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Method for detecting porcine epidemic diarrhea virus and B-type porcine enterovirus by using double real-time fluorescene quantification RT-PCR

A porcine epidemic diarrhea, real-time fluorescence quantitative technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganism determination/inspection, etc. Specific duplex real-time fluorescence quantitative RT-PCR amplification, high specificity and good repeatability

Pending Publication Date: 2019-11-12
SHENZHEN COMBINED BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no laboratory method for the differential diagnosis of PEDV and PEV-B at home and abroad.

Method used

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  • Method for detecting porcine epidemic diarrhea virus and B-type porcine enterovirus by using double real-time fluorescene quantification RT-PCR
  • Method for detecting porcine epidemic diarrhea virus and B-type porcine enterovirus by using double real-time fluorescene quantification RT-PCR
  • Method for detecting porcine epidemic diarrhea virus and B-type porcine enterovirus by using double real-time fluorescene quantification RT-PCR

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1 The design of specific primers and probes for PEDV and PEV-B detection

[0063] The present invention has completed the type B porcine enterovirus Ch-SDbz-W2Y strain (preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee (CGMCC, address: China. Beichen, Chaoyang District, Beijing) on ​​March 2, 2015. No. 3, No. 1 Yard, West Road, Institute of Microbiology, Chinese Academy of Sciences), preservation number: CGMCC No.10405, classification and designation: Type B porcine enterovirus, Porcine enterovirus B) On the basis of whole genome sequencing, the identification of the virus strain Each gene (5'UTR, 3'UTR, VP4, VP3, VP2, VP1, 2A, 2B, 2C, 3A, 3B, 3C, 3D) and the different types of enteroviruses that have been entered in GenBank (GenBank accession number: Y14459(PEV-9, UKG), AF363455(PEV-10, LP54), HM131607(PEV-9, Ch-ah-f1), JQ818253(PEV-B, KOR), HQ702854(PEV-3H, K23), AF363453 (PEV-9, UKG) nucleotide homo...

Embodiment 2

[0083] The detection kit of embodiment 2 PEDV and PEV-B

[0084] The present embodiment provides the kit for the double real-time fluorescent quantitative RT-PCR detection of PEDV and PEV-B, and this kit comprises following components: 2 * RT-PCR reaction solution, enzyme mixture (comprising reverse transcriptase and DNA polymerase), primer-probe mixture (concentrations of primers PEDV-F, PEDV-R, PEV-F and PEV-R are all 10 μM, probes PEDV-P and PEV-P are all 5 μM), nucleic acid-free Enzyme purified water, positive and negative controls.

[0085] Among them, the 2×RT-PCR reaction solution and the enzyme mixture solution are all commercially available products from Treasure Bioengineering (Dalian) Co., Ltd. (product code RR064A).

[0086] The positive control is the cell culture supernatant of the inactivated PEDV strain and the Ch-SDbz-W2Y strain, and its preparation method is as follows: the vero cells are cultured to a single layer, and the Ch-SDbz-W2Y virus of PEDV and PEV-...

Embodiment 3

[0091] Example 3 The double real-time fluorescent quantitative RT-PCR detection method of PEDV and PEV-B

[0092] The present embodiment provides a double real-time fluorescent quantitative RT-PCR detection method for PEDV and PEV-B, specifically as follows:

[0093] (1) Preparation of test samples

[0094] Take suspected infection samples (cell cultures, diarrhea samples, intestinal tissue grinds, etc.), centrifuge at 8000r / min for 5 minutes, take the supernatant, and use a commercial RNA / DNA rapid extraction kit to extract total nucleic acid substances.

[0095] (2) Establishment and amplification of the reaction system for dual real-time fluorescent quantitative RT-PCR

[0096] With the nucleic acid extracted in step (1) as a detection template, the sequence obtained by screening in Example 1 is used as primers and probes shown in SEQ ID NO.1-6 to carry out double real-time fluorescent quantitative RT-PCR detection:

[0097] Take a fluorescent quantitative PCR reaction tu...

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Abstract

The invention relates to the technical field of virus detection, in particular to a method for detecting porcine epidemic diarrhea virus and B-type porcine enterovirus by using double real-time fluorescene quantification RT-PCR, and provides a primer and a probe set for double real-time fluorescene quantification RT-PCR detection of the porcine epidemic diarrhea virus and the B-type porcine enterovirus, a detection kit comprising the primer and the probe set and a detection method. The detection method has high detection specificity, good repeatability and high sensitivity, and has the advantages of simple and convenient operation, short detection time and the like, differential diagnosis and elimination detection of the porcine epidemic diarrhea virus and the B-type porcine enterovirus can be realized, and higher application value is achieved in rapid detection of the porcine epidemic diarrhea virus and the B-type porcine enterovirus.

Description

technical field [0001] The invention relates to the technical field of virus detection, in particular to specific primers and probes for double real-time fluorescent quantitative RT-PCR detection of porcine epidemic diarrhea virus and type B porcine enterovirus, and the invention also relates to the use of the specific primers 1. A method and kit for double real-time fluorescent quantitative RT-PCR detection or identification of porcine epidemic diarrhea virus and type B porcine enterovirus with probes. Background technique [0002] With the development of the pig industry, piglet diarrhea has caused serious harm to the pig industry. The causes of piglet diarrhea include viral, bacterial, feed and environmental factors. Among them, viral diarrhea is particularly harmful to piglets. Early and rapid differential diagnosis of the causative agent of viral diarrhea is critical for successful treatment and prevention of disease outbreaks. [0003] Porcine epidemic diarrhea (Porci...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6851C12Q2600/16C12Q2531/113C12Q2537/143C12Q2561/113C12Q2563/107C12Q2521/107Y02A50/30
Inventor 王金良胡绍良邓凤林魏凤于新友陈金龙
Owner SHENZHEN COMBINED BIOTECH
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