MLPA (multiplex ligation-dependent probe amplification) identification kit capable of detecting multiple pathogens causing sudden death of pigs

A technology for multiple ligation of probes and kits, applied in the field of inspection and quarantine, can solve problems such as identification, and achieve the effects of high sensitivity and good specificity

Active Publication Date: 2018-06-29
ZHEJIANG FORESTRY UNIVERSITY
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Problems solved by technology

Although there are corresponding methods and strategies for the factors that cause sudden death in pi

Method used

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  • MLPA (multiplex ligation-dependent probe amplification) identification kit capable of detecting multiple pathogens causing sudden death of pigs
  • MLPA (multiplex ligation-dependent probe amplification) identification kit capable of detecting multiple pathogens causing sudden death of pigs
  • MLPA (multiplex ligation-dependent probe amplification) identification kit capable of detecting multiple pathogens causing sudden death of pigs

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Example Embodiment

[0087] Preparation of in vitro transcribed RNA of swine fever virus E2 gene: RT-PCR amplifies swine fever virus (SM strain) E2 gene and recovers the amplified product. The length is 345bp, which is cloned into pGEM-5zf vector (purchased from Promega) and sequenced After confirmation, a positive recombinant plasmid was obtained and named pGEM-CSFV-E2. Using the purified plasmid as a template, the plasmid was linearized with MluI enzyme, and then transcribed in vitro with Ambion’s MEGAscript T7 kit. The in vitro transcription product was precipitated with LiCl, washed with 70% ethanol and dissolved in RNase-free ddH 2 In O, nucleic acid electrophoresis detects the completeness and correctness of RNA synthesis, and determines the RNA concentration. The in vitro transcribed RNA of porcine reproductive and respiratory syndrome virus M gene was named CSFV-E2-RNA. The E2 gene sequence of classical swine fever virus is shown in SEQ ID NO: 25 in the sequence table.

[0088] Preparation o...

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Abstract

The invention relates to a biological detection technology and aims to provide an MLPA (multiplex ligation-dependent probe amplification) identification kit capable of detecting multiple pathogens causing sudden death of pigs. The kit comprises an RT (reverse transcription) and pre-amplification primer mixed liquid, a probe mixed liquid, an MLPA buffer solution, a ligation buffer solution A, a ligation buffer solution B, ligase Ligase-65, a PCR (polymerase chain reaction) mixed liquid, SALSA polymerase, negative control and positive control. Compared with the prior art, the kit has the characteristics of high throughput, good specificity and high sensitivity. The kit can detect 6 pathogens causing sudden death of pigs simultaneously and can detect 96 samples simultaneously each time by useof a full-automatic nucleic acid analyzer. Through probe design, sequence comparison and blast analysis, a probe is ensured to be only bound with a target gene. During detection, all that is requiredis to amplify a target fragment in a corresponding size from a corresponding template, and other pathogens are not amplified. The target gene is enriched through the added step of RT-PCR, and one copied target gene can be detected to the minimum.

Description

technical field [0001] The invention provides a multiplex connection probe amplification for detecting porcine reproductive and respiratory syndrome virus, swine fever virus, Nipah virus, African swine fever virus, Clostridium perfringens and Actinobacillus pleuropneumoniae The identification detection kit, primers and probes can simultaneously detect 6 kinds of pathogens that cause sudden death in pigs, and belong to the field of inspection and quarantine. Background technique [0002] Porcine reproductive and respiratory syndrome virus (PRRSV), swine fever virus (CSFV), Nipah virus (NiPV), African swine fever virus (ASFV), Clostridium perfringens and Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae) is a common pathogen causing sudden death syndrome in pigs. Although African swine fever and porcine Nipah disease have not been found in my country, the reports of existing diseases in neighboring countries and Asian regions should also be paid attention to, a...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/689C12Q1/6844C12Q1/04C12N15/11C12R1/145C12R1/04
CPCC12Q1/6844C12Q1/689C12Q1/701C12Q2537/143C12Q2521/107C12Q2531/113
Inventor 宋厚辉周莹珊王晓杜邵春艳陈琳章先程昌勇杨杨
Owner ZHEJIANG FORESTRY UNIVERSITY
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