Animal epidemic disease three-color fluorescence RT-PCR detection kit and detection method thereof

A technology of RT-PCR and detection kits, which is applied in the field of three-color fluorescent RT-PCR detection kits, can solve the problems of synchronous detection of bronchitis and other problems, and achieve the goals of saving production costs and detection costs, shortening time, and improving detection efficiency Effect

Inactive Publication Date: 2013-01-02
NINGBO ACAD OF SCI & TECH FOR INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, at present, there are no relevant research reports on the simultaneous detection of avian influenza, Newcastle disease and avian infectious bronchitis at home and abroad.

Method used

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  • Animal epidemic disease three-color fluorescence RT-PCR detection kit and detection method thereof

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Experimental program
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Effect test

specific Embodiment 1

[0044] Avian Influenza Virus, Newcastle Disease Virus Wild Virus and Avian Infectious Bronchitis Virus Wild Virus Three-color Fluorescence RT-PCR Detection Kit

[0045] 1. RT-PCR detection kit composition

[0046] This kit includes multiple fluorescent RT-PCR reaction master solution, positive control substance, negative control substance, AMV reverse transcriptase, Taq polymerase. The multiple fluorescent RT-PCR reaction mother solution contains multiple 10× fluorescent RT-PCR reaction buffer, avian influenza primers, avian influenza probes, Newcastle disease primers, Newcastle disease probes, avian infectious bronchitis primers, avian infectious bronchitis Probe, bovine serum albumin; multiplex 10× fluorescent RT-PCR reaction buffer contains tris hydrochloride, potassium chloride, glycerin, four kinds of mononucleotide monomers, magnesium chloride.

[0047] Wherein the multiple 10×PCR reaction buffer consists of: the final concentration of Tris hydrochloride is 100mM, the f...

specific Embodiment 2

[0073] Three-color fluorescence RT-PCR detection method for wild virus of avian influenza virus, wild virus of Newcastle disease virus and wild virus of avian infectious bronchitis virus. A positive control group and a negative control group should be set up for each detection

[0074] 1. Sample processing

[0075] If the sample is a live poultry, it is recommended to take a throat swab and a cloacal swab: when taking a throat swab, put the swab deep into the mouth of the throat and palate and scrape back and forth several times to get the throat secretions; Turn the genital cavity around to pick up feces. Then put the swab together into a test tube containing 2ml of phosphate (PBS) buffer (containing antibiotics), wash the swab thoroughly, then squeeze out the liquid on the tube wall, and transfer it to a sterile centrifuge tube for later use; if the sample is fresh For muscle or viscera, take 2g of the sample to be tested and thoroughly grind it in a mortar that has been wa...

specific Embodiment 3

[0090] Sensitivity and specificity experiment of three-color fluorescent RT-PCR detection kit

[0091] 1. Experimental materials:

[0092] The selected pathogenic microorganisms include the following: experimental group: avian influenza virus, virulent Newcastle disease virus, avian infectious bronchitis virus; control group: human influenza A virus, human influenza B virus, Newcastle disease vaccine strain and avian infectious bronchitis virus. Bronchitis vaccine strains, the above pathogenic strains were all from the Southeast Poultry Research Laboratory of the Agricultural Research Institute of the United States Department of Agriculture, and the vaccine strains were purchased from Ningbo Animal Disease Prevention and Control Center

[0093] 2. Sensitivity and specificity analysis of the detection kit:

[0094] The avian influenza virus, Newcastle disease virus wild virus and avian infectious bronchitis virus wild virus three-color fluorescent RT-PCR detection kit in speci...

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Abstract

The invention discloses an animal epidemic disease three-color fluorescence RT-PCR detection kit and a detection method thereof, and is characterized in that the kit comprises multiple fluorescence RT-PCR reaction mother liquor, a positive control, a negative control, an AMV reverse transcriptase, and a Taq polymerase, wherein the multiple fluorescence RT-PCR reaction mother liquor contains a multiple 10*fluorescence RT-PCR reaction buffer, an avian influenza primer and a probe, a newcastle disease virus primer and a probe, an avian infectious bronchitis primer and a probe, and bovine serum albumin, wherein sequences of the forward primers, reverse primers, and specific probes are as shown in SEQIDNO. 1, NO. 2, NO.3, NO.4, NO.5, NO.6, NO.7. NO.8, NO.9, and NO.10. The advantages of the invention are that avian influenza, newcastle disease, and avian infectious bronchitis viruses can be detected simultaneously in a same reaction tube; the specificity is strong; the sensitivity is high, is rapid and accurate.

Description

technical field [0001] The invention relates to a fluorescent PCR kit in the field of biotechnology, in particular to a three-color fluorescent RT-PCR detection kit used for bird flu, wild virus of Newcastle disease virus and wild virus of avian infectious bronchitis virus. Background technique [0002] Avian influenza and Newcastle disease are both Class A severe poultry infectious diseases designated by the International Veterinary Bureau. They have the characteristics of rapid spread, acute onset, and high mortality. They are both listed as Class I animal diseases in the National Animal Epidemic Prevention Law. Avian infectious bronchitis is an acute, high-contact respiratory disease caused by a coronavirus that occurs only in poultry. The symptoms of avian influenza (denoted as AIV), Newcastle disease (denoted as NDV) and avian infectious bronchitis (denoted as AIBV) are similar, the pathological process is indistinguishable, and the social hazards are very different: th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 周冬根孙大为曹雪春李红张升王燕岑晨霞
Owner NINGBO ACAD OF SCI & TECH FOR INSPECTION & QUARANTINE
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