CN103233082A - Kit for testing H7N9 bird flu virus using real time fluorescence quantitative PCR - Google Patents
Kit for testing H7N9 bird flu virus using real time fluorescence quantitative PCR Download PDFInfo
- Publication number
- CN103233082A CN103233082A CN2013101293024A CN201310129302A CN103233082A CN 103233082 A CN103233082 A CN 103233082A CN 2013101293024 A CN2013101293024 A CN 2013101293024A CN 201310129302 A CN201310129302 A CN 201310129302A CN 103233082 A CN103233082 A CN 103233082A
- Authority
- CN
- China
- Prior art keywords
- probe
- quantitative pcr
- fluorescence quantitative
- time fluorescence
- reagent
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Abstract
The invention discloses a kit for testing H7N9 bird flu virus using real time fluorescence quantitative PCR, which is characterized in that the kit comprises: (1) a RNA extraction reagent, a reverse transcription reagent and a real time fluorescence quantitative PCR amplification reaction reagent; (2) two pairs of primers H7-F, H7-R and N9-F and N9-R for amplification of sample, and corresponding probes N7-probe and N9-probe. The kit for testing H7N9 bird flu virus nucleic acid of the present invention can test H7N9 bird flu virus by virus RNA extraction, reverse transcription and real time fluorescence quantitative PCR, and has the advantages of high accuracy, high sensitivity, high singularity and short detection window period, etc., and can provide early diagnosis and early treatment of epidemic disease and reduced case fatality rate and time for epidemic situation control.
Description
Technical field
The invention belongs to life science and biological technical field, particularly a kind of test kit for detection of the H7N9 avian influenza virus.
Background technology
Influenza virus belongs to orthomyxoviridae family (orthomyxoviridae), and coating, segmented genomic single strand RNA virus are arranged.Antigenicity according to the M1 albumen of nucleoprotein and coating medial surface is divided first (A), second (B), third (C), three types with influenza virus.Influenza A virus again according to coating surface hemagglutinin (hemagglutinin, HA) and neuraminidase (neuraminidase, NA) antigenicity of two kinds of furcellas is divided some hypotypes, comprises H1～H16 hypotype at present, NA comprises N1～N9 hypotype.All human influenza viruses can cause the bird influenza, but not every avian influenza virus can cause people's parainfluenza, and in the avian influenza virus, H3, H5, H7, H9 can infect to the people, and wherein H5 is highly pathogenic.H3 suffers from altogether for the people dog, can be divided into HxNx totally 135 kinds of hypotypes according to the influenza virus feature, and the H7N9 subtype avian influenza virus is wherein a kind of.Biology characteristics, virulence, transmissibility that this is viral also have no basis and carry out analysis and judgement.
The H7N9 subtype avian influenza virus is a kind of in the influenza A, previously only finds between fowl, does not find remarkable infection conditions.But Chinese people infects the H7N9 bird flu to take the lead in finding in Shanghai and two places, Anhui in by the end of March, 2013.The bird flu of H7N9 type is the new subtype influenza virus that the whole world is found first, does not include China's statutory report monitoring of infectious disease reporting system as yet in, and does not have vaccine to release as yet at the beginning of 2013 4 months.
China national health and Family Planning Committee printed and distributed the people and infect H7N9 bird flu diagnosis and treatment scheme, prevention and control scheme and Infection Prevention and control technique guideline on March 3rd, 2013.Infect H7N9 bird flu diagnosis and treatment scheme (2013 the 1st edition) according to the people, it is still indeterminate at present that the people infects H7N9 bird flu contagium, according to experience and this case epidemiology survey in the past, supposition may be bird and secretory product or the movement that carries the H7N9 avian influenza virus.The route of transmission is through respiratory infectious, and bird secretory product that also can be by close contact infection or movement etc. are infected, and directly contacting virus also can be infected.Now still there is not the interpersonal definite evidence of propagating.The present stage high risk population is engaged in that bird is cultured, sells, slaughters, the processer, and premorbid contacted bird person in 1 week.The diagnosis and treatment scheme points out that the people infects the H7N9 bird flu and is generally in 7 days latent period.The patient generally shows as influenza-like symptom, as heating, and cough, few phlegm can be with headache, sore muscle and general malaise.Patient with severe symptoms's PD is rapid, shows as severe pneumonia, and body temperature continues mostly more than 39 ℃, expiratory dyspnea occurs, can be with spitting of blood phlegm; But adult respiratory distress syndrome, mediastinal emphysema, Sepsis, shock, the disturbance of consciousness and acute injury of kidney etc. appear in rapid progress.
According to epidemiology contact history, clinical manifestation and laboratory examination result, can make the diagnosis that the people infects the H7N9 bird flu.Under the not quite clear situation of epidemiological history, according to clinical manifestation, auxiliary examination and laboratory detection result, particularly from patient respiratory road secretory product sample, isolate the H7N9 avian influenza virus, or the H7N9 avian influenza virus detection of nucleic acids positive, can diagnose.
Summary of the invention
In view of detection of nucleic acids is directly to detect pathogen nucleic acid, have high sensitivity, advantages such as high special and short detection window phase are for eqpidemic disease diagnosis early, early treatment, reduction case fatality rate and control epidemic situation are raced against time.The invention provides the test kit that a kind of real-time fluorescence quantitative PCR detects the H7N9 avian influenza virus, for detection of H7N9 avian influenza virus nucleic acid.
A kind of test kit that utilizes real-time fluorescence quantitative PCR to detect the H7N9 avian influenza virus is characterized in that described test kit comprises:
(1) RNA extracts reagent, reverse transcription reagent, real-time fluorescence quantitative PCR amplification reaction reagent;
(2) for primer and the probe of amplified sample cDNA, comprising:
Detect primer and the probe of H7N9 avian influenza virus coating surface hemagglutinin H7:
H7-F: GGGWTTCACMTACAGYGGAATAAG
H7-R: ACAGGARCCATTTCATCTCYGC
H7-probe: FAM-CAACSAGTGCATGTAGGAGATCAGGWTCTTC-TAMARA
Detect primer and the probe of H7N9 avian influenza virus neuraminidase N9:
N9-F: TGARTGCAGGTTCTATGCTCTCA
N9-R: TGTATACTGTGGGYGGTGATGA
N9-Probe: HEX-CTCAAACGGAACAATACACGATAGGTCCCA-TAMARA。
Further, described RNA extraction reagent comprises QIAamp Viral RNA mini Kit; Described reverse transcription reagent comprises 5 * RT-Buffer, 100uM Primer Mix, 100U/ul ReverTra Ace reversed transcriptive enzyme and DEPC water; Described real-time fluorescence quantitative PCR amplification reaction reagent comprises qPCR Mix, 50 * ROX, amplimer and probe and aqua sterilisa.
Further, the using method of described test kit may further comprise the steps:
(1) extracts the RNA that reagent extracts the respiratory tract sample with RNA;
(2) with reverse transcription reagent the RNA that obtains in the step (1) being carried out reverse transcription is cDNA;
(3) cDNA that uses primer H7-F, H7-R and N9-F, N9-R and correspondent probe H7-probe, N9-probe in single tube PCR reaction system step (2) to be obtained carries out the real-time fluorescence quantitative PCR detection.
Wherein, real-time fluorescence quantitative PCR response procedures: 95 ℃ of pre-sex change of 10min; 95 ℃ of 15sec, 58 ℃ of 35sec, 39 circulations.
The concrete extraction step of step (1) is as follows:
1) draw the ready buffer AVL(of 560ul and contain carrier RNA) in the 1.5ml centrifuge tube.(actual amount is per sample adjusted buffer AVL-carrier RNA in proportion).
2) with 140ul blood plasma, serum, urine, culturing cell supernatant liquor or acellular body fluid joins in the centrifuge tube that buffer AVL-carrier RNA is housed.Mediated mixing 15 seconds.(for guaranteeing lysis efficiency, thoroughly mixing forms uniform solution.The sample that only dissolved once is also still available).
3) room temperature is placed 10min.(10 minutes enough, and time expand can not increase product quality.Buffer AVL can make potential pollution and Rnase inactivation).
4) instantaneous centrifugal, the drop on the lid got rid of return pipe at the bottom of.
5) add 560ul dehydrated alcohol (96%～100%), the abundant mixing of mediation 15s in the sample.Instantaneous centrifugal then, the drop on the lid got rid of return pipe at the bottom of.(only to use ethanol, because other alcohol can reduce output and purity.Can not use methylated spirits, because wherein contain other materials.If sample size greater than 140ul, increases the amount of ethanol in proportion.This step is wanted abundant mixing, forms uniform solution, to guarantee output).
6) the careful adding column(of solution during absorption 630ul upward goes on foot has been encased in the 2ml centrifuge tube) in, the edge of pillar is not run in attention.Cover lid, 6000 * g(8000rpm) centrifugal 1min.Column is put into new 2ml centrifuge tube, discard old collection tube.(each column will cover, with anti-cross-contamination.The speed of 8000rpm is in order to limit noise, centrifugally at full speed also is fine, and can not influence product.If solution does not see through film fully, speed recentrifuge that can be higher sees through fully up to solution).
7) the careful lid of opening column repeated for the 6th step.(if sample size has surpassed 140ul, repeats this step so, up to all lysates upper prop all).
8) the careful column lid of opening adds 500ul buffer AW1.Cover lid, 8000rpm is centrifugal, 1min.Column is put into new 2ml collection tube (Kit provides), discard old collection tube.(even sample size also need not to increase the amount of buffer AW1 greater than 140ul).
9) the careful column lid of opening adds 500ul buffer AW2.Cover lid, centrifugal (14000rpm) at full speed, 3min.Then carried out for the 11st step, perhaps carried out for the 10th step earlier, avoiding buffer AW2 residual, and then carried out for the 11st step.(buffer AW2 is residual can to influence the downstream experiment.Some whizzer can vibrate when slowing down, and causes refluxing, and makes buffer AW2 touch column.Also may cause this phenomenon to produce when column and collection tube are taken out in the whizzer, therefore, preferably carry out for the 10th step earlier).
10) recommend: column is put into new 2ml collection tube (Kit does not provide), discard old collection tube, at full speed centrifugal 1min.
11) column is placed in the 1.5ml centrifuge tube (not providing among the kit).Discard old collection tube.The careful column that opens adds the buffer AVE of 60ul room temperature.Cover lid, room temperature is placed 1min.The centrifugal 1min of 8000rpm.
The reverse transcription concrete steps of step (2) are as follows: add 1ul 100uM Primer Mix in the 10ul RNA, with mixed solution as for 65 ℃, behind the 5min, ice bath 2min; Add 4ul 5 * RT-Buffer to above-mentioned mixed solution, 4ul DEPC water, 1ul 100U/ul ReverTra Ace reversed transcriptive enzyme, mixing, 37 ℃ of 60min, 95 ℃ of 5min, obtain cDNA-20 ℃ standby.
In step (3) real-time fluorescence quantitative PCR detection method, two probes by different fluorescent marks (FAM, HEX), finish a routine sample and in a pipe PCR reaction system, detect first type avian influenza virus coating surface hemagglutinin (HA) gene and neuraminidase (NA) gene simultaneously, both can detect H7 hypotype and the N9 hypotype of H7N9 avian influenza virus simultaneously.
According to the real-time fluorescence quantitative PCR detected result, can whether infect the H7N9 avian influenza virus to the detection sample and identify.Concrete grammar is: the positive quality control at H7 positive quality control and N9 has amplification curve, and under the prerequisite of Ct value≤38,1) detects in the pipe PCR reaction system of sample, if the probe of the probe of FAM mark and HEX mark all produces amplification curve, and Ct value≤38 judge that then sample is the H7N9 virus-positive; 2) if detect in the pipe PCR reaction system of sample, the probe of the probe of FAM mark and HEX mark does not all have obvious amplification curve, has only perhaps wherein that a probe has amplification curve, judges that then sample is the H7N9 feminine gender.
Beneficial effect: the test kit for detection of H7N9 avian influenza virus nucleic acid of the present invention, by to the viral RNA extracting, reverse transcription and real-time fluorescence quantitative PCR can be identified the H7N9 avian influenza virus.The result of this test kit gained has high accuracy, hypersensitivity, and advantages such as high specific and short detection window phase are for eqpidemic disease diagnosis early, early treatment, reduction case fatality rate and control epidemic situation are raced against time.
Description of drawings
Fig. 1 is sample A real-time fluorescence quantitative PCR detected result.As seen from the figure, this sample H7 and N9 produce amplification curve, so sample A is the H7N9 avian influenza virus positive.
Fig. 2 is sample B real-time fluorescence quantitative PCR detected result.This sample has only H7 to produce amplification curve, so sample B is H7N9 avian influenza virus feminine gender.
Fig. 3 is sample C real-time fluorescence quantitative PCR detected result.This sample has only N9 to produce amplification curve, so sample C is H7N9 avian influenza virus feminine gender.
Fig. 4 is sample D real-time fluorescence quantitative PCR detected result.This sample H7 and N9 do not have amplification curve, so sample D is H7N9 avian influenza virus feminine gender.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be noted that, unaccounted normal condition and method among the embodiment, usually according to the conventional employing method of affiliated field experimenter: for example, Ao Sibai and James Kingston chief editor's " fine works molecular biology experiment guide " the 4th edition, perhaps step and the condition of advising according to manufacturer.
Test kit for detection of H7N9 avian influenza virus nucleic acid comprises:
(i) RNA extracts reagent: QIAamp Viral RNA mini Kit;
(ii) reverse transcription reagent: 5 * RT-Buffer, 100uM Primer Mix, 100U/ul ReverTra Ace reversed transcriptive enzyme, DEPC water;
(iii) real-time fluorescence quantitative PCR reagent: qPCR Mix, 50 * ROX is used for 2 pairs of primers and the corresponding probe of amplified sample cDNA, aqua sterilisa, wherein:
Detect primer and the probe of H7N9 avian influenza virus coating surface hemagglutinin H7:
H7-F: GGGWTTCACMTACAGYGGAATAAG
H7-R: ACAGGARCCATTTCATCTCYGC
H7-probe: FAM-CAACSAGTGCATGTAGGAGATCAGGWTCTTC-TAMARA
Detect primer and the probe of H7N9 avian influenza virus neuraminidase N9:
N9-F: TGARTGCAGGTTCTATGCTCTCA
N9-R: TGTATACTGTGGGYGGTGATGA
N9-Probe: HEX-CTCAAACGGAACAATACACGATAGGTCCCA-TAMARA
The using method of described test kit may further comprise the steps:
(1) extracts serum sample rna to be checked with QIAamp Viral RNA mini Kit;
(2) with reverse transcription reagent the RNA that obtains in the step (1) being carried out reverse transcription is cDNA;
(3) cDNA that uses 2 pairs of primers and correspondent probe in single tube PCR reaction system step (2) to be obtained carries out the real-time fluorescence quantitative PCR detection; The PCR detection architecture is 25ul, and comprising 12.5ul qPCR Mix, each 0.4ul(probe of 10 uM probes is respectively H7-probe, N9-probe); Each 0.8ul(forward primer of 10uM forward primer is respectively H7-F, N9-F); Each 0.8ul(reverse primer of 10uM reverse primer is respectively H7-R, N9-R); 50 * ROX 0.5ul, cDNA 2ul, ddH
2O 6ul; Real-time fluorescence quantitative PCR response procedures: 95 ℃ of pre-sex change of 10min; 95 ℃ of 15sec, 58 ℃ of 35sec, 39 circulations.
According to the real-time fluorescence quantitative PCR detected result, can whether infect the H7N9 avian influenza virus to the detection sample and identify.Concrete grammar is: the positive quality control at H7 positive quality control and N9 has amplification curve, and under the prerequisite of Ct value≤38,1) detects in the pipe PCR reaction system of sample, if the probe of the probe of FAM mark and HEX mark all produces amplification curve, and Ct value≤38 judge that then sample is the H7N9 virus-positive; 2) if detect in the pipe PCR reaction system of sample, the probe of the probe of FAM mark and HEX mark does not all have obvious amplification curve, has only perhaps wherein that a probe has amplification curve, judges that then sample is the H7N9 feminine gender.
Embodiment 2: detection method
1. the extraction of the RNA of sample to be detected
1) draw the ready buffer AVL(of 560ul and contain carrier RNA) in the 1.5ml centrifuge tube.(actual amount is per sample adjusted buffer AVL-carrier RNA in proportion).
2) with 140ul blood plasma, serum, urine, culturing cell supernatant liquor or acellular body fluid joins in the centrifuge tube that buffer AVL-carrier RNA is housed.Mediated mixing 15 seconds.(for guaranteeing lysis efficiency, thoroughly mixing forms uniform solution.The sample that only dissolved once is also still available).
3) room temperature is placed 10min.(10 minutes enough, and time expand can not increase product quality.Buffer AVL can make potential pollution and Rnase inactivation).
4) instantaneous centrifugal, the drop on the lid got rid of return pipe at the bottom of.
5) add 560ul dehydrated alcohol (96%～100%), the abundant mixing of mediation 15s in the sample.Instantaneous centrifugal then, the drop on the lid got rid of return pipe at the bottom of.(only to use ethanol, because other alcohol can reduce output and purity.Do not use methylated spirits, because wherein contain other materials.If sample size greater than 140ul, increases the amount of ethanol in proportion.This step is wanted abundant mixing, forms uniform solution, to guarantee output).
6) the careful adding column(of solution during absorption 630ul upward goes on foot has been encased in the 2ml centrifuge tube) in, the edge of pillar is not run in attention.Cover lid, 6000 * g(8000rpm) centrifugal 1min.Column is put into new 2ml centrifuge tube, discard old collection tube.(each column will cover, with anti-cross-contamination.The speed of 8000rpm is in order to limit noise, centrifugally at full speed also is fine, and can not influence product.If solution does not see through film fully, speed recentrifuge that can be higher sees through fully up to solution).
7) the careful lid of opening column repeated for the 6th step.(if sample size has surpassed 140ul, repeats this step so, up to all lysates upper prop all).
8) the careful column lid of opening adds 500ul buffer AW1.Cover lid, 8000rpm is centrifugal, 1min.Column is put into new 2ml collection tube (Kit provides), discard old collection tube.(even sample size also need not to increase the amount of buffer AW1 greater than 140ul).
9) the careful column lid of opening adds 500ul buffer AW2.Cover lid, centrifugal (14000rpm) at full speed, 3min.Then carried out for the 11st step, perhaps carried out for the 10th step earlier, avoiding buffer AW2 residual, and then carried out for the 11st step.(buffer AW2 is residual can to influence the downstream experiment.Some whizzer can vibrate when slowing down, and causes refluxing, and makes buffer AW2 touch column.Also may cause this phenomenon to produce when column and collection tube are taken out in the whizzer, therefore, preferably carry out for the 10th step earlier).
10) recommend: column is put into new 2ml collection tube (Kit does not provide), discard old collection tube, at full speed centrifugal 1min.
11) column is placed in the 1.5ml centrifuge tube (not providing among the kit).Discard old collection tube.The careful column that opens adds the buffer AVE of 60ul room temperature.Cover lid, room temperature is placed 1min.The centrifugal 1min of 8000rpm.
2. be cDNA with the gained RNA in the step 1 with the random primer reverse transcription, re-use 2 pairs of primers and probe the cDNA that obtains is carried out the real-time fluorescence quantitative PCR detection.
The concrete grammar of reverse transcription wherein: in 10ul RNA, add 1ul 100uM Primer Mix, with mixed solution as for 65 ℃, behind the 5min, ice bath 2min.Add 4ul 5*RT-Buffer to above-mentioned mixed solution, 4ul DEPC water, 1ul 100U/ul ReverTra Ace reversed transcriptive enzyme, mixing, 37 ℃ of 60min, 95 ℃ of 5min, obtain cDNA-20 ℃ standby.
The concrete grammar that detects of real-time fluorescence quantitative PCR wherein: the cDNA that uses 2 pairs of primers and correspondent probe in single tube PCR reaction system step (2) to be obtained carries out real-time fluorescence quantitative PCR detection (in every batch of detection, need carry out the detection of H7 and N9 positive quality control); The PCR detection architecture is 25ul, and comprising 12.5ul qPCR Mix, each 0.4ul(probe of 10 uM probes is respectively H7-probe, N9-probe); Each 0.8ul(forward primer of 10uM forward primer is respectively H7-F, N9-F); Each 0.8ul(reverse primer of 10uM reverse primer is respectively H7-R, N9-R); 50 * ROX 0.5ul, cDNA 2ul, ddH
2O 6ul.
Real-time fluorescence quantitative PCR response procedures: 95 ℃ of pre-sex change of 10min; 95 ℃ of 15sec, 58 ℃ of 35sec, 39 circulations.
3. interpretation of result, concrete grammar is: the positive quality control at H7 positive quality control and N9 has amplification curve, and under the prerequisite of Ct value≤38,1) detects in the pipe PCR reaction system of sample, if the probe of the probe of FAM mark and HEX mark all produces amplification curve, and Ct value≤38 judge that then sample is the H7N9 virus-positive; 2) if detect in the pipe PCR reaction system of sample, the probe of the probe of FAM mark and HEX mark does not all have obvious amplification curve, has only perhaps wherein that a probe has amplification curve, judges that then sample is the H7N9 feminine gender
Fig. 1 is sample A real-time fluorescence quantitative PCR detected result.As seen from the figure, this sample H7 and N9 produce amplification curve, so sample A is the H7N9 avian influenza virus positive.
Fig. 2 is sample B real-time fluorescence quantitative PCR detected result.This sample has only H7 to produce amplification curve, so sample B is H7N9 avian influenza virus feminine gender.
Fig. 3 is sample C real-time fluorescence quantitative PCR detected result.This sample has only N9 to produce amplification curve, so sample C is H7N9 avian influenza virus feminine gender.
Fig. 4 is sample D real-time fluorescence quantitative PCR detected result.This sample H7 and N9 do not have amplification curve, so sample D is H7N9 avian influenza virus feminine gender.
SEQUENCE LISTING
＜110〉Hangzhou Ai Dikang medical test center company limited
＜120〉a kind of test kit that utilizes real-time fluorescence quantitative PCR to detect the H7N9 avian influenza virus
<130>
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 24
<212> DNA
＜213〉artificial sequence
<400> 1
gggwttcacm tacagyggaa taag 24
<210> 2
<211> 22
<212> DNA
＜213〉artificial sequence
<400> 2
acaggarcca tttcatctcy gc 22
<210> 3
<211> 31
<212> DNA
＜213〉artificial sequence
<400> 3
caacsagtgc atgtaggaga tcaggwtctt c 31
<210> 4
<211> 23
<212> DNA
＜213〉artificial sequence
<400> 4
tgartgcagg ttctatgctc tca 23
<210> 5
<211> 22
<212> DNA
＜213〉artificial sequence
<400> 5
tgtatactgt gggyggtgat ga 22
<210> 6
<211> 30
<212> DNA
＜213〉artificial sequence
<400> 6
ctcaaacgga acaatacacg ataggtccca 30
Claims (4)
1. test kit that utilizes real-time fluorescence quantitative PCR to detect the H7N9 avian influenza virus is characterized in that described test kit comprises:
(1) RNA extracts reagent, reverse transcription reagent, real-time fluorescence quantitative PCR amplification reaction reagent;
(2) for primer and the probe of amplified sample cDNA, comprising:
Detect primer and the probe of H7N9 avian influenza virus coating surface hemagglutinin H7:
H7-F: GGGWTTCACMTACAGYGGAATAAG
H7-R: ACAGGARCCATTTCATCTCYGC
H7-probe: FAM-CAACSAGTGCATGTAGGAGATCAGGWTCTTC-TAMARA
Detect primer and the probe of H7N9 avian influenza virus neuraminidase N9:
N9-F: TGARTGCAGGTTCTATGCTCTCA
N9-R: TGTATACTGTGGGYGGTGATGA
N9-Probe: HEX-CTCAAACGGAACAATACACGATAGGTCCCA-TAMARA。
2. test kit as claimed in claim 1 is characterized in that, described RNA extracts reagent and comprises QIAamp Viral RNA mini Kit; Described reverse transcription reagent comprises 5 * RT-Buffer, 100uM Primer Mix, 100U/ul ReverTra Ace reversed transcriptive enzyme and DEPC water; Described real-time fluorescence quantitative PCR amplification reaction reagent comprises qPCR Mix, 50 * ROX, amplimer and probe and aqua sterilisa.
3. with claim 1 or 2 described test kits, it is characterized in that the using method of described test kit may further comprise the steps:
(1) extracts the RNA that reagent extracts the respiratory tract sample with RNA;
(2) with reverse transcription reagent the RNA that obtains in the step (1) being carried out reverse transcription is cDNA;
(3) cDNA that uses primer H7-F, H7-R and N9-F, N9-R and correspondent probe H7-probe, N9-probe in single tube PCR reaction system step (2) to be obtained carries out the real-time fluorescence quantitative PCR detection.
4. test kit as claimed in claim 3 is characterized in that, the real-time fluorescence quantitative PCR response procedures: 95 ℃ of pre-sex change of 10min; 95 ℃ of 15sec, 58 ℃ of 35sec, 39 circulations.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310129302.4A CN103233082B (en) | 2013-04-12 | 2013-04-12 | Kit for testing H7N9 bird flu virus using real time fluorescence quantitative PCR |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310129302.4A CN103233082B (en) | 2013-04-12 | 2013-04-12 | Kit for testing H7N9 bird flu virus using real time fluorescence quantitative PCR |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410639459.6A Division CN104388589A (en) | 2013-04-12 | 2013-04-12 | Primers and method for detecting H7N9 avian influenza virus by using real-time fluorescence quantitative PCR |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103233082A true CN103233082A (en) | 2013-08-07 |
| CN103233082B CN103233082B (en) | 2014-12-17 |
Family
ID=48881146
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310129302.4A Active CN103233082B (en) | 2013-04-12 | 2013-04-12 | Kit for testing H7N9 bird flu virus using real time fluorescence quantitative PCR |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103233082B (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104404172A (en) * | 2014-12-15 | 2015-03-11 | 中国医学科学院医学实验动物研究所 | Liquid chip detection kit for H7N9 subtype influenza viruses |
| CN105331745A (en) * | 2015-09-14 | 2016-02-17 | 北京佰鸥创投生物科技有限公司 | Double droplet type digital PCR absolute quantification detection kit for AIV H7N9 subtypes |
| CN105755174A (en) * | 2016-04-08 | 2016-07-13 | 广东省农业科学院动物卫生研究所 | Primer group, kit and method for quickly identifying H7N9 avian influenza virus |
| CN107541570A (en) * | 2017-04-21 | 2018-01-05 | 中国科学院微生物研究所 | Detect the primer and probe and its kit of highly pathogenic H7N9 mutated viruses |
| CN111257555A (en) * | 2020-04-03 | 2020-06-09 | 安徽省疾病预防控制中心（省健康教育所） | Rapid detection method and test strip for lateral chromatography colloidal gold of new coronavirus nucleic acid recombinase mediated isothermal amplification |
| CN112626066A (en) * | 2021-01-19 | 2021-04-09 | 武汉艾迪康医学检验所有限公司 | Method for concentrating extracted virus nucleic acid |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101058833A (en) * | 2007-02-08 | 2007-10-24 | 中国检验检疫科学研究院动植物检疫研究所 | Primer system and method for detecting and analyzing avian influenza virus |
-
2013
- 2013-04-12 CN CN201310129302.4A patent/CN103233082B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101058833A (en) * | 2007-02-08 | 2007-10-24 | 中国检验检疫科学研究院动植物检疫研究所 | Primer system and method for detecting and analyzing avian influenza virus |
Non-Patent Citations (2)
| Title |
|---|
| CHINA CDC: "Real-time RT-PCR protocol for the detection of A(H7N9) influenza virus", 《WHO》, 8 April 2013 (2013-04-08), pages 1 - 2 * |
| 简报: "H7N9型禽流感检测方法", 《中国地方病防治杂志》, vol. 28, no. 2, 5 April 2013 (2013-04-05), pages 132 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104404172A (en) * | 2014-12-15 | 2015-03-11 | 中国医学科学院医学实验动物研究所 | Liquid chip detection kit for H7N9 subtype influenza viruses |
| CN105331745A (en) * | 2015-09-14 | 2016-02-17 | 北京佰鸥创投生物科技有限公司 | Double droplet type digital PCR absolute quantification detection kit for AIV H7N9 subtypes |
| CN105755174A (en) * | 2016-04-08 | 2016-07-13 | 广东省农业科学院动物卫生研究所 | Primer group, kit and method for quickly identifying H7N9 avian influenza virus |
| CN105755174B (en) * | 2016-04-08 | 2019-05-14 | 广东省农业科学院动物卫生研究所 | A kind of primer sets, kit and the method for Rapid identification H7N9 avian influenza virus |
| CN107541570A (en) * | 2017-04-21 | 2018-01-05 | 中国科学院微生物研究所 | Detect the primer and probe and its kit of highly pathogenic H7N9 mutated viruses |
| CN111257555A (en) * | 2020-04-03 | 2020-06-09 | 安徽省疾病预防控制中心（省健康教育所） | Rapid detection method and test strip for lateral chromatography colloidal gold of new coronavirus nucleic acid recombinase mediated isothermal amplification |
| CN112626066A (en) * | 2021-01-19 | 2021-04-09 | 武汉艾迪康医学检验所有限公司 | Method for concentrating extracted virus nucleic acid |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103233082B (en) | 2014-12-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111057797B (en) | Novel coronavirus 2019-nCoV real-time fluorescent quantitative PCR detection primer, probe, kit and method | |
| CN103233082B (en) | Kit for testing H7N9 bird flu virus using real time fluorescence quantitative PCR | |
| CN104232798B (en) | The multiple fluorescence quantitative PCR method of DHAV detection and Gene A type and the discriminating of C type and test kit | |
| CN103275862A (en) | Fluorescent quantitative reverse transcription-polymerase chain reaction (RT-PCR) kit for detecting influenza A virus subtype H7N9 | |
| JP7064473B2 (en) | How to detect influenza A virus and influenza B virus | |
| CN103290144A (en) | Primer for detecting H7N9 subtype avian influenza virus infected by humans though RT-LAMP (reverse transcription loop-mediated isothermal amplification) and application of primer | |
| CN109517927A (en) | A kind of A type, influenza B virus rapid typing detection reagent box and its application | |
| CN105296673A (en) | Influenza A virus molecular detection kit and preparation method thereof | |
| Zhang et al. | Simultaneous detection of eight swine reproductive and respiratory pathogens using a novel GeXP analyser-based multiplex PCR assay | |
| CN101649356B (en) | Fluorescent quantitative detection kit of H1N1 influenza virus A and detection method thereof | |
| CN102251061A (en) | Nucleic acid dual fluorescence PCR (Polymerase Chain Reaction) detection kit for influenza A/B virus | |
| CN103243181B (en) | H7N9 influenza virus A detection kit and detection method | |
| CN104846121A (en) | Virus triple fluorescence quantitative RT-PCR detection method and virus triple fluorescence quantitative RT-PCR detection kit | |
| CN110699492A (en) | Yonganhe virus real-time fluorescent quantitative PCR detection primer, probe, detection kit, detection method and application thereof | |
| CN102180927A (en) | Method for obtaining influenza virus nucleic acid in influenza sample and special primer thereof | |
| CN108239677A (en) | A kind of influenza A genes parting detecting reagent | |
| Golabi et al. | Development of reverse transcription loop-mediated isothermal amplification assay for rapid and on-site detection of avian influenza virus | |
| CN102146485B (en) | One-step RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection kit for influenza virus | |
| Li et al. | Simultaneous detection of hemagglutinin and neuraminidase genes of novel influenza A (H7N9) by duplex real-time reverse transcription polymerase chain reaction | |
| CN104388589A (en) | Primers and method for detecting H7N9 avian influenza virus by using real-time fluorescence quantitative PCR | |
| CN102154514A (en) | One-step real-time fluorescence quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) kit for influenza A virus H1N1 | |
| CN111004867B (en) | Influenza A virus detection primer, probe and kit thereof | |
| CN104073571B (en) | H7N9 subtype avian influenza virus double fluorescent RT-PCR primer, probe and detection kit thereof and detection method | |
| CN101712999B (en) | Dual real-time fluorescent RT-PC detection kit for subtypes of flu viruses N1 and N2 | |
| CN105039591B (en) | A kind of method of quick discriminating detection N1, N2, N6, N8 subtype influenza virus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |