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Newcastle disease virus/avian influenza virus H9 subtype/infectious bronchitis virus triplex fluorescence quantification detection reagent and detection method

A technology of avian influenza virus and Newcastle disease virus, applied in the direction of microbe-based methods, biochemical equipment and methods, microbiological determination/inspection, etc.

Inactive Publication Date: 2016-06-15
山东省动物疫病预防与控制中心 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are many single fluorescent RT-PCR methods reported at home and abroad, and the triple fluorescent RT-PCR technology for simultaneous detection of Newcastle disease virus, avian influenza virus H9 subtype and chicken infectious bronchitis virus has not been reported

Method used

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  • Newcastle disease virus/avian influenza virus H9 subtype/infectious bronchitis virus triplex fluorescence quantification detection reagent and detection method
  • Newcastle disease virus/avian influenza virus H9 subtype/infectious bronchitis virus triplex fluorescence quantification detection reagent and detection method
  • Newcastle disease virus/avian influenza virus H9 subtype/infectious bronchitis virus triplex fluorescence quantification detection reagent and detection method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0094] The design of embodiment 1 primer, probe

[0095]According to the published M gene sequence of Newcastle disease virus (Genbank accession No. Genbank accession number KJ435285.1) designed general primers and probes for Newcastle disease virus, primers and probes for H9 subtype of avian influenza virus and primers and probes for chicken infectious bronchitis virus. The 5' end of the Newcastle disease virus universal probe sequence is labeled with the reporter fluorescent group FAM (6-carboxy-fluorescein), and the 3' end is labeled with the quencher group TAMRA; the 5' end of the avian influenza virus H9 subtype probe sequence is labeled The reporter fluorescent group HEX (5-hexachloro-fluorescein), the 3' end is labeled with the quencher fluorescent group BHQ1; the 5' end of the chicken infectious bronchitis virus probe sequence is labeled with the fluorescent reporter group CY5, and the 3' end is labeled with the quencher Inactivate the fluorophore BHQ2. Through analy...

Embodiment 2

[0105] Example 2 Column type extraction operation method extracts RNA

[0106] 1. Take a sterilized 1.5mL centrifuge tube, add 500mL of BufferA, 200mL of samples (sample to be tested and negative and positive control samples), mix well, and place at room temperature for 10 minutes;

[0107] 2. Take the same amount of RNase-Free adsorption column as the centrifuge tube; transfer the solution and flocculent precipitate in the centrifuge tube to the RNase-Free adsorption column, and cover the adsorption column with a collection tube (to avoid clogging the adsorption column, try not to absorb the suspension Impurities (impurities in the sample); centrifuge at 13,000 rpm for 30 seconds at room temperature; discard the liquid in the collection tube, and put the adsorption column back into the collection tube;

[0108] 3. Add 600uL BufferB to the adsorption column, centrifuge at 13,000 rpm for 30 seconds; discard the liquid in the collection tube, and put the adsorption column back i...

Embodiment 3

[0117] Embodiment 3 configuration PCR amplification reaction system

[0118] Add 20μL fluorescent RT-PCR reaction solution and 1.0μL enzyme mixture into a small tube of appropriate volume, mix well, take 20μL as the detection reagent; put the detection reagent into the PCR tube, then add 5ul template RNA, and finally make 25ul reaction system (1 part).

[0119] (1) Fluorescent RT-PCR reaction solution (1 portion, 20 μL)

[0120] ;

[0121] 10×PCR buffer, 25mmol / LMgCl 2 Purchased from Promega; 5×RT-buffer, 10mmol / LdNTP were purchased from Dalian Baobio Bioengineering Co., Ltd. Primers and probes were synthesized by Dalian Baobiobioengineering Co., Ltd. The active ingredients of 10×PCR buffer are: 500mmol / LKCl, 100mmol / L Tris-HCl (pH9.0, 25°C), 1.0% TritonX-100. The active ingredients of 5×RT-buffer are 375mmol / LKCl, 15mmol / LMgCl 2 , 50mmol / LDTT, 250mmol / LTris-HCl (pH8.3, 25°C). The DEPC water: tap water is distilled twice, purified by a MilliporeMILLI-QPFPLUS pure wate...

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Abstract

The invention relates to a newcastle disease virus / avian influenza virus H9 subtype / infectious bronchitis virus triplex fluorescence quantification detection reagent and a detection method and belongs to the technical field of animal quarantine. A newcastle disease virus M gene coding region specific sequence, an avian influenza virus H9 subtype H gene coding region specific sequence and a chicken infectious bronchitis virus M gene coding region specific sequence are selected as target regions, and on the basis of multi-sequence comparison, primer and probe design is conducted. The length of primers is about 20 basic groups, the GC content is 50-60%, a two-stage structure and repeatability do not exist in the primers, no complementary sequence exists between the primers or in the primers, and the melting temperature (Tm value) difference between the primers is smaller than 5 DEG C. In order to guarantee universal use of a newcastle disease virus probe, the length of the probe is only 13 basic groups, the probe is modified by LAN, and the Tm value of the probe is increased. The lengths of the other two virus probes are both about 25 basic groups, and the Tm values are about 5 DEG C higher than those of the primers.

Description

technical field [0001] The invention relates to a Newcastle disease virus / avian influenza virus H9 subtype / infectious bronchitis virus triple fluorescent quantitative RT-PCR detection reagent and a detection method, belonging to the technical field of animal quarantine. Background technique [0002] Newcastle disease, avian influenza and chicken infectious bronchitis are important infectious diseases that endanger the poultry industry, and belong to acute and highly contagious respiratory infectious diseases. Newcastle disease is an infectious disease that is included in the daily immunization procedures of poultry farms. Although the vaccine covers a wide range, due to various reasons, Newcastle disease occurs frequently, but the mortality rate is significantly lower than that without vaccines. Avian influenza H9 subtype has a greater impact on the poultry industry, and vaccines are widely used. However, due to the rapid mutation of avian influenza H9 subtype strains, new m...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/6851C12Q1/701C12Q2531/113C12Q2537/143C12Q2563/107C12Q2545/113
Inventor 陈静徐聪张向东王苗利杨鹏马慧玲王贵升孙圣福张月孔祥华于青海李玉杰徐鸿刘丽平蔺晓月
Owner 山东省动物疫病预防与控制中心
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