CN105803111A - Double-target real-time fluorescent PCR (Polymerase Chain Reaction) detection method for human infected H7N9 avian influenza virus - Google Patents
Double-target real-time fluorescent PCR (Polymerase Chain Reaction) detection method for human infected H7N9 avian influenza virus Download PDFInfo
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Abstract
The invention relates to rapid and sensitive double-target real-time fluorescent PCR (Polymerase Chain Reaction) detection method for human infected H7N9 avian influenza virus. According to the detection method disclosed by the invention, HA genes and NA genes of the human infected H7N9 avian influenza virus serve as double target genes to perform amplification. The detection method comprises the following steps of: performing specimen treatment, performing RNA nucleic acid extraction, carrying out RT-PCR amplified reaction and performing fluorescence signal detection, wherein a specific primer and a probe of the RT-PCR amplified reaction are designed in a specific primer amplification area aiming at the two targets such as the HA genes and NA genes and can distinguish other virus strains. By adopting the PCR and TaqMan fluorescence probe technology, an H7N9 specific nucleotide sequence in an infected person specimen suffering from suspected human infected H7N9 avian influenza virus can be subjected to double-target gene amplification and detection, so that the infection condition of H7N9 is determined. According to the method disclosed by the invention, the detection efficiency of the first-line inspection and quarantine personnel at the entry and exit port can be greatly improved, the workload can be reduced, and the positive missing detection problem which possibly exists in the traditional detection method can be solved to the greatest degree, so that input of major avian influenza viruses is prevented to the greatest degree.
Description
Technical field
The present invention relates to external diagnosis reagent technical field, be specifically related to a kind of people and infect double target genes of H7N9 bird flu virus in fact
Time fluorescence PCR detecting method.
Background introduction
Current respiratory infectious disease wide-scale distribution in the world is with popular, and new sick kind is constantly found, and popular region is continuous
Extension, popular frequency constantly strengthens, and the safe and sanitary of frontier port is caused serious threat, therefore to respiratory infectious disease
Monitoring be the important process of frontier port health quarantine.For frontier port, do not require nothing more than pathogen " inspection draws ",
Also requirement " can be examined accurate, examine soon ", could meet the on-the-spot needs quickly disposing epidemic situation in port.
Influenza virus (Influenza virus) is divided into first, second, the third three types.A type (A type) influenza infection birds,
People and other mammals；B-mode (Type B) influenza virus only infects the mankind, the generation of disease relatively influenza A virus temperature
With；Third type (c-type) influenza virus only infects the mankind, can't cause serious disease.Therefore influenza A virus is current
Main object of study.Influenza A virus belongs to orthomyxoviridae family, Influenza Virus, belongs to the sub-thread strand RNA that gene is segmented
Virus.This fragment occurs the most heavily to join during natural infection, causes virus antigenicity, particularly surface glycoprotein HA
Quick variation antigenic with NA so that the immune surveillance of viral escape body, causes infecting and morbidity, is used vaccine simultaneously
Epidemic prevention brings difficulty.Influenza A virus is divided into many hypotypes according to HA with NA antigen is different, and HA can be divided into 16
Hypotype (H1～H16), NA has 9 hypotypes (N1～N9).Have now been found that infect the mankind hypotype mainly have H1N1, H2N2,
H3N2, H5N1, H7N7, H7N2 and H9N2 etc., the natural reservoir (of bird flu viruses) of other many hypotypes is multiple birds and animal.People infects
H7N9 bird flu virus is the new influenza subtype sent out virus, and on March 31st, 2013, national health and Family Planning Committee lead to
Report, finds that in Shanghai and Anhui 3 example people infect H7N9 bird flu case.This is that global range finds human infection H7N9 first
Virus, and this virus the most only finds between fowl.Therefore the monitoring to H7N9 bird flu virus is national each port monitoring of infectious disease
Focus.For the epidemiologic feature of port immigration heating personnel, strengthen temperature monitoring and the medical science of port key population
Inspection, strengthens the propaganda strength to traveller, can effectively prevent H7N9 bird flu virus from propagating through port, protection population health peace
Entirely.
Along with China's commerce and trade, the fast development of tourist industry, local and overseas personnel contacts are day by day close, and the impact such as the import of animal,
People infects the danger of H7N9 bird flu virus input and day by day increases.Therefore, set up the detection of sensitivity fast and convenient, special as early as possible
The input that strick precaution people is infected H7N9 bird flu virus by method is significant.
Summary of the invention
The technical problem to be solved is to provide a kind of quick, sensitive people and infects double target genes of H7N9 bird flu virus
Real-time fluorescence PCR detection method.
The people of the present invention infects double target gene real-time fluorescence PCR detection methods of H7N9 bird flu virus, including following detecting step:
(1) sample disposal: for next step nucleic acid extraction after specimen mixing.
(2) RNA nucleic acid extraction: utilize cracking process lytic virus, utilizes centrifugal column absorption to carry out nucleic acid extraction.
(3) RT-PCR amplified reaction:
A.RT-PCR reaction system includes: RT-PCR reactant liquor, and two pairs of forward and reverse primers of specificity and two specific probes mix
Close liquid, reverse transcriptase/archaeal dna polymerase, RNase inhibitor, DEPC water, RT-PCR quality-control product, specimen RNA nucleic acid；Wherein,
Described specific primer and probe are design, Neng Gouqu in the primer amplified district for two targets of HA and NA gene
Not other Strain.
Described specific primer probe specificity primer and probe be:
H7-Forward 5’-AGAAATGAAATGGCTCCTGTCAA-3’SEQ ID NO1；
H7-Reverse 5’-GGTTTTTTCTTGTATTTTTATATGACTTAG-3’SEQ ID NO2；
H7-Probe 5’-FAM-AGATAATGCTGCATTCCCGCAGATG-TAMRA-3’SEQ ID NO3；
N9-Forward 5’-TAGCAATGACACACACTAGTCAAT-3’SEQ ID NO4；
N9-Reverse 5’-ATTACCTGGATAAGGGTCATTACACT-3’SEQ ID NO5；
N9-Probe 5’-VIC-AGACAATCCCCGACCGAATGACCC-TAMRA-3’SEQ ID NO6。
B. the above-mentioned reactant liquor mixed is put into ABI7500Fast instrument and carries out PCR amplification and fluorescence signal detection；
(4) result judges: according to amplification, it is determined that infection conditions.
RNA described in step (2) is extracted as viral RNA and extracts test kit extraction, takes 140ul oropharyngeal swab specimen, after cracking,
RNA is extracted according to the extraction step in test kit description.
RT-PCR reaction system described in step (3) is: 2X RT-PCR reactant liquor 10.0ul, primed probe mixed liquor 1.8ul,
Reverse transcriptase/archaeal dna polymerase 0.2ul, RNase inhibitor 0.4ul, DEPC H2O 3.6ul, specimen nucleic acid 4.0ul.
The reaction condition of the RT-PCR amplification described in step (3) is: 45 DEG C of reverse transcription 10min, 95 DEG C of degeneration 2min,
95 DEG C of degeneration 5s, 60 DEG C of annealing and extension 26s, 40 circulations.
The fluorescent labeling of the probe N-Probe described in step (3) is HEX.
The present invention is directed to double target genes of H7N9 virus, utilize thermal starting and quick enzymatic amplification, it is achieved Rapid Circulation, greatly shorten
Circulation extension of time, whole detection completed in 78 minutes.Import-export ports one X-ray inspection X can be increased substantially by the present invention
The detection efficiency of quarantine functionary, not only can reduce workload, but also can solve the traditional detection method positive that may be present to greatest extent
Missing inspection problem, thus prevent the generation of external Introduced cases infectious disease to greatest extent.
Accompanying drawing explanation
Fig. 1 embodiment 1 people infects the double target gene real-time PCR detection positive control result of H7N9 bird flu virus HA and NA.
Fig. 2 embodiment 1 people infects the double target gene real-time PCR detection result of H7N9 bird flu virus HA and NA.
In figure, curve is the most respectively: positive control, and positive sample 1:10 dilutes, and positive sample 1:100 dilutes, positive
Sample 1:1000 dilutes, and negative control is straight line.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that these embodiments be merely to illustrate the present invention and not
For limiting the scope of the present invention.In addition, it is to be understood that after having read the content that the present invention lectures, those skilled in the art can
To make various changes or modifications the present invention, these equivalent form of values fall within the application appended claims limited range equally.
Embodiment 1 people infects the double target gene real-time PCR detection of H7N9 bird flu virus specimen
One, experimental procedure
1. material, reagent, instrument
Primed probe is synthesized by Jin Weizhi bio tech ltd, Suzhou, and PCR reactant liquor is purchased from Bioline Reagents
Limited, fluorescent PCR instrument is ABI 7500Fast, and viral RNA extracts test kit purchased from QIAGEN company.
2. specimen prepares
Positive sample is behaved and is infected inactivation of viruses after the titration of H7N9 bird flu virus, then carries out the dilution of different multiples with DEPC water,
Negative control specimen is the throat swab of Healthy People.
The extraction of 3.RNA
Use viral RNA to extract test kit (centrifugal column type) and extract specimen RNA.Take 140ul specimen, according in test kit description
Operating procedure extract RNA, finally with 60ul elution RNA.
4. the amplification of target gene
4.1 primers and the design of probe
According to strain AShanghai4664T2013 (H7N9) sequential design specific primer and probe, specific primer and probe it is
For designing in the primer amplified district of HA gene and two targets of NA gene, other Strain can be distinguished, it is respectively
Two pairs of specific primers and two specific probes, specific primer and probe be:
H7-Forward 5’-AGAAATGAAATGGCTCCTGTCAA-3’SEQ ID NO1；
H7-Reverse 5’-GGTTTTTTCTTGTATTTTTATATGACTTAG-3’SEQ ID NO2；
H7-Probe 5’-FAM-AGATAATGCTGCATTCCCGCAGATG-TAMRA-3’SEQ ID NO3；
N9-Forward 5’-TAGCAATGACACACACTAGTCAAT-3’SEQ ID NO4；
N9-Reverse 5’-ATTACCTGGATAAGGGTCATTACACT-3’SEQ ID NO5；
N9-Probe 5’-VIC-AGACAATCCCCGACCGAATGACCC-TAMRA-3’SEQ ID NO6。
4.2 positive control
Positive control in this method is two synthetic gene fragments of HA gene and NA gene, is connected to pUC57 plasmid vector
On.The DNA concentration extracted is about 2ug/uL, then carries out 10 with DEPC water6-107Again after dilution, eventually serve as we
Positive control in method.
4.3 RT-PCR react platform
RT-PCR reaction system is: 2X RT-PCR reactant liquor 10.0ul, primed probe mixed liquor 1.8ul, reverse transcriptase/DNA
Polymerase 0.2ul, RNase inhibitor 0.4ul, DEPC H2O 3.6ul, specimen nucleic acid 4.0ul.Concrete as shown in table 1:
Table 1 H7N9 RT-PCR reaction system
| Component | Volume (ul) |
| 2xSensiFASTMix | 10.0 |
| Forward primer F (10uM) | 0.8 |
| Reverse primer R (10uM) | 0.8 |
| Probe P (10uM) | 0.2 |
| Reversetranscriptase | 0.2 |
| RNaseInhibitor | 0.4 |
| dH2O | 3.6 |
| SampleRNA | 4 |
| Total | 20 |
The reaction condition of RT-PCR amplification is: 45 DEG C of reverse transcription 10min；95 DEG C of degeneration 2min；95 DEG C of degeneration 5s, 60 DEG C are moved back
Fire and extension 26s, 40 circulations.
Two, experimental result
Single tube double-colored H7N9 molecular detection kit for two targets of HA and NA gene, have preferably amplification kinetics with
Linear relationship, and there is relatively low Viral diagnosis limit.This detection kit may be used for H7N9 Molecular Detection.To H1N1, H3N2,
H5N1, Flu B, HCoV, PIV1, PIV2, PIV3, HRV, HMPV, RSVA, RSVB etc. are viral all without specific reaction,
Positive and negative match-rate all reaches 100%, illustrates that test kit susceptiveness is preferable.
1) to positive control plasmid, proceed by 10 times of dilutions from 2ug/ul concentration, as different test samples, test
H7N9 reacts, and finally selects 106Being diluted to final positive control concentration again, Ct value is about 21.Result is shown in Fig. 1.
2) detection negative sample, positive control, and the people of 3 10 times dilutions infect H7N9 viral nucleic acid sample, three samples
Being 1:10 respectively, 1:100,1:1000 positive diluted sample, testing result is shown in Fig. 2, it can be seen that 3 10 times dilutions
People infect H7N9 viral nucleic acid sample standard deviation and be detected.
The present embodiment is surveyed testing result display H7N9 viral cultures and is shown as positive when utilizing this detection method.
Claims (7)
1. a people infects double target gene real-time fluorescence PCR detection methods of H7N9 bird flu virus, it is characterised in that: include with
Lower detecting step:
(1) sample disposal: for next step nucleic acid extraction after specimen mixing；
(2) RNA nucleic acid extraction: utilize cracking process lytic virus, utilizes centrifugal column absorption to carry out nucleic acid extraction；
(3) RT-PCR amplified reaction:
A.RT-PCR reaction system includes: RT-PCR reactant liquor, and two pairs of forward and reverse primers of specificity and two specificitys are visited
Pin mixed liquor, reverse transcriptase/archaeal dna polymerase, RNase inhibitor, DEPC water, RT-PCR quality-control product, specimen RNA nucleic acid；
Wherein, described specific primer and probe are to set in the primer amplified district for two targets of HA and NA gene
Count, other Strain can be distinguished；
Described specific primer probe specificity primer and probe be:
H7-Forward 5’-AGAAATGAAATGGCTCCTGTCAA-3’SEQ ID NO1；
H7-Reverse 5’-GGTTTTTTCTTGTATTTTTATATGACTTAG-3’SEQ ID NO2；
H7-Probe 5’-FAM-AGATAATGCTGCATTCCCGCAGATG-TAMRA-3’SEQ ID NO3；
N9-Forward 5’-TAGCAATGACACACACTAGTCAAT-3’SEQ ID NO4；
N9-Reverse 5’-ATTACCTGGATAAGGGTCATTACACT-3’SEQ ID NO5；
N9-Probe 5’-VIC-AGACAATCCCCGACCGAATGACCC-TAMRA-3’SEQ ID NO6；
B. the above-mentioned reactant liquor mixed is carried out PCR amplification and fluorescence signal detection；
(4) result judges: according to amplification, it is determined that infection conditions.
People the most according to claim 1 infects double target gene real-time fluorescence PCR detection methods of H7N9 bird flu virus, its
Being characterised by, described viral H7N9 Strain is AShanghai4664T2013.
People the most according to claim 1 infects double target gene real-time fluorescence PCR detection methods of H7N9 bird flu virus, its
It is characterised by: the RNA described in step (2) extracts and viral RNA can be used to extract test kit extraction, takes 140ul specimen,
RNA is extracted, finally with 60ul elution RNA according to the operating procedure in test kit description.
People the most according to claim 1 infects double target gene real-time fluorescence PCR detection methods of H7N9 bird flu virus, its
Being characterised by: the PCR reaction system described in step (3) is: 2X RT-PCR reactant liquor 10.0ul, primed probe mixes
Close liquid 1.8ul, reverse transcriptase/archaeal dna polymerase 0.2ul, RNase inhibitor 0.4ul, DEPC H2O 3.6ul, specimen nucleic acid
4.0ul。
People the most according to claim 1 infects double target gene real-time fluorescence PCR detection methods of H7N9 bird flu virus, its
Being characterised by: in the PCR reaction system described in step (3), the concentration of the forward and reverse primer of specificity and probe is 10uM.
People the most according to claim 1 infects double target gene real-time fluorescence PCR detection methods of H7N9 bird flu virus, its
It is characterised by: the reaction condition of the RT-PCR amplification described in step (3) is: 45 DEG C of reverse transcription 10min；95 DEG C of degeneration
2min；95 DEG C of degeneration 5s, 60 DEG C of annealing and extension 26s, 40 circulations.
People the most according to claim 1 infects double target gene real-time fluorescence PCR detection methods of H7N9 bird flu virus, its
It is characterised by: the fluorescent labeling of the probe N9-Probe described in step (3) is HEX.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107043829A (en) * | 2017-04-06 | 2017-08-15 | 中国疾病预防控制中心病毒病预防控制所 | Influenza virus rRT PCR detection primers and probe and the method for detecting H7N9 subtype influenza virus |
| CN110317903A (en) * | 2019-06-20 | 2019-10-11 | 上海伯杰医疗科技有限公司 | The detection kit and detection method of influenza A virus H8N7 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103305634A (en) * | 2013-05-28 | 2013-09-18 | 天津出入境检验检疫局动植物与食品检测中心 | Isothermal amplication rapid detection method of H7N9 avian influenza virus |
| CN104711269A (en) * | 2015-03-10 | 2015-06-17 | 普生（天津）科技有限公司 | Gene sequence of H7N9 type avian influenza virus |
-
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- 2015-12-11 CN CN201510924371.3A patent/CN105803111A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103305634A (en) * | 2013-05-28 | 2013-09-18 | 天津出入境检验检疫局动植物与食品检测中心 | Isothermal amplication rapid detection method of H7N9 avian influenza virus |
| CN104711269A (en) * | 2015-03-10 | 2015-06-17 | 普生（天津）科技有限公司 | Gene sequence of H7N9 type avian influenza virus |
Non-Patent Citations (2)
| Title |
|---|
| CHINESE NATIONAL INFLUENZA CENTER: "Real-time RT PCR (rRT-PCR）Protocol for the Detection of A(H7N9) Avian Influenza Virus", 《WHO COLLABORATING CENTER FOR REFERENCE AND RESEARCH ON INFLUENZA》 * |
| JUN CHENG等: "Laboratory diagnosis of avian influenza virus H7N9 infection in a renal transplant recipient", 《INT J CLIN EXP MED》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107043829A (en) * | 2017-04-06 | 2017-08-15 | 中国疾病预防控制中心病毒病预防控制所 | Influenza virus rRT PCR detection primers and probe and the method for detecting H7N9 subtype influenza virus |
| CN107043829B (en) * | 2017-04-06 | 2020-09-29 | 中国疾病预防控制中心病毒病预防控制所 | Influenza virus rRT-PCR detection primer and probe and method for detecting H7N9 subtype influenza virus |
| CN110317903A (en) * | 2019-06-20 | 2019-10-11 | 上海伯杰医疗科技有限公司 | The detection kit and detection method of influenza A virus H8N7 |
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Application publication date: 20160727 |