GeXP rapid detection kit for identifying eight types of porcine viral diseases and primer group thereof

A detection kit and a technology for swine virus disease, which are applied in the field of swine virus disease detection, can solve the problems of reagents or kits that have not yet been reported for various infectious disease pathogens, and achieve the resolution of amplification preference, high sensitivity and application prospects. expansive effect

Active Publication Date: 2014-08-13
GUANGXI VETERINARY RES INST
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, no reagents or kits based on the GeXP system that can detect multiple infectious disease pathogens of porcine virus disease at the same time have been reported.

Method used

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  • GeXP rapid detection kit for identifying eight types of porcine viral diseases and primer group thereof
  • GeXP rapid detection kit for identifying eight types of porcine viral diseases and primer group thereof
  • GeXP rapid detection kit for identifying eight types of porcine viral diseases and primer group thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1, the design of specific primer pair

[0020]Using the primer sequence published by GenBank as a reference, download the known sequence from NCBI and use DNASTAR software for sequence alignment, select highly conserved and specific gene fragments for each target gene, and design 9 specific primers by primer premier5.0 software , and a GeXP universal primer (Table 1) was added to the 5' end of the primer.

[0021] Table 1 Primer Information

[0022]

[0023] In Table 1, the codes of the merged bases are R=A / G, D=A / G / T, Y=C / T, and the underline indicates the sequence of the upstream and downstream universal primers; the fluorescent dye Cy5 marks the label of the upstream universal primer, and the downstream primer Not marked. According to the different strains of the pathogens actually detected and the error of the GeXP system (such as the GenomeLabTM GeXP Genetic Analysis System capillary electrophoresis instrument), the length of the actual amplified pr...

Embodiment 2

[0025] Embodiment 2, the specific detection of PCR primer pair

[0026] 1. Template preparation

[0027] 1. Extraction of viral RNA and acquisition of cDNA

[0028] 1) Extraction of viral RNA

[0029] Using DNA / RNA extraction kits (all purchased from Beijing Quanshijin Biotechnology Co., Ltd., catalog number ER201), according to the kit instructions, respectively extract swine influenza virus (H1N1, H1N2, H3N2), porcine Japanese encephalitis virus (JEV ), the RNA of American porcine reproductive and disorder syndrome virus (PRRSV), classical swine fever virus (CSFV)

[0030] 2) Acquisition of cDNA

[0031] The RNA samples obtained in step 1) were reverse-transcribed according to the following reaction system and reaction conditions to obtain cDNA; DEPC water was used as the control of total RNA.

[0032] Reaction system (25 μL): 5×Reverse Transcriptase Buffer 5 μL, 50 pmol Random Primer (9mer) 1 μL, dNTP Mixture (10 mM) 2 μL, 40U Ribonuclease Inhibitor 0.5 μL, 5U / μL AMV Re...

Embodiment 3

[0080] Embodiment 3, the sensitivity detection of PCR primer pair

[0081] 1. Preparation of monoclonal plasmid standards containing target genes

[0082] Swine influenza virus (H1N1, H1N2, H3N2), porcine pseudorabies virus, swine fever virus, American type porcine reproductive and disorder syndrome virus, porcine encephalitis virus, porcine parvovirus, porcine The cDNA or DNA sample of circovirus type 2 is used as a template, and the M gene of swine influenza virus obtained by PCR amplification, the HA gene of H1 subtype swine influenza virus, the HA gene of H3 subtype swine influenza virus, the GD gene of porcine pseudorabies virus, The E2 gene of classical swine fever virus, the NSP2 gene of American type porcine reproductive and disorder syndrome virus, the M protein gene of porcine encephalitis virus, the VP2 gene of porcine parvovirus, and the ORF1 gene of porcine circovirus type 2 were respectively connected with the vector pMD18-T vector ( Purchased from Dalian Bao Bi...

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Abstract

The invention discloses a GeXP rapid detection kit for identifying eight types of porcine viral diseases and a primer group thereof. Nine pairs of specific primers and a pair of universal primers are designed by the inventor based on research of a GeXP system, accordingly, a GeXP rapid detection method for identifying the eight types of porcine viral diseases is established, and the corresponding detection kit is prepared. The GeXP rapid detection method and the detection kit have the advantages of good specificity, high sensitivity, convenience, high efficiency and the like and can be used for simultaneously detecting and identifying eight types of pathogens. Compared with identification results of conventional experiment methods such as virus isolation and hemagglutination inhibition test, the coincidence rate of the GeXP rapid detection method reaches 100%. The GeXP rapid detection method and the detection kit can be used for effectively solving amplification preference in a multiplex-PCR (polymerase chain reaction) process, provide simple, convenient and high-throughput detection means for detection of main porcine common infectious diseases and pathogens thereof, meet practical requirements and have a broad application prospect.

Description

technical field [0001] The invention belongs to the technical field of porcine virus disease detection, and in particular relates to a GeXP rapid detection kit and a primer set for distinguishing eight kinds of porcine virus diseases. Background technique [0002] H1, H3 subtype swine influenza virus, porcine pseudorabies virus, swine fever virus, porcine reproductive and disorder syndrome virus, porcine encephalitis virus, porcine parvovirus, and porcine circovirus type 2 are the eight main infections that seriously harm pigs disease. With the development of pig breeding industry, the incidence of porcine infectious viral diseases is gradually increasing, which has become an important factor restricting the development of pig farming industry. Traditional methods for the differential diagnosis of these porcine infectious diseases mainly include isolation and identification of pathogens and serological tests, etc., but these methods are often limited by the freshness of cli...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/686C12Q1/70C12Q2531/113C12Q2537/143C12Q2565/125
Inventor 谢芝勋张民秀谢丽基罗思思刘加波谢志勤邓显文庞耀珊范晴
Owner GUANGXI VETERINARY RES INST
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