PCR-HRM (Polymerase Chain Reaction-High Resolution Melting) primer for authenticating swine acute diarrhoea syndrome virus and porcine epidemic diarrhea virus, method for authenticating swine acute diarrhoea syndrome and porcine epidemic diarrhea, and application of PCR-HRM primer

A PCR-HRM, porcine epidemic diarrhea technology, applied in microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve problems such as timely diagnosis and treatment of unfavorable diseases, cumbersome operation methods, economic losses, etc., and achieve rapid differentiation. , good repeatability, shorten the time effect

Active Publication Date: 2020-07-24
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The clinical symptoms and anatomical symptoms of porcine acute diarrhea syndrome virus and porcine epidemic diarrhea virus are very similar, and it is difficult to distinguish clinically and histopathologically. The differential diagnosis usually requ

Method used

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  • PCR-HRM (Polymerase Chain Reaction-High Resolution Melting) primer for authenticating swine acute diarrhoea syndrome virus and porcine epidemic diarrhea virus, method for authenticating swine acute diarrhoea syndrome and porcine epidemic diarrhea, and application of PCR-HRM primer
  • PCR-HRM (Polymerase Chain Reaction-High Resolution Melting) primer for authenticating swine acute diarrhoea syndrome virus and porcine epidemic diarrhea virus, method for authenticating swine acute diarrhoea syndrome and porcine epidemic diarrhea, and application of PCR-HRM primer
  • PCR-HRM (Polymerase Chain Reaction-High Resolution Melting) primer for authenticating swine acute diarrhoea syndrome virus and porcine epidemic diarrhea virus, method for authenticating swine acute diarrhoea syndrome and porcine epidemic diarrhea, and application of PCR-HRM primer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1PCR-HRM primer design

[0042] A primer pair P-F and P-R for amplifying partial gene sequences of porcine acute diarrhea syndrome virus and porcine epidemic diarrhea virus were designed according to the conserved gene sequences of porcine acute diarrhea syndrome virus and porcine epidemic diarrhea virus, and their base sequences are as follows:

[0043] P-F: GATGGTGGTTGTAASACTAT;

[0044] P-R: ACAGTRGCACCTATGTAGCC.

[0045] One degenerate base S (equivalent to G / C) was introduced into primer P-F, and one degenerate base R (equivalent to A / G) was introduced into P-R.

Embodiment 2

[0046] The preparation of embodiment 2 standard sample and PCR-HRM analysis thereof

[0047] (1) Extraction of viral RNA: use AxyPrep TM The bodily fluid virus DNA / RNA preparation kit (product serial number: PID0320425) was used to extract the RNA of porcine acute diarrhea syndrome virus and porcine epidemic diarrhea virus in diseased material samples (collected from a pig farm in Guangdong Province in 2019). Disease material samples are mainly intestinal and other tissue samples.

[0048] (2) Reverse transcription to obtain cDNA: use the RNA obtained in the previous step as a template, add reverse transcription reagents, and put the PCR tube into the PCR instrument for the reaction system shown in Table 1, and reverse transcribe at 37°C for 15 minutes; Inactivate at 85°C for 5s, and store at -20°C for later use.

[0049] Table 1 reverse transcription PCR reaction system

[0050]

[0051] For the reverse transcription system, first add the first three reagents to the PCR t...

Embodiment 3

[0068] Embodiment 3 specificity experiment

[0069] Extract the RNA of viruses that commonly cause diarrhea in other pigs, such as porcine transmissible gastroenteritis virus (Porcine Transmissible Gastroenteritis Virus, TGEV) (TGEV-SY strain, which has been published in the literature "You Liuyang et al., Porcine Transmissible Gastroenteritis Virus SY strain Separation and identification of S gene and sequence analysis of S gene. China Veterinary Science, 2016.46(05): pp. 579-585." published in), Porcine Deltacoronavirus (Porcine Deltacoronavirus, PDCoV) (H223 strain, has been published in the literature "He Dongsheng et al., Isolation and identification of porcine delta virus H223 strain and continuous passage and genetic evolution analysis of N gene. Porcine Science, 2019.36(07): pp. 72-74.") and porcine rotavirus (Porcine Rotavirus, PoRV ) (porcine transmissible gastroenteritis, porcine epidemic diarrhea, porcine rotavirus (G5 type) triple live vaccine (attenuated Sinoviru...

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Abstract

The invention discloses a PCR-HRM (Polymerase Chain Reaction-High Resolution Melting) primer for authenticating a swine acute diarrhoea syndrome virus and a porcine epidemic diarrhea virus, a method for authenticating a swine acute diarrhoea syndrome and porcine epidemic diarrhea, and application of the PCR-HRM primer. A primer pair provided by the invention has good specificity, only one pair ofprimers needs to be used for carrying out PCR amplification on the SADS-CoV (Swine Acute Diarrhoea Syndrome Coronavirus) and the PEDV (Porcine Epidemic Diarrhea Virus), a bonding situation of a double-stranded DNA fluorescent dye and a PCR amplification product in a temperature rise process can be monitored in real time, and fluorescent data is collected and is authenticated according to the difference of two virus melting curves. The detection method provided by the invention does not need the separation and identification of viruses, virus authentication time is shortened, gel electrophoresis is not required for observing a result, and software can be used for analyzing the result when the PCR is finished, cost is low, a specific probe is not required, and the fluorescent saturable dye is cheap and can be easily obtained. The PCR-HRM primer has the characteristics of good specificity, high sensitivity and good repeatability, can accurately and quickly distinguish viruses, and can beprompted and used in clinical detection.

Description

technical field [0001] The invention belongs to the technical field of virus detection, and in particular relates to a PCR-HRM primer, a method and an application thereof for distinguishing porcine acute diarrhea syndrome virus and porcine epidemic diarrhea virus. Background technique [0002] There are four main subgroups of coronaviruses, including Alpha, Beta, Gamma, and Delt coronaviruses, which have caused enormous harm and economic losses to humans and livestock. In 2017, a novel bat-like coronavirus HKU-2 strain was identified in an outbreak of acute diarrheal disease on a pig farm in South China, and was subsequently identified as Swine Acute Diarrhea Syndrome Coronavirus (SADS-CoV). ). The clinical symptoms of porcine acute diarrhea syndrome are very similar to those caused by other known porcine enteric coronaviruses, manifested as acute vomiting and diarrhea, and rapid weight loss in newborn piglets leading to acute death. The disease can infect pigs of all ages...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/686C12Q1/701C12Q2527/107C12Q2563/107C12Q2561/113
Inventor 贺东生司广斌马沐林陈志飞梁文清陈一波
Owner SOUTH CHINA AGRI UNIV
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