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A multiplex connection probe amplification identification kit capable of detecting various porcine sudden death disease pathogens

A technology of multiple connection probes and sudden death syndrome, which is applied in the field of inspection and quarantine, can solve problems such as identification, and achieve high sensitivity and good specificity

Active Publication Date: 2021-08-13
ZHEJIANG FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although there are corresponding methods and strategies for the factors that cause sudden death in pigs, there is no method that can identify multiple diseases that cause sudden death in pigs at one time

Method used

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  • A multiplex connection probe amplification identification kit capable of detecting various porcine sudden death disease pathogens
  • A multiplex connection probe amplification identification kit capable of detecting various porcine sudden death disease pathogens
  • A multiplex connection probe amplification identification kit capable of detecting various porcine sudden death disease pathogens

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preparation example Construction

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

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Abstract

The invention relates to biological detection technology, and aims to provide a multiple connection probe amplification identification kit capable of detecting various porcine sudden death disease pathogens. The kit includes: reverse transcription and preamplification primer mix, probe mix, MLPA buffer, ligation buffer A, ligation buffer B, ligase Ligase-65, PCR reaction mix, SALSA polymerase, Negative control, positive control. Compared with the prior art, the invention has the characteristics of high throughput, good specificity and high sensitivity. It can detect 6 kinds of pathogens that cause sudden death in pigs at the same time, and 96 samples can be detected at the same time by using the automatic nucleic acid analyzer. Through probe design, sequence alignment and blast analysis, it is ensured that the probe only binds to the target gene. During the detection, only the target fragment of the corresponding size is amplified from the corresponding template, and there is no amplification for other pathogens. By adding the step of RT‑PCR to enrich the target gene, a minimum of 1 copy of the target gene can be detected.

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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Patent Type & Authority Patents(China)
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