CN105986044B - Avian influenza virus nucleic acid General rapid detection method - Google Patents
Avian influenza virus nucleic acid General rapid detection method Download PDFInfo
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- CN105986044B CN105986044B CN201610066605.XA CN201610066605A CN105986044B CN 105986044 B CN105986044 B CN 105986044B CN 201610066605 A CN201610066605 A CN 201610066605A CN 105986044 B CN105986044 B CN 105986044B
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
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Abstract
The invention belongs to biological technical fields, it establishes avian influenza virus nucleic acid General rapid detection method, containing there are three technical essentials：Establish the required primer sequence of detection of nucleic acids；Establish detection reactive species；Establish detection reaction system and reaction condition.The nucleic acid detection method can be used for the scientific research of each subtype avian influenza virus, can be used for animal or the clinical diagnosis detection of people.
Description
Technical field
The invention belongs to biological technical fields；Particularly, the present invention establishes the general quick detection side of avian influenza virus
Method, for the quick detection of each subtype avian influenza virus, in vitro the fields such as diagnosis, animal doctor, food security, bio-safety have compared with
Big use value.
Background technology
Bird flu can be divided into highly pathogenic bird flu, low pathogenicity bird flu and without pathogenic according to pathogenic difference
Property bird flu.Wherein, highly pathogenic bird flu is the disease as caused by some strains of H5 and H7 hypotypes.
Viral nucleic acid detection is one of viral diagnosis important method.At present, it is general to avian influenza virus various both at home and abroad
Nucleic acid detection method, including agricultural industry criteria or national standard, the consuming time is all long, such as agricultural industry criteria（Fowl is flowed
Influenza Virus RT-PCR detection method, NY/T 772-2013）At least need 2 h, national standard（Avian influenza virus universal fluorescent RT-
PCR detection method, GB/T 19438.1-2004）1.2 h are at least needed, currently without the detection more efficient than these methods
Method.
The content of the invention
The present invention is directed to the deficiency of the above-mentioned existing general detection technique of avian influenza virus, has carried out further investigation and test,
Establish the avian influenza virus nucleic acid General rapid detection method based on recombinase polymeric enzymatic amplification technology, it is only necessary to 20 min
Complete detection.This method includes following three aspect contents：
（One）The required primer sequence of detection of nucleic acids is established, for conserved region in each HA Gene of H 9 Subtype AIV
Primer sequence is designed in domain, and wherein upstream primer sequence contains 30 bases, and sequence is
ggtagatgttgaaagatgagtcttctaacc（SEQ ID NO.1 i.e. in sequence table）, downstream upstream primer sequence contains 30
A base, sequence gaatacaaatcccaagatccctttagtcag（SEQ ID NO.2 i.e. in sequence table）；
（Two）Establish the rapid detection method based on recombinase polymeric enzymatic amplification technology；
（Three）Detection reaction system and reaction condition are established, pure water 9.2 is sequentially added in PCR pipe
μ l, 29.5 μ l of reaction buffer（Contain four kinds of nucleotide such as dATP, dGTP, dTTP, dCTP）, upstream synthesized by the first step
Primer 2 .4 μ l（Concentration is 10 μm of ol/L）, 2.4 μ l of anti-sense primer synthesized by the first step（Concentration is 10 μm of ol/L）, enzyme
[reverse transcriptase can combine single-chain nucleic acid to mixture（Oligonucleolide primers）Recombinase, single-stranded DNA binding protein（SSB）And chain
Displacement archaeal dna polymerase] 1.0 μ l, 1.0 μ l of RNase inhibitor, need the 2.0 μ l of influenza nucleic acids that detect（From clinical sample
It is extracted in product or other samples with nucleic acid extraction kit）, magnesium acetate（280 mmol/L）2.5 μl；Then by reaction system
It is closed, it is placed in thermostat（Or water-bath）On reacted, reaction condition be 40 DEG C of 20 min；After reaction, produced in reaction
It is added in object containing coloured nucleic acid electrophoresis buffer solution, carries out agarose nucleic acid gel electrophoresis, be about 207 if there is size
The nucleic acid electrophoresis band of bp, then be judged as the positive, be otherwise judged as feminine gender.
Specific embodiment
Below by embodiment, illustrate technical scheme, but protection scope of the present invention is not limited to this implementation
Example.
The present embodiment recombinase polymeric enzymatic amplification technology carries out nucleic acid to more subtype avian influenza virus and quickly detects, wraps
Include following steps：
The first step（Synthetic primer）：The nucleotide sequence specified according to the present invention（SEQ ID NO.1 and SEQ i.e. in sequence table
ID NO.2）, artificial synthesized recombinase polymeric enzymatic amplification reacts required sense primer and anti-sense primer；
Second step（Configure reaction system）：9.2 μ l of pure water, reaction buffering are sequentially added in PCR pipe
29.5 μ l of liquid（Contain four kinds of nucleotide such as dATP, dGTP, dTTP, dCTP）, 2.4 μ l of sense primer synthesized by the first step
（Concentration is 10 μm of ol/L）, 2.4 μ l of anti-sense primer synthesized by the first step（Concentration is 10 μm of ol/L）, enzymatic mixture [reverse
Record enzyme can combine single-chain nucleic acid（Oligonucleolide primers）Recombinase, single-stranded DNA binding protein（SSB）It polymerize with strand displacement DNA
Enzyme] 1.0 μ l, 1.0 μ l of RNase inhibitor, need the 2.0 μ l of influenza nucleic acids that detect（From clinical sample or other samples
It is middle with nucleic acid extraction kit extract）, magnesium acetate（280 mmol/L）2.5 μl；
3rd step（Reaction）：After reaction system that second step has been configured is closed, thermostat is placed in（Or water-bath）It is enterprising
Row reaction, reaction condition are 40 DEG C of 20 min；
4th step（As a result detect）：It is added in the reaction product of the 3rd step containing coloured nucleic acid electrophoresis buffer solution, into
Row agarose nucleic acid gel electrophoresis, the nucleic acid electrophoresis band for being about 207 bp if there is size are then judged as the positive, otherwise sentence
Break as feminine gender.
Result of practical application：480 parts of H5, H6, H7, H9, H10 subtype avian influenza virus standard positive clinical samples are carried out
Detection and 300 parts of standard female samples, are detected, as a result with the above-mentioned general detection technique of avian influenza virus Rapid nucleic acid
The sensitivity for showing the technology is 100.0%, and specificity is 100.0%.
Claims (1)
1. a kind of avian influenza virus nucleic acid primer is to preparing to detect the examination of H5, H6, H7, H9, H10 subtype avian influenza virus
Application in agent, it is characterised in that：The sequence of the primer is respectively 5'-3'ggtagatgttgaaagatgagtcttctaacc
And 5'-3'gaatacaaatcccaagatccctttagtcag；
The avian influenza virus nucleic acid primer is to preparing to detect the reagent of H5, H6, H7, H9, H10 subtype avian influenza virus
In application reacted using recombinase polymeric enzymatic amplification；
The avian influenza virus nucleic acid primer is to preparing to detect the reagent of H5, H6, H7, H9, H10 subtype avian influenza virus
In application in detect reaction system and reaction condition it is as follows：9.2 μ L of pure water, anti-is sequentially added in PCR pipe
29.5 μ L of buffer solution, the concentration synthesized by the first step are answered as the concentration synthesized by 2.4 μ L of sense primer of 10 μm of ol/L, the first step
The influenza virus core detected for 2.4 μ L of anti-sense primer of 10 μm of ol/L, 1.0 μ L of enzymatic mixture, 1.0 μ L of RNase inhibitor, needs
2.0 μ L of acid, concentration are 2.5 μ L of 280mmol/L magnesium acetates；Then it is reaction system is closed, it is placed in thermostat or water-bath is enterprising
Row reaction, reaction condition are 40 DEG C of 20min；After reaction, add in and buffered containing coloured nucleic acid electrophoresis in the reaction product
Liquid carries out agarose nucleic acid gel electrophoresis, and the nucleic acid electrophoresis band for being about 207bp if there is size is then judged as the positive, no
Then it is judged as feminine gender；
The reaction buffer contains tetra- kinds of nucleotide of dATP, dGTP, dTTP, dCTP；
The enzymatic mixture includes：Reverse transcriptase can be put with reference to recombinase, single-stranded DNA binding protein and the chain of single-chain nucleic acid
Change archaeal dna polymerase；
The influenza nucleic acids are to be extracted from clinical sample or other samples with nucleic acid extraction kit.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105296670B (en) * | 2015-11-09 | 2018-10-19 | 山西省农业科学院畜牧兽医研究所 | A kind of H1, H3 and H9 type avian influenza virus detection kit and detection method |
| CN108624714B (en) * | 2018-04-28 | 2021-03-26 | 佛山科学技术学院 | RPA-LFD visual kit for detecting avian influenza virus and application thereof |
| CN109234462A (en) * | 2018-11-13 | 2019-01-18 | 中国动物卫生与流行病学中心 | A kind of reverse transcription recombinase-mediated isothermal amplification detection method of avian influenza virus |
| CN113699279A (en) * | 2021-09-26 | 2021-11-26 | 上海海关动植物与食品检验检疫技术中心 | Kit for detecting avian influenza virus and detection method thereof |
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| CN104762413A (en) * | 2015-01-04 | 2015-07-08 | 中国动物卫生与流行病学中心 | General nucleic acid detection method for influenza viruses |
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| CN104762413A (en) * | 2015-01-04 | 2015-07-08 | 中国动物卫生与流行病学中心 | General nucleic acid detection method for influenza viruses |
Non-Patent Citations (3)
| Title |
|---|
| Development of reverse transcription recombinase polymerase amplification assay for avian influenza H5N1 HA gene detection;Nahed Yehia et al.;《Journal of Virological Methods》;20150728;第223卷;摘要第4-5行、第8行 * |
| Diagnostics-in-a-Suitcase: Development of a portable and rapid assay for the detection of the emerging avian influenza A (H7N9) virus;Ahmed Abd El Wahed et al.;《Journal of Clinical Virology》;20150831;第69卷;第16-21页 * |
| 禽流感病毒 RT-LAMP检测技术的建立;彭宜等;《动物医学进展》;20121231;第33卷(第12期);摘要第1-2行，第44页左栏第2段 * |
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