virus. The fluorescence quantitative PCR kit comprises a primer pair shown as SEQ ID NO: 1-2 and used for amplifying porcine epidemic diarrhea virus genes. The invention further provides a use of the kit and a method for detecting the porcine epidemic diarrhea virus. The kit disclosed by the invention can accurately and effectively detect the porcine epidemic diarrhea virus, and is high in sensitivity, strong in specificity, good in repeatability, fast in detection and good in application prospects.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a fluorescent quantitative PCR kit for detecting porcine epidemic diarrhea virus and its application. Background technique [0002] Porcine epidemic diarrhea (Porcine epidemic diarrhea, PED) is caused by porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus, PEDV), and is characterized by diarrhea, vomiting, dehydration and high lethality to suckling piglets. sexually transmitted intestinal diseases. As of 2015, PED has spread to most pig raising countries, and has become a worldwide pig disease in Europe, Southeast Asia, the Americas (United States, Canada, Mexico), Japan, South Korea and other places, causing serious damage to the global pig industry. Huge economic loss, PED has become the common concern of pig industry in the world and needs to be solved urgently. [0003] Since the current immune prevention and control effect on PED is not v...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/701C12Q1/6851C12Q2531/113C12Q2563/107
Inventor 黄小波李亚青曹三杰文心田伍锐文翼平张雨迪
Owner SICHUAN AGRI UNIV
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