CN101942525B - One-tube method with multiplex detection for human Influenza A and B and new Influenza A H1N1 virus and kit - Google Patents
One-tube method with multiplex detection for human Influenza A and B and new Influenza A H1N1 virus and kit Download PDFInfo
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Abstract
The invention provides a one-tube method with multiplex fluorescent PCR detection for human Influenza A and B and new Influenza A H1N1 virus. The method adopts the primers of which the sequence is shown in SEQ ID NO: 1-6, and also adopts the probes of which the sequence is shown in SEQ ID NO: 7-9. The invention also provides a kit for one-tube method with complex fluorescent PCR detection for human Influenza A and B and new Influenza A H1N1 virus. The kit comprises the primers and the probes. The invention adopts InfA/InfB/A (H1N1) specific primers and Taqman probes, and uses FAM/JOE/TAMRA multiplex fluorescein labels to realize the multiplex detection for the human Influenza A and B and new Influenza A H1N1 virus. The invention has the advantages of high specificity, high sensitivity, high speed, simple and convenient operation, low cost and the like, can be used as a multiple-detection reagent for scientific research and clinical application.
Description
Technical field
The present invention relates to the molecular Biological Detection technical field, specifically human influenza A, second type influenza virus and the new H1N1virus one many detecting methods of pipe and test kit.
Background technology
The human influenza virus is that a kind of mankind of causing suffer from grippal RNA viruses; on taxonomy, influenza virus belongs to Orthomyxoviridae family, and it can cause acute upper respiratory tract infection; and propagate rapidly by air, often have periodically all over the world and be very popular.
The human influenza virus is divided into A type, Type B and C type influenza virus (claiming again first, second and influenza virus C) with the antigenicity of its nucleoprotein.In three kinds of influenza viruses that infect the mankind, influenza A virus has extremely strong variability, B-mode taking second place, and the antigenicity of influenza virus C is highly stable.So cause and extensive popular generally be front two kinds.
Influenza virus can cause more serious symptom the weak old man of immunizing power or the patient of children and some immune disorders, as pneumonia or cardiopulmonary exhaustion etc.From EPDML angle, influenza causes that worldwide being very popular is influenza antigen variation and the interactional result of community immunity barrier.Four flu outbreaks appear in 20 th Centuries altogether, are all to be caused by the influenza A virus that makes a variation, and cause great personnel and property damage.The new H1N1virus of being found by Mexico at first in 2009 just contains the gene fragment of porcine influenza, bird flu and three kinds of influenza viruses of human influenza, comes in global bamboo telegraph, causes popular on a large scale.Various countries, the whole world are the new first stream of prevention and control H1N1 at present, all doing positive effort, have obtained simultaneously significant effect.
The serious respiratory tract disease that the mankind is caused in view of the new H1N1virus in the first and second type influenza viruses and present influenza A, particularly in children, be diagnosis and treatment and prevention and control needs, this just needs medical institutions that the detection method of accurate rapid sensitive is provided, quick diagnosis, fast treating.With regard to present circumstances, to the main following several method of the diagnosis of the first and second types and new H1N1virus: the culture of isolated that (1) is viral; (2) serology detects special IgG and IgM; (3) RT-PCR method and fluorescence quantifying PCR method.The several method of comparing, front two kinds of classical ways that method is all early development, PCR method is the detection method for pathogen nucleic acid, the cycle length, the not high problem of sensitivity that exist in the method for two kinds of fronts have been overcome, the particularly introduction of fluorescence quantifying PCR method, solved PCR method and easily produced false-positive problem, made it obtain development fast, the multiple advantage such as the method has high specific, highly sensitive, the cycle is short, simple to operate, cost is lower.
But the influenza virus somatotype reagent of exploitation is substantially all the substance fluorescence quantitative PCR detection reagent at present, can not detect simultaneously these three kinds of influenza viruses; As detecting simultaneously, need to carry out respectively its complex operation, much time power in three pipes.
Summary of the invention
In order to overcome above-mentioned technological deficiency, one of purpose of the present invention is to provide a kind of human influenza A, second type influenza virus and new H1N1virus one pipe multiple fluorescence PCR detection method, improving at present existing method, thus make detect reach sensitive, fast, accurately, the purpose of economical with materials and reagent.
Human influenza A of the present invention, second type influenza virus and new H1N1virus one pipe multiple fluorescence PCR detection method comprise preparation and the multiplex PCR amplification step of sample nucleic acid, wherein, adopted sequence primer as follows (seeing sequence table SEQ ID NO:1-6) in the multiplex PCR amplification step:
IFAF：GAGGTCGAAACGTATGTTCTCTCTATC；
IFAR：TCTTCAAGTCTCTGCGCGATT；
IFBF：CCCTGCTTGCTCGTAGTATGG；
IFBR：GCTTATGGGAAGCACCACTTTG；
AH1F：GGTTTGAGATATTCCCCAAGACA；
AH1R：GAGGACATGCTGCCGTTACA。
Wherein, primer I FAF and IFAR are used for detecting the influenza virus A hominis, and we are referred to as InfA group primer; Primer I FBF and IFBR are used for detecting Influenza B virus, are referred to as InfB group primer in the present invention; Primer AH1F and AH1R are used for detecting new H1N1virus, and we are referred to as A (H1N1) group primer.
In aforesaid method, also adopted sequence probe as follows (seeing sequence table SEQ ID NO:7-10):
IFAP：CATCAGGCCCCCTCAAAGCCG；
IFBP：CGTTGTTAGGCCCTCTGTGGCGA；
AH1P：TTCATGGCCCAATCATGACTCGAACA。
Preferably, adopt the reaction pattern of the quantitative fluorescent PCR of Taqman probe in detecting, the fluorescent mark of above-mentioned probe is specific as follows:
IFA-TAMRA：TAMRA-CATCAGGCCCCCTCAAAGCCG-BHQ2；
IFB-FAM：FAM-CGTTGTTAGGCCCTCTGTGGCGA-BHQ1；
AH1-JOE：JOE-TTCATGGCCCAATCATGACTCGAACA-BHQ1。
Wherein, IFA-TAMRA is the probe that detects influenza A virus, and IFB-FAM is the probe that detects Influenza B virus, and AH1-JOE is the probe that detects new H1N1virus.Above-mentioned three kinds of fluorescently-labeled probes are referred to as FAM/JOE/TAMRA multi-fluorescence element label probe in the present invention.
When the present invention adopts a multiple mode of pipe to detect influenza A virus, Influenza B virus and new H1N1virus, above-mentioned InfA, InfB and three groups of primers of A (H1N1) and three Taqman probes are blended in a reaction tubes, use the RT-PCR damping fluid of ' PrimeScript single stage method RT-PCR test kit (Ver.2) ' (TAKARA ' PrimeScript One Step RT-PCR Kit Ver.2 ') to carry out the reagent preparation, wherein the primer usage quantity is 7-15pmol, and the probe usage quantity is 0.5-5pmol.The reaction cumulative volume is 25 μ l; The 18 μ l/ reactions of reagent preparation volume (are reserved 2 μ l and are used for adding enzyme mixture; Reserve 5 μ l and be used for template), the reaction conditions of the method be 50 ℃ 30 minutes, 94 ℃ 2 minutes, thermal cycling be 94 ℃ 10 seconds, 55 ℃ 35 seconds, totally 40 circulations.Need to prove, the said multiple mode of pipe of the present invention is included in any two-strain or the three kinds of viruses that detect simultaneously in same reaction tubes in influenza A virus, Influenza B virus and new H1N1virus.
Two of purpose of the present invention has been to provide primer and the probe for detection of influenza A virus, Influenza B virus and new H1N1virus.Primer sequence be selected from following sequence at least a identical, similar or complementary sequence (seeing sequence table SEQ ID NO:1-6):
IFAF：GAGGTCGAAACGTATGTTCTCTCTATC；
IFAR：TCTTCAAGTCTCTGCGCGATT；
IFBF：CCCTGCTTGCTCGTAGTATGG；
IFBR：GCTTATGGGAAGCACCACTTTG；
AH1F：GGTTTGAGATATTCCCCAAGACA；
AH1R：GAGGACATGCTGCCGTTACA。
Probe for detection of influenza A virus, Influenza B virus and new H1N1virus provided by the present invention be can with the sequence of the nucleic acid array hybridizing that obtains with above-mentioned primer amplification.
Preferably, described probe be selected from as at least a identical, the similar or complementary sequence in sequence SEQ ID NO:7-10.Described similar sequences refers to and there are indivedual different bases in above-mentioned sequence, such as 1-5 base, however affect expanding effect; Different base performances can be the modes such as insertion on original sequence basis, disappearance, replacement.
The present invention also provides a kind of human influenza A, second type influenza virus and new H1N1virus one pipe multiple fluorescence PCR detection reagent box, and this test kit comprises the primer of sequence as shown in SEQ ID NO:1-6.
This test kit also comprises following fluorescence labeling probe:
RSV-FAM：FAM-TATGAATGCCTATGGTGCAGGGCAAG-BHQ；
ADV-TAMRA：TAMRA-AAGAGGCCACTCTTGAGTGCCAGCG-BHQ2；
HMP1-JOE：JOE-AGAGATGTAGGCACCACAAC-MGB；
HMP2-JOE：JOE-TGGCCAATTGCCCCAATTTTGC-BHQ1；
HMP3-JOE：JOE-CTAGCCAACTGTCCCAACTTTGCA-BHQ1。
Compared with prior art, present method adopts InfA/InfB/A (H1N1) special primer and the Taqman probe that self designs, use FAM/JOE/TAMRA multi-fluorescence element mark, the Multiple detection of realization to infA, infB, A (H1N1) (referred to as M-ABH1), have the multiple advantages such as specificity is high, highly sensitive, quick, simple to operation, with low cost, can be used as human influenza A, the reagent that second type influenza virus and new H1N1virus detect is used for scientific research and clinical application.
Embodiment
For making the present invention easier to understand, below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention, NM concrete experimental technique in the following example carries out according to the normal experiment method usually.
Embodiment 1: the specificity experiment of primer, probe
Use the biosoftware of information biology and design primer probe to reach new H1N1virus sequence data to first, Influenza B virus, respectively the conserved regions of first, Influenza B virus is carried out the primer probe design, conserved regions to the H1 gene of new H1N1virus is carried out the primer probe design, and its middle probe adopts respectively TAMRA, FAM, JOE mark.Detailed sequence is shown in Table 1, and table 1 is human influenza A, second type influenza virus and new H1N1virus primer and probe sequence.
Table 1
For verifying the specificity of designed primer and probe, at first design the substance Fluorescence PCR, primer and probe are assessed, namely detect respectively three groups of primers of InfA, InfB and A (H1N1) and the correspondent probe specificity of reaction separately.Experiment adopts the positive strain of culture of isolated to carry out, positive strain derives from Guangzhou Inst. of Respiratory Diseases, the experiment of respiratory disease state key, select respectively influenza A, influenza B, use in addition parainfluenza 1, parainfluenza 2, parainfluenza 3, respiratory syncytial virus RSV, adenovirus ADV, rhinovirus HRV, metapneumovirus HMP, HCoV-229E, OC43, mycoplasma pneumoniae MP, Chlamydia pneumoniae CP as negative control.100 μ l culture samples are got respectively in experiment, extract by conventional RNA/DNA extracting method, obtain 50 μ l nucleic acid product, use DEPC water to 100 times of dilutions of nucleic acid extraction product after as template (wherein H5N1 does not dilute, 10 times of coronavirus and the dilutions of metapneumovirus product).Use the RT-PCR damping fluid in ' PrimeScript single stage method RT-PCR test kit (Ver.2) ' (TAKARA ' PrimeScript One Step RT-PCR KitVer.2 ') to carry out the reagent preparation, wherein the primer usage quantity is 7-15pmol, and the probe usage quantity is 0.5-5pmol.the reaction cumulative volume is 25 μ l, the 18 μ l/ reactions of reagent preparation volume (are reserved 2 μ l and are used for adding enzyme mixture, reserve 5 μ l and be used for adding template), add respectively 18 μ L influenza A in eight combs, influenza B, the substance reaction solution of new first stream H1N1, be positioned in quantitative real time PCR Instrument at last and react, reaction conditions is as follows: 50 ℃ 30 minutes, 94 ℃ 2 minutes, thermal cycling be 94 ℃ 10 seconds, 55 ℃ 35 seconds, totally 40 circulations, table 2 is substance infA, infB, A (H1N1) reagent detects the situation of 80 strain positive-virus strains, wherein influenza A (H3N213 strain, common H1N110 strain, new first stream H1N111 strain, 1 strain of H5N1 deactivation sample, H9 hypotype strain 1 strain), influenza B 20 strains, parainfluenza 1 is arranged in addition, parainfluenza 2, parainfluenza 3, respiratory syncytial virus RSV, adenovirus ADV, rhinovirus HRV, metapneumovirus HMP, HCoV-229E, OC43, mycoplasma pneumoniae MP, each 2 strains of Chlamydia pneumoniae CP are as negative control.
Table 2
| Substance reagent detected result | InfA | InfB | A(H1N1) |
| Positive | 36 | 20 | 11 |
| Negative | 44 | 60 | 69 |
| Amount to | 80 | 80 | 80 |
In experimental result, positive findings meets fully with expection, with other common respiratory pathogen parainfluenza 1, parainfluenza 2, parainfluenza 3, respiratory syncytial virus RSV, adenovirus ADV, rhinovirus HRV, metapneumovirus HMP, HCoV-229E, OC43, the no cross reactions such as mycoplasma pneumoniae MP, Chlamydia pneumoniae CP, also no cross reaction between infA-infB, A (H1N1)-infB, infA-A (H1N1) no cross reaction when common influenza A, specificity is high.
The specificity experiment of embodiment 2:M-ABH1 Multiple detection reagent
Multiple detection reagent uses TAKARA ' PrimeScript single stage method RT-PCR test kit (Ver.2) ' (TAKARA ' PrimeScript One Step RT-PCR Kit Ver.2 ') RT-PCR damping fluid carries out the reagent preparation, wherein the usage quantity of primer is 7-15pmol, and the usage quantity of probe is 0.5-5pmol.InfA/InfB/A in table 1 (H1N1) primer and each probe are formulated as a tube reaction liquid (M-ABH1).The reaction cumulative volume is 25 μ l/ reactions, and the 18 μ l/ reactions of reagent preparation volume are reserved 2 μ l and are used for adding enzyme mixture; Reserve 5 μ l and be used for adding template.Use respectively single positive template (common influenza A; Influenza B or new first stream H1N1); Two positive templates (common influenza A-influenza B; The new first stream of common influenza A-H1N1; The new first stream of influenza B-H1N1); Three positive templates (the new first stream of common influenza A-influenza B-H1N1) are tested Multiple detection reagent, use simultaneously substance fluorescent PCR reagent to compare.Experimental result sees Table 3, and table 3 is contrasts that in various template situations, multiple M-ABH1 influenza virus detection reagent and substance detect.
Table 3
Can find out from the experimental data of table 3, the specificity of Multiple detection reagent is compared with substance does not have any difference substantially, and simultaneously from the performance of CT value, multiple M-ABH1 influenza virus detection reagent and substance detect basically identical single check reagent that even is better than.
The sensitivity experiments of embodiment 3:M-ABH1 Multiple detection reagent
Susceptibility for further testing reagent, human influenza A (selecting the H3N2 type), second type influenza virus and new H1N1virus positive are carried out 10 times of gradient dilutions, be designated as extent of dilution 1～6, represent respectively 10～106 times of infA-H3N2, infB, A (H1N1) viral dilution, compare with multiple influenza virus detection reagent and substance detection respectively, carry out simultaneously virus culture and detect.The experimental result of quantitative PCR sees Table 4.The appraisable scope of virus culture is at extent of dilution 4, and it is cloudy detecting with culture method during extent of dilution 5, and sensitivity is far below substance or multiple influenza quantitative PCR detection.Table 4 be multiple and substance parainfluenza quantitative PCR detecting reagent to the contrast and experiment data sheet of the virus of 10 times of gradient dilutions.
Table 4
From top experimental result, do respectively typical curve according to the multiple of its dilution, can find out the linearly dependent coefficient (R of typical curve
2) all more than 0.99, better linear, multiple influenza test reagent separates far above virus culture with substance detection reagent detection sensitivity, is applicable to human influenza A, the detection of second type influenza virus and new H1N1virus.Multiple parainfluenza detection reagent can substitute the substance detection simultaneously, and does not affect experimental result.
Embodiment 4:M-ABH1 Multiple detection reagent clinical samples test experience
Use substance and multiple influenza quantitative PCR detecting reagent in July, 2009-throat swab clinical samples in 427 parts of October (Specimen origin is in The Second Affiliated Hospital of Guangzhou Medical School, Guangzhou Inst. of Respiratory Diseases, State Key Laboratory of Respiratory Diseases), carry out simultaneously the virus culture isolation identification.The results are shown in following table 5.427 parts of clinical respiratory tract throat swab samples of table 5 expression use the result of substances, multiple and virus culture separation detection:
Table 5
Show from top experiment, pipe multiple human influenza A, second type influenza virus and the new H1N1virus detection reagent that this experiment is researched and developed, highly sensitive in virus culture evaluation (also visible experimental example 3), can be used for scientific research and clinical position, detect human influenza A with a tube reaction, second type influenza virus and new H1N1virus reach purpose sensitive, quick, accurate, that save.
Last institute should be noted that; above embodiment is only in order to illustrate technical scheme of the present invention but not limiting the scope of the invention; although with reference to preferred embodiment, the present invention has been done detailed description; those of ordinary skill in the art is to be understood that; can modify or be equal to replacement technical scheme of the present invention, and not break away from essence and the scope of technical solution of the present invention.
Sequence table
＜120〉people's first, second type influenza virus reaches the new H1N1virus one many detecting methods of pipe and test kit
<160>9
<210>1
<211>27
＜212〉artificial sequence IFAF
<400>1
gaggtcgaaa cgtatgttct ctctatc 27
<210>1
<211>21
＜212〉artificial sequence IFAR
<400>2
tcttcaagtc tctgcgcgat t 21
<210>1
<211>21
＜212〉artificial sequence IFBF
<400>3
ccctgcttgc tcgtagtatg g 21
<210>1
<211>22
＜212〉artificial sequence IFBR
<400>4
gcttatggga agcaccactt tg 22
<210>1
<211>23
＜212〉artificial sequence AH1F
<400>5
ggtttgagat attccccaag aca 23
<210>1
<211>20
＜212〉artificial sequence AH1R
<400>6
gaggacatgc tgccgttaca 20
<210>1
<211>21
＜212〉artificial sequence IFAP
<400>7
catcaggccc cctcaaagcc g 21
<210>1
<211>23
＜212〉artificial sequence IFBP
<400>8
cgttgttagg ccctctgtgg cga 23
<210>1
<211>26
＜212〉artificial sequence AH1P
<400>9
ttcatggccc aatcatgact cgaaca 26
Claims (1)
1. people's first, second type influenza virus reaches new H1N1virus one many check reagent of pipe box, it is characterized in that, described many check reagent box comprises the primer of sequence as shown in SEQ ID NO:1-6, has also adopted following fluorescently-labeled probe in described many check reagent box:
IFA-TAMRA：TAMRA-CATCAGGCCCCCTCAAAGCCG-BHQ2；
IFB-FAM：FAM-CGTTGTTAGGCCCTCTGTGGCGA-BHQ1；
AH1-JOE：JOE-TTCATGGCCCAATCATGACTCGAACA-BHQ1 。
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| CN102154514A (en) * | 2011-03-18 | 2011-08-17 | 武汉大学 | One-step real-time fluorescence quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) kit for influenza A virus H1N1 |
| CN102146485B (en) * | 2011-03-24 | 2012-12-12 | 武汉大学 | One-step RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection kit for influenza virus |
| CN102251061A (en) * | 2011-08-05 | 2011-11-23 | 江苏硕世生物科技有限公司 | Nucleic acid dual fluorescence PCR (Polymerase Chain Reaction) detection kit for influenza A/B virus |
| CN103614495B (en) * | 2013-12-02 | 2014-12-10 | 上海之江生物科技股份有限公司 | Joint detection kit for influenza viruses A and B and application thereof |
| CN104032034A (en) * | 2014-03-07 | 2014-09-10 | 王全意 | Method for simultaneously detecting influenza A virus, influenza B virus and influenza C virus and kit |
| CN105219876A (en) * | 2015-11-05 | 2016-01-06 | 江苏省疾病预防控制中心 | A kind of people's seasonal influenza Viral typing detection kit and using method thereof |
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