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PCR (Polymerase Chain Reaction) method for simultaneously detecting four bovine infectious disease RNA (Ribose Nucleic Acid) viruses by single tube

A bovine parainfluenza virus and virus technology, applied in the field of fluorescent quantitative PCR, can solve problems such as vacancy and unsatisfaction

Inactive Publication Date: 2013-12-25
SHANGHAI ZHUORUN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The detection of pathogens in animal husbandry needs to be timely and accurate, easy to operate, and a large number of samples can be processed at one time to facilitate the prevention and control of epidemics; however, the existing level of pathogen detection cannot meet this need
One reaction in the same PCR tube can detect the presence or absence of pathogens of several common livestock diseases, especially the pathogens of bovine infectious diseases. The results are quickly given and are applicable to large-scale automatic analysis instruments to guide the next step of epidemic prevention work. And products are currently vacant in the market

Method used

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  • PCR (Polymerase Chain Reaction) method for simultaneously detecting four bovine infectious disease RNA (Ribose Nucleic Acid) viruses by single tube
  • PCR (Polymerase Chain Reaction) method for simultaneously detecting four bovine infectious disease RNA (Ribose Nucleic Acid) viruses by single tube
  • PCR (Polymerase Chain Reaction) method for simultaneously detecting four bovine infectious disease RNA (Ribose Nucleic Acid) viruses by single tube

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0113] 1. Nucleic acid extraction

[0114] Utilizing the technical means of genetic engineering, bovine parainfluenza virus, foot-and-mouth disease virus, bovine diarrhea virus, and bovine reovirus strains are made into corresponding pseudovirus particles for nucleic acid extraction.

[0115]The extraction of nucleic acid is carried out according to the conventional magnetic bead extraction method, and the method is as follows: add 90 μl of nucleic acid extraction solution (formulation and final concentration: guanidine isothiocyanate 12M, Sodium amine tetraacetate (pH8.0) 10mM, Tween-202% (W / W), sodium perchlorate 1.0M, ethanol 40% (V / V), Tris-HCl (pH8.0) 10mM), in Incubate at 42°C for 10min, then add 10μl Mag Magnetic bead suspension (50 mg / mL, purchased from Beijing Aibigen Biotechnology Co., Ltd.), after oscillating and mixing evenly, put it on a magnetic rack to apply a magnetic field, discard the liquid, and then add 200 μl of washing solution A (formulation and final ...

Embodiment 2

[0172] Embodiment 2 kit specific detection

[0173] Using total RNA from samples such as rinderpest virus, bovine leukemia virus, bovine epidemic fever virus, bovine respiratory syncytial virus, bovine parainfluenza virus, foot-and-mouth disease virus, bovine diarrhea virus, bovine reovirus, etc. The primers and probes were subjected to real-time fluorescent PCR reaction. As a result, only bovine parainfluenza virus, foot-and-mouth disease virus, bovine diarrhea virus, and bovine reovirus produced fluorescent signals. The amplification conditions are shown in Table 1. It is proved that the method is specific for the detection of bovine parainfluenza virus, foot-and-mouth disease virus, bovine diarrhea virus and bovine reovirus.

[0174] Table 1 Single-tube detection of four kinds of bovine RNA virus method-specific fluorescent PCR amplification results

[0175]

[0176] The results showed that the detection kit had high specificity and had no crossover with various factors...

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Abstract

The invention discloses a PCR (Polymerase Chain Reaction) method for simultaneously detecting four bovine infectious disease RNA (Ribose Nucleic Acid) viruses by a single tube. The PCR method is used for detecting a bovine parainfluenza virus, a foot and mouth disease virus, a bovine diarrhea virus and a bovine reovirus. The PCR method is rapid in operation without one step. In addition, the invention also relates to a reagent related in the PCR method, which is used in a detection kit of the method and preparation and application of the detection kit.

Description

technical field [0001] The invention belongs to the field of pathogen detection, in particular, it relates to a multiplex fluorescent quantitative PCR detection method for common bovine infectious disease pathogens, more specifically, it relates to a method for quickly and parallelly detecting four kinds of bovine infectious disease RNAs in the same PCR tube Fluorescent quantitative PCR method for viruses (bovine parainfluenza virus, foot-and-mouth disease virus, bovine diarrhea virus, bovine reovirus). Background technique [0002] Bovine parainfluenza virus, foot-and-mouth disease virus, bovine diarrhea virus, and bovine reovirus are four common causative agents of bovine infectious diseases. [0003] Bovine parainfluenza virus belongs to parainfluenza virus type 3 (PIV3), which is an RNA virus. Under natural conditions, it only infects cattle. Sick cattle and infected cattle are the main source of infection. It can be transmitted through contact between cattle and can als...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
Inventor 王奕江曹振国齐洁婷刘明霞
Owner SHANGHAI ZHUORUN BIOTECH
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