Composite kit for detecting avian influenzas and Newcastle disease viruses and detection method

A technology for Newcastle disease virus and avian influenza virus, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, fluorescence/phosphorescence, etc., to achieve good repeatability, eliminate false negative results, and simple operation

Inactive Publication Date: 2010-08-11
INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a simple, accurate test kit and test method for compound detection of avian

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1. The kit of compound detection avian influenza and Newcastle disease virus

[0041] The reagents of the kit include:

[0042] (1) Sample processing reagent: 100% Trizol, purchased from Takara Company, product number D312;

[0043] (2) Nucleic acid amplification reagents:

[0044] 1. RT-PCR reaction solution: containing 25mmol / L MgCl 2 , 10mmol / L dNTP Mixture;

[0045] 2. Enzyme mixture: AMV reverse transcriptase and Taq enzyme at a concentration of 5 U / μL were mixed and divided at a ratio of 1:1. Both enzymes were purchased from Takara Company;

[0046]3. Primer-probe mixture: the upstream and downstream primers for detection of avian influenza virus and the upstream and downstream primers for detection of Newcastle disease virus are mixed in equal proportions at a final concentration of 20 pmol / L, and the detection of avian influenza virus probes and Newcastle disease virus probes is carried out at a final concentration of 10pmol / L was mixed in equal p...

Embodiment 2

[0090] Embodiment 2. The method for detecting avian influenza and Newcastle disease virus of the present invention

[0091] 1. Sample processing

[0092] (1) Take n (n = number of samples + 1 tube of negative control + 1 tube of positive control) 1.5 mL sterilized centrifuge tubes and mark them.

[0093] (2) Add 600uL of lysate, then add 200μL of the collected poultry sample, negative control, and positive control respectively (change a tip for each sample), then add 200μL of chloroform, shake and mix on the mixer for 5sec (not too much Strong, so as not to produce an emulsified layer; can also be mixed by hand upside down). Centrifuge at 12000rpm for 15min (centrifuge at 4°C if possible).

[0094] (3) Take another 1.5 mL sterilized centrifuge tube and add 400 μL of isopropanol (pre-cooled at -20°C) for marking. Pipette at least 500 μL of the supernatant from each tube in the previous step (be careful not to suck out the middle layer), add it to the corresponding tube, and ...

Embodiment 3

[0120] Embodiment 3, the composite detection method of the present invention detects the specificity test and sensitivity test of avian influenza virus

[0121] The established composite detection method was used to detect chicken embryo infected allantoic fluid with different dilutions and compared with chicken embryo isolation method to evaluate the sensitivity of the method; the composite detection method was used to detect common avian viruses to evaluate the specificity of the method.

[0122] 1. Materials

[0123] 1. the test kit (embodiment 1) of compound detection avian influenza and Newcastle disease virus

[0124] 2. Avian influenza virus H5N1 subtype strain, avian influenza virus H7N2 subtype strain, avian influenza virus H9N2 subtype strain, avian reovirus (ARV), infectious bronchitis virus (IBV), infectious laryngovirus Tracheitis virus (ILTV), infectious anemia virus (CAV), chicken infectious bursal virus (IBDV), duck viral enteritis virus (DEV), duck viral hepa...

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PUM

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Abstract

The invention relates to a composite kit for detecting avian influenzas and Newcastle disease viruses and a detection method, characterized in that the kit comprises a lysis solution, an RT-PCR buffer solution, primers and a probe mixture solution, particularly a primer and a probe sequenc for detecting various subtype avian influenzas viruses, a primer and a probe sequenc for detecting Newcastle medium and strong viruses, enzyme mixture, DEPC water and negative and positive contrasts. The detection method comprises the following steps of: carrying out an RT-PCR reaction and judging a quality control standard and results. The invention has the advantages of high sensitivity, good specificity and simple operation. The process from processing samples to getting a result only needs 4 hours. The invention overcomes the defects of single fluorescence and realizes the aim of simultaneously detecting two viruses by one real-time fluorescent quantification PCR (Polymerase Chain Reaction) reaction.

Description

technical field [0001] The invention relates to a kit and a testing method for detecting avian influenza virus and Newcastle disease virus, in particular to a kit and a detection technology for compound detection of virulence in various subtypes of avian influenza virus and Newcastle disease virus by double TaqMan probe method. Background technique [0002] Avian influenza (AI) and Newcastle disease (Newcastle disease, ND) are viral diseases that seriously harm poultry. They often infect poultry alone or in mixed infection, with high morbidity and mortality. In addition to infecting poultry, bird flu can also infect many animals such as humans, pigs and horses. Both diseases can cause symptoms of respiratory illness in birds. There are many similarities in epidemiology, clinical symptoms, pathological changes, etc., so there are great difficulties in clinical differential diagnosis. When these two viruses co-infect poultry, it is more difficult to correctly judge and preve...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
Inventor 孙涛徐彪朱来华凌宗帅肖西至邓明俊张太翔文峰
Owner INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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