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

A swine flu virus and virus technology, applied in the field of inspection and quarantine, can solve the problems of long time, difficult to judge the results, heavy detection workload and other problems

Active Publication Date: 2014-03-12
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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1. Preparation and use of kit

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

[0077] Table 4 The composition of the kit

[0078] Composition (30Tests / box)

Quantity

MLPA buffer

600μL×1 tube

Probe mix

600μL×1 tube

Connection 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] Among them, the MLPA buffer was purchased from The MRC-Holland Company, which includes KCl, Tris-HCl, EDTA and PEG-6000, and has a pH of 8.5.

[0080] Probe mixture, which includes long and short probes of swine influenza virus, porcine reproductive and respiratory syndrome virus, pseudorabies virus, transmissible gastroenteritis virus and foot-and-mouth disease virus. The sequence of each probe is shown in Table 1. The 5'ends of the SEQ ID NO: 2, ...

Embodiment 2

[0111] Example 2. Sensitivity test of the kit

[0112] 1. Material:

[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 strains are preserved in the laboratory, O type foot and mouth disease is inactivated Live virus was provided by Lanzhou Veterinary Research Institute of 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: recovery of the RT-PCR amplification product of swine influenza virus A / swine / 2003 (H1N1) strain M gene, length of 982bp, and pGEM-T vector (purchased from PromeGA) Ligation, transformation of JM109 competent cells, alkaline lysis method to extract plasmid DNA, PCR and restriction enzyme digestion to obtain a positive recomb...

Embodiment 3

[0132] Example 3. Specificity test of the kit

[0133] 1. Material

[0134] Table 6 Viruses and nucleic acids used in the study of specificity tests

[0135] Virus

Source

Swine flu virus A / Swine / 2003(H1N1)

Saved in this laboratory

Porcine reproductive and respiratory syndrome JXA1 strain inactivated virus

Saved in this laboratory

Nanyang strain of pseudorabies virus

Saved in this laboratory

Swine transmissible gastroenteritis purdue115 international standard strain

Saved in this laboratory

O-type foot-and-mouth disease inactivated virus

Lanzhou Veterinary Research Institute

Porcine parvovirus

Saved in this laboratory

Swine fever inactivated virus

Provided by China Veterinary Drug Supervision Institute

Porcine epidemic diarrhea virus

Saved in this laboratory

[0136] 2. Method

[0137] 2.1 Use any set of probes among the five viruses of swine influenza virus, porcine reproductive and respiratory syndrome virus, pseudorabies virus, porcine transmissible gast...

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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 , It can realize the purpose of one sampling, one analysis, and simultaneous detection of 5 swine diseases, 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), foot-and-mouth disease virus (FMDV) are causes of respiratory diseases, reproductive disorders and The main common pathogens of gastrointestinal diseases are the most serious pathogens that harm the pig industry. They are designated by the World Organization for Animal Health as transboundary pathogens, and they are also...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70
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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