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Multiplex ligation-dependent probe amplification detection kit for simultaneously detecting five swine disease viruses, primers and probes

A technology for multiple ligation of probes and swine influenza virus, applied in the field of inspection and quarantine, can solve the problems of difficult to achieve differential diagnosis of diseases with similar symptoms, difficult to determine results, and large detection workload.

Active Publication Date: 2013-02-27
PEOPLES REPUBLIC OF CHINA BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the currently established methods are single detection techniques for one pathogen. In the actual detection, the purpose of detecting different pathogens needs to be repeated many times. need
And it is difficult to realize the differential diagnosis of a large number of mixed infections in field samples and diseases with similar symptoms
While establishing a separate disease detection technology, veterinary institutions in various countries have also successively developed multiplex PCR and multiplex fluorescent PCR that can simultaneously detect common pig viruses. Fluorescence PCR is due to the mutual interference of the fluorescence emitted by the fluorescent groups used by different probes, and the limitation of fluorescent PCR instrument to the fluorescence resolution of different wavelengths also limits the development of fluorescent PCR multiple detection technology

Method used

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  • Multiplex ligation-dependent probe amplification detection kit for simultaneously detecting five swine disease viruses, primers and probes
  • Multiplex ligation-dependent probe amplification detection kit for simultaneously detecting five swine disease viruses, primers and probes
  • Multiplex ligation-dependent probe amplification detection kit for simultaneously detecting five swine disease viruses, primers and probes

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Embodiment 1, preparation and use of kit

[0076] 1. The composition of the kit is shown in Table 4.

[0077] Table 4 Kit preparation composition

[0078] Composition (30Tests / box)

quantity

MLPA buffer

600μL×1 tube

probe mix

600μL×1 tube

Ligation reaction solution

1240μL×1 tube

Ligase-65 ligase (5U / μL)

115μL×1 tube

PCR reaction solution (including universal primers)

750μL×1 tube

SALSA polymerase (5U / μL)

65μL×1 tube

DEPC water

1mL×3 tubes

negative control

1mL×3 tubes

positive control

1mL×3 tubes

[0079] Wherein, MLPA buffer solution is purchased from The MRC-Holland Company, which includes KCl, Tris-HCl, EDTA and PEG-6000, pH 8.5.

[0080] Probe mixture, it comprises the long and short probe of swine influenza virus, porcine reproductive and respiratory syndrome virus, pseudorabies virus, porcine transmissible gastroenteritis virus and foot-and-mouth ...

Embodiment 2

[0111] Embodiment 2, the sensitivity test of kit

[0112] 1. Materials:

[0113] Swine influenza virus A / swine / 2003 (H1N1), porcine reproductive and respiratory syndrome JXA1 strain inactivated virus, pseudorabies virus Nanyang strain, porcine transmissible gastroenteritis virus purdue115 international standard strain are preserved in the laboratory, O-type foot-and-mouth disease is eliminated Live virus was provided by Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0114] 2. Method

[0115] 1) Preparation of in vitro transcribed RNA and plasmid DNA

[0116] Preparation of swine influenza virus M gene in vitro transcription RNA: Recover the RT-PCR amplification product of swine influenza virus A / swine / 2003 (H1N1) strain M gene, the length is 982bp, and carry out with pGEM-T vector (purchased from PromeGA company) Connect and transform JM109 competent cells, extract plasmid DNA by alkaline lysis, and obtain a positive recombinant plasmid a...

Embodiment 3

[0132] Embodiment 3, the specificity test of kit

[0133] 1. Materials

[0134] Table 6 Viruses and nucleic acids used in specificity test research

[0135] Virus

source

Swine Influenza Virus A / Swine / 2003(H1N1)

The lab saves

Porcine Reproductive and Respiratory Syndrome JXA1 Strain Inactivated Virus

The lab saves

Pseudorabies virus Nanyang strain

The lab saves

Porcine transmissible gastroenteritis purdue115 international standard strain

The lab saves

Type O foot and mouth disease inactivated virus

Lanzhou Veterinary Research Institute

porcine parvovirus

The lab saves

Inactivated swine fever virus

Provided by China Veterinary Drug Administration

porcine epidemic diarrhea virus

The lab saves

[0136] 2. Method

[0137] 2.1 Use any group of probes in the five viruses of swine influenza virus, porcine reproductive and respiratory syndrome virus, pseudorabies virus, porcine...

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Abstract

The invention discloses a multiplex ligation-dependent probe amplification detection kit for simultaneously detecting five swine disease viruses, primers and probes. The multiplex ligation-dependent probes are shown in sequence tables SEQ ID NO:1 to SEQ ID NO:10; and the primers are shown in sequence tables SEQ ID NO:11 to SEQ ID NO:12. By using the primers, the probes and / or the multiplex ligation-dependent probe amplification detection kit containing the primers and the probes, five important swine disease pathogens such as a swine influenza virus, a swine reproductive and respiratory syndrome virus, a pseudorabies virus, a swine transmissible gastroenteritis virus and a foot-and-mouth disease virus can be simultaneously detected, thereby saving the detection time and cost and being beneficial to accurately diagnosing the epidemic diseases in time.

Description

technical field [0001] The invention provides a multiple connection probe amplification detection kit, primers and probes for detecting swine influenza virus, porcine reproductive and respiratory syndrome virus, pseudorabies virus, porcine transmissible gastroenteritis virus and foot-and-mouth disease virus , can realize the purpose of one sampling, one analysis, and detection of 5 kinds of pig diseases at the same time, which belongs to the field of inspection and quarantine. Background technique [0002] Swine influenza virus (SIV), porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (PRV), porcine transmissible gastroenteritis virus (TGEV), and foot-and-mouth disease virus (FMDV) are respiratory diseases, reproductive disorders and The main common pathogens of digestive tract diseases are the most serious pathogens that harm the pig industry. They are defined as cross-border pathogens by the World Organization for Animal Health and are also impo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 马贵平史喜菊乔彩霞郭志红张伟
Owner PEOPLES REPUBLIC OF CHINA BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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