CN103525949A - RT-LAMP (Reverse Transcription Loop-Mediated Isothermal Amplification) primer combination and kit used for detecting HA (Hemagglutinin) gene and NA (Neutrophil Antigen) gene of H7N9 virus - Google Patents
RT-LAMP (Reverse Transcription Loop-Mediated Isothermal Amplification) primer combination and kit used for detecting HA (Hemagglutinin) gene and NA (Neutrophil Antigen) gene of H7N9 virus Download PDFInfo
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Abstract
The invention discloses an RT-LAMP (Reverse Transcription Loop-Mediated Isothermal Amplification) primer combination and a kit used for detecting HA (Hemagglutinin) gene and NA (Neutrophil Antigen) gene of H7N9 virus. By using the composition and the kit disclosed by the invention, the HA gene and NA gene of the H7N9 virus can be quickly, conveniently and efficiently detected with high specificity and high sensitivity under an isothermal condition; complex instruments are not added; the detection for clinical specimens of the H7N9 can be met well; a novel technology platform is provided for quickly screening the H7N9 virus; the urgent need of detection of the H7N9 virus at present can be met well; the composition and the kit are applicable to fast detection of the grass-root health and medical units and various disease prevention and control centers, easy to popularize and apply in a large range, board in market prospect and high in economic and social benefits.
Description
Technical field
The invention belongs to health care check field, relate to a kind of combination of primers and test kit for detection of H7N9 virus HA gene and NA gene, also relate to a kind of RT-LAMP test kit and detection method and application for detection of H7N9 virus HA and NA gene.
Background technology
In March, 2013, in Shanghai City, China and Anhui Province, find respectively that 3 examples take the patient that serious lower respiratory infection is feature, CDC is defined as novel avian influenza virus (H7N9) and infects, by the end of on April 20th, 2013,96 routine H7N9 cases of infection have been made a definite diagnosis in China, wherein 18 examples are dead, and number of the infected is still increasing at present.
The isothermal amplification (LAMP) of ring mediation is by T.Notomi (Notomi T, Okayama H, MasubuchiH, et al.Loop-mediated isothermal amplification of DNA.Nucleic Acids Res2000; 28 (12): 63.) a kind of novel nucleic acids amplification technique of invention, this technology relies on primer and a kind of archaeal dna polymerase with strand displacement characteristic of 4 special designs, can be efficiently under isothermal condition, quick, high amplified target sequence specifically.In recent years, this technology is widely used in to pathogen detection abroad.People (the Imai M such as Masaki Imai; Ninomiya A, Minekawa H Rapid diagnosis of H5N1avian influenza virus infection by newly developed influenza H5hemagglutinin gene-specific loop-mediated isothermal amplification method.[J] Virol Methods.2007May; 141 (2): 173-80.) utilize LAMP to set up the Laboratory Diagnosed system of avian influenza virus.People (Hayashi N, Arai R, Tada S, Taguchi H, the Ogawa Y Food Microbiol.2007Oct-Dec such as Nobuyuki Hayashi; 24 (7-8): 778-85Detection and identification of Brettanomyces/Dekkera sp.yeasts with a loop-mediated isothermal amplification method.) for the ITS sequences Design of four kinds of cordiale yeast LAMP Auele Specific Primer, set up efficient LAMP detection system.The RT-LAMP technology of setting up based on LAMP technology can detect other virus relevant to the mankind, as viral hemorrhagic septicemia (VHS), cytomegalovirus (CMV), Ebola virus (EBOV), chronic burkitt's lymphoma virus (EBV), irido virus, mankind's herpus vivus 8 types, hematopoietic tissue necrosis virus (HHNV), tomato spotted wilf virus, tomato yellow leaf curl virus etc.At present, be showed no both at home and abroad and be useful on the RT-LAMP test kit of detection H7N9 virus and the report of application in H7N9 virus detects thereof.
Summary of the invention
First object of the present invention is to provide the specificity RT-LAMP combination of primers for detection of H7N9 virus HA gene and NA gene.
For achieving the above object, first from U.S. gene database GenBank retrieval, obtain H7N9 virus HA gene (No. GenBank: KC885956.1) and NA gene (No. GenBank: KC885958.1) sequence, by BLAST software, carry out homology analysis, find specific conservative target sequence, the nucleotide sequence of the conservative target sequence of HA gene specific is as shown in sequence in sequence table 13, the nucleotide sequence of the conservative target sequence of NA gene specific is as shown in sequence in sequence table 14, the more conservative target sequence of basis carries out RT-LAMP design of primers.
Specifically, the specificity RT-LAMP combination of primers for detection of H7N9 virus HA gene and NA gene provided by the present invention, comprises 6 primers for detection of HA gene and 6 primers for detection of NA gene, and concrete sequence is as follows:
The described specificity RT-LAMP combination of primers for detection of H7N9 virus HA gene, comprises two outer primer H7-11F3 and H7-11B3, two inner primer H7-11FIP and H7-11BIP and two ring primer H7-11LF and H7-11LB; Outer primer: inner primer: ring primer in molar ratio 5:40:20 mixes.
The described specificity RT-LAMP combination of primers for detection of H7N9 virus N A gene, comprises two outer primer N9-37F3 and N9-37B3, two inner primer N9-37F3FIP and N9-37F3BIP and two ring primer N9-37F3LF and N9-37F3LB; Outer primer: inner primer: ring primer in molar ratio 5:40:20 mixes.
Another object of the present invention is to provide a kind of have highly sensitive, high specific, the RT-LAMP detection kit for detection of H7N9 virus HA gene and NA gene simple to operate.
RT-LAMP detection kit for detection of H7N9 virus HA gene and/or NA gene provided by the present invention, comprises by above-mentioned 6 primers for detection of HA gene and/or 6 RT-LAMP combination of primers that form for detection of the primer of NA gene.
Test kit of the present invention also comprises RT-LAMP reaction solution, jointly forms LAMP detection system with RT-LAMP combination of primers.
In detection system, each material concentration is: 20mM Tris-HCl (pH8.8), 10mM KCl, 10mM (NH
4)
2sO
4, 0.1%Tween20,0.8M trimethyl-glycine (betaine), 8mM MgSO
4, 1.4mM dNTPs (above material is the reaction mother liquor mixing), 8U Bst archaeal dna polymerase, 8U AMV reversed transcriptive enzyme, 40pmol primers F IP and BIP, 20pmol primer LF and LB, 5pmol primers F 3 and B3.
The present invention also provides the above-mentioned RT-LAMP test kit of a kind of use to detect the method for H7N9 virus HA and NA gene, can comprise the following steps:
1) by aforementioned component and concentration, prepare 23 μ L detection system, while detecting HA gene, use outer primer H7-11F3& H7-11B3, inner primer H7-11FIP& H7-11BIP and ring primer H7-11LF& H7-11LB, uses outer primer N9-37F3& while detecting NA gene; N9-37B3, inner primer N9-37FIP& N9-37BIP and ring primer N9-37LF& N9-37LB;
2) extract according to a conventional method the nucleic acid in testing sample;
3) get 2 μ L nucleic acid extraction liquid and add step 1) preparation detection system in, making end reaction volume is 25 μ L, mixes reaction solution;
4) put 60-65 ℃ of constant temperature 45min and carry out RT-LAMP amplification;
5) carry out result interpretation, method is as follows:
5.1) amplification is gone to and in reaction solution, is added 1 μ L fluorexon, can be according to the colour-change judged result of reaction solution: green, shows to exist in testing sample HA gene or the NA gene (result is positive) of H7N9 virus; Safran, represents not exist in testing sample HA gene or the NA gene (result is negative) of H7N9 virus;
5.2) do not add fluorexon and directly with turbidimeter, according to turbidity, change judged result: turbidity (reduced turbidity >=0.3) rises, show to exist in testing sample HA gene or the NA gene (result is positive) of H7N9 virus; Turbidity is unchanged, shows not exist in testing sample HA gene or the NA gene (result is negative) of H7N9 virus.
The invention provides a kind of RT-LAMP combination of primers and test kit for detection of H7N9 virus HA gene and NA gene.The present invention has the following advantages:
1) high specific: use 6 combination of primers the identification in 8 special regions of target sequence to be guaranteed to the high degree of specificity of RT-LAMP amplification, RT-LAMP can, from differing the gene sample of a Nucleotide only, find out corresponding target sequence and increase.The specificity that H1N1virus, seasonal H1N1 influenza virus, H3N2 influenza virus and human genome in 2009 are template detection system is take in the present invention, result shows, by combination of primers of the present invention and test kit, detect, the HA gene of the H7N9 virus that can only increase and NA gene nucleic acid, other non-target viral nucleic acid can not be detected, confirm to utilize RT-LAMP combination primer of the present invention and test kit to detect and there is higher specificity.
2) highly sensitive: RT-LAMP sensitivity and RT-PCR are equal to.With 10 times of gradient dilutions, containing the template of H7N9 virus HA gene and NA gene, carry out respectively RT-LAMP and RT-PCR detection, the relatively sensitivity of two kinds of detection methods, result demonstration detects by RT-LAMP combination of primers of the present invention and test kit, can detect 10
1the nucleic acid of concentration, has identical sensitivity with RT-PCR detection method.
3) it is simple to operate: once whether RT-LAMP design of primers success, as long as will detect sample (target nucleic acid) and reagent is put into 60-65 ℃ of thermostat water bath together, just can judge amplification after 45 minutes.
4) efficiently amplification fast: whole amplified reaction can complete in a hour, and productive rate can reach 0.5mg/mL.
5) result interpretation is easy: when adding the fluorescence dyes such as fluorexon in reaction solution, if there is amplification, by visual inspection, if reaction solution becomes green, positive, and if reaction solution is safran, the explanation reaction that is negative; Also can directly use turbidimeter judged result.
In sum, use combination of primers of the present invention and the test kit can be under isothermal condition, fast, convenient, efficiently, high specific, HA gene and the NA gene of H7N9 virus detected in high sensitivity, do not need complex instrument, can better meet the clinical sample of H7N9 is detected, for rapid screening H7N9 virus provides new technology platform, can better meet at present H7N9 virus is detected in the urgent need to, for basic health medical institutions, the rapid detection of each disease prevention and control center, be easy to apply on a large scale, there is wide market outlook and higher economy, social benefit.
Below in conjunction with specific embodiment, the present invention is described in further details.
Accompanying drawing explanation
Fig. 1 is that RT-LAMP experimental result picture is judged in fluorexon dyeing.Green positive (No. 1-6), safran negative (No. 7-8).
Fig. 2 is the specific detection result of H7N9 virus HA gene and NA gene RT-LAMP detection system.A is HA gene specific detected result, and B is NA gene specific detected result.
Fig. 3 is the sensitivity detected result of H7N9 virus HA gene and NA gene RT-LAMP detection system.A is HA gene sensitivity detected result, and B is NA gene sensitivity detected result.
Embodiment
In following embodiment, method therefor is ordinary method if no special instructions, concrete steps can be referring to: " Molec μ Lar Cloning:A Laboratory Manual " (Sambrook, J., Russell, David W., Molec μ Lar Cloning:A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor).
Described percentage concentration is mass/volume (g/100ml of W/V， unit) percentage concentration or volume/volume (V/V) percentage concentration if no special instructions.
The approach that obtains of the various biomaterials that are described in embodiment is only to provide approach that a kind of experiment obtains to reach concrete disclosed object, should not become the restriction to biological material source of the present invention.In fact, the source of biomaterial used is widely, any keep on the right side of the law and the moral ethics biomaterial that can obtain can be replaced and use according to the prompting in embodiment.
The primer is synthetic by the Shanghai biological company limited of raw work.
Embodiment implements take technical solution of the present invention under prerequisite, has provided detailed embodiment and concrete operating process, and embodiment will contribute to understand the present invention, but protection scope of the present invention is not limited to following embodiment.
The present invention, for detection of the preparation method of the RT-LAMP test kit of H7N9 virus HA gene and NA gene, comprises the following steps:
1) from gene data library searching, obtain HA and NA gene order, by BLAST software, carry out homology analysis, obtain the specific conservative target sequence of HA and NA gene;
2) according to the conservative target sequence of step 1) gained, design RT-LAMP primer;
3) according to step 2) the RT-LAMP primer of gained configuration RT-LAMP reaction solution, form detection system.
First from U.S. gene database GenBank retrieval, obtain H7N9 virus HA gene (No. GenBank: KC885956.1) and NA gene (No. GenBank: KC885958.1) sequence, by BLAST software, carry out homology analysis, find specific conservative target sequence, the nucleotide sequence of the conservative target sequence of HA gene specific is as shown in sequence in sequence table 13, the nucleotide sequence of the conservative target sequence of NA gene specific is as shown in sequence in sequence table 14, according to conservative target sequence, with LAMP primer-design software Primer design V4 (http://primerexplorer.jp/e/), carry out RT-LAMP design of primers again.
Specifically, the present invention, for detection of the specificity RT-LAMP combination of primers of H7N9 virus HA gene and NA gene, comprises 6 primers for detection of HA gene and 6 primers for detection of NA gene, and concrete sequence is as follows:
Table 1: for detection of the specificity RT-LAMP primer of H7N9 virus HA gene and NA gene
Wherein, combination of primers for detection of HA gene, comprise two outer primer H7-11F3 and H7-11B3(holding the 89th bit base and holding the 277th bit base position sequence design from 5 ' from 5 ' for the conservative target sequence of HA gene specific), article two, inner primer H7-11FIP and H7-11BIP(guard holding the 177th bit base and holding the 302nd bit base position sequence design from 5 ' from 5 ' of target sequence for HA gene specific), and two rings primer H7-11LF and H7-11LB(holding the 130th bit base and holding the 230th bit base position sequence design from 5 ' from 5 ' for the conservative target sequence of HA gene specific),
Combination of primers for detection of NA gene, comprise two outer primer N9-37F3 and N9-37B3(holding the 1012nd bit base and holding the 1205th bit base position sequence design from 5 ' from 5 ' for the conservative target sequence of NA gene specific), article two, inner primer N9-37F3FIP and N9-37F3BIP(guard holding the 1050th bit base and holding the 1211st bit base position sequence design from 5 ' from 5 ' of target sequence for NA gene specific), and two rings primer N9-37F3LF and N9-37F3LB(holding the 1058th bit base and holding the 1144th bit base position sequence design from 5 ' from 5 ' for the conservative target sequence of NA gene specific).
While detecting HA or NA gene, 6 primers of correspondence need be mixed to use, blending ratio is outer primer: inner primer: ring primer is 5:40:20 in molar ratio.Each primer consumption in the detection system of 25 μ L, two outer primer F3, B3 are respectively 0.1 μ L, and two inner primer FIP, BIP are respectively 0.8 μ L, and two ring primer LF, LB are respectively 0.4 μ L.
The foundation of the RT-LAMP detection method of embodiment 2, H7N9 virus HA gene and NA gene
1, extract the RNA of testing sample
With QIAamp viral RNA mini kit (Qiagen, Hilden, Germany) commercialization RNA, extract test kit and extract the nucleic acid in testing sample, concrete extracting method comprises the following steps:
1.1 draw the ready buffer AVL(of 560 μ L contains carrier RNA, refers in QIAamp viral RNA mini kit commercialization RNA and extracts test kit specification sheets) in 1.5mL centrifuge tube.Actual amount is per sample adjusted in proportion buffer AVL-carrier RNA(and is referred in QIAamp viral RNA mini kit commercialization RNA extraction test kit specification sheets)
1.2 join the testing samples such as 140 μ L blood plasma, serum, urine, culturing cell supernatant liquor or acellular body fluid in the centrifuge tube that buffer AVL-carrier RNA is housed, and mediate 15 seconds, mix.
1.3 room temperatures are placed 10min.
1.4 is instantaneous centrifugal, by lid and tube wall on drop get rid of return pipe at the bottom of.
1.5 directly add 560 μ L dehydrated alcohols (96%-100%) in upper step pipe, and mediation 15s fully mixes, then instantaneous centrifugal, at the bottom of the drop on lid is got rid of to return pipe.
1.6 draw careful the adding in adsorption column (column has been encased in 2mL centrifuge tube) of solution in step on 630 μ L, note not encountering the edge of pillar, cover lid, 6000 * g(8000rpm) centrifugal 1min, puts into new 2mL centrifuge tube by column, discards old centrifuge tube.
The 1.7 careful lids of opening column, repeat the 1.6th step.
The 1.8 careful column lids of opening, add 500 μ L buffer AW1(to refer in QIAamp viral RNA mini kit commercialization RNA and extract test kit specification sheets), cover lid, 8000rpm is centrifugal, 1min.Column is put into new 2mL collection tube (Kit provides), discard old collection tube.
The 1.9 careful column lids of opening, add 500 μ L buffer AW2(to refer in QIAamp viral RNA mini kit commercialization RNA and extract test kit specification sheets), cover lid, at full speed centrifugal (14000rpm) 3min.
1.10 column are provided in 1.5mL centrifuge tube (not providing in kit), discard old centrifuge tube, and the careful column that opens adds the buffer AVE of 60 μ L room temperatures.Cover lid, room temperature is placed 1min, the centrifugal 1min of 8000rpm, in centrifuge tube, liquid is the RNA of the testing sample of extraction.
2, RT-LAMP reaction conditions
Respectively in different temperature (45 ℃, 46.8 ℃, 49.9,54.3 ℃, 60.3 ℃, 65 ℃, 68.1 ℃, 70 ℃) isothermal reactions (15min, 25min, 35min, 45min, 55min, 65min, 75min), termination reaction, the final time occurring by amplification positive findings, obtain optimum response parameter.Because amplification under the condition at 60-65 ℃ of constant temperature 45min is the fastest, preferably react with this understanding.
3, RT-LAMP reaction system
At 60-65 ℃ of constant temperature 45min, the reaction system of the inner primer of different concns, outer primer and ring primer (referring to table 1) is optimized to reaction, the time occurring by amplification positive findings is as the ultimate density of reaction, two outer primer F3 of result, B3 concentration are respectively 5pmol, article two, inner primer FIP, BIP concentration are respectively 40pmol, article two, ring primer LF, LB concentration are respectively mixed under the condition of using for 20pmol, increase the fastest.Therefore the detection system, being optimized (23 μ L reaction solutions and primer+2 μ L template ribonucleic acid) is as follows:
In 25 μ L detection system, each material concentration is: 20mM Tris-HCl (pH8.8), 10mM KCl, 10mM (NH
4)
2sO
4, 0.1%Tween20,0.8M trimethyl-glycine (betaine), 8mM MgSO
4, 1.4mM dNTPs (above material is the reaction mother liquor mixing), 8U Bst archaeal dna polymerase, 8U AMV reversed transcriptive enzyme, masterplate RNA2 μ L, each 0.8 μ L of 40pmol primers F IP and BIP(), each 0.4 μ L of 20pmol primer LF and LB(), each 0.1 μ L of 5pmol primers F 3 and B3().
3, result interpretation
On the LA230 of company of Eiken Chemical model instrument, carry out isothermal amplification and record result, RT-LAMP reaction conditions is 60-65 ℃ of constant temperature 45min, and result interpretation method is as follows:
1) amplification is gone to and in reaction solution, is added 1 μ L fluorexon, can be according to the colour-change judged result of reaction solution: (A and B are twice parallel test result) as shown in Figure 1, green (No. 1-6), shows to exist in testing sample HA gene or the NA gene (result is positive) of H7N9 virus; Safran (No. 7-8), represents not exist in testing sample HA gene or the NA gene (result is negative) of H7N9 virus;
2) do not add fluorexon and directly with turbidimeter, according to turbidity, change judged result: turbidity (reduced turbidity >=0.3) rises, show to exist in testing sample HA gene or the NA gene (result is positive) of H7N9 virus; Turbidity is unchanged, shows not exist in testing sample HA gene or the NA gene (result is negative) of H7N9 virus.
The RT-LAMP of embodiment 3, H7N9 virus HA gene and NA gene detects and specificity
Take respectively negative control (distilled water), containing the solution of H7N9 positive sample, H1N1virus, seasonal H1N1 influenza virus, H3N2 influenza virus and human genome in 2009, be contrast testing sample, by the method in embodiment 2 respectively with primer (the outer primer H7-11F3& of detection HA gene; H7-11B3, inner primer H7-11FIP& H7-11BIP, ring primer H7-11LF& H7-11LB) and detect primer (the outer primer N9-37F3& of NA gene; N9-37B3, inner primer N9-37FIP& N9-37BIP, ring primer N9-37LF& N9-37LB) carry out RT-LAMP detection, to detect the specificity of detection reagent of the present invention and detection method.
As Fig. 2, (A is HA gene specific detected result to specific detection result, B is NA gene specific detected result) shown in, result shows, the specificity of RT-LAMP detection system of the present invention and detection method is good, can be in numerous influenza viruses specific HA gene and the NA gene that H7N9 virus detected, and can not detect other containing the sample of H7N9 virus HA gene and NA gene.
The sensitivity of the RT-LAMP detection method of embodiment 4, H7N9 of the present invention virus HA gene and NA gene
The H7N9 positive sample template of 10 times of gradient dilutions of take is testing sample, by the method in embodiment 2 respectively with primer (the outer primer H7-11F3& that detects HA gene; H7-11B3, inner primer H7-11FIP& H7-11BIP, ring primer H7-11LF& H7-11LB) and detect primer (the outer primer N9-37F3& of NA gene; N9-37B3, inner primer N9-37FIP& N9-37BIP, ring primer N9-37LF& N9-37LB) carry out RT-LAMP detection, (25 μ L detection system comprise: 8U Bst archaeal dna polymerase with primer B3 and F3, to carry out RT-PCR detection, 8U AMV reversed transcriptive enzyme, masterplate RNA2 μ L, each 0.8 μ L of 40pmol primers F IP and BIP, each 0.4 μ L of 20pmol primer LF and LB, each 0.1 μ L of 5pmol primers F 3 and B3, distilled water 6.9 μ L, RM12.5 μ L,, testing conditions is 55 ℃, 30 circulations) detect, to detect the sensitivity of RT-LAMP detection method of the present invention and RT-PCR detection method.
As shown in Figure 3, wherein A is HA gene sensitivity detected result to the sensitivity detected result of RT-LAMP detection method, and B is NA gene sensitivity detected result; Curve 1 is 10
6copy, curve 2 is 10
5copy, curve 3 is 10
4copy, curve 4 is 10
3copy, curve 5 is 10
2copy, curve 6 is 10
1copy.Result demonstration, H7N9 virus HA gene is identical with RT-PCR detection method with the sensitivity of NA gene RT-LAMP detection system.
The RT-LAMP detection kit of embodiment 5, H7N9 virus HA gene and/or NA gene
One of test kit provided by the present invention, the RT-LAMP detection kit for for detection of H7N9 virus HA gene, wherein comprises 6 primers for detection of HA gene shown in table 1.
Two of test kit provided by the present invention, is the RT-LAMP detection kit for detection of H7N9 virus N A gene, wherein comprises 6 primers for detection of NA gene shown in table 1.
Three of test kit provided by the present invention, for the RT-LAMP detection kit for detection of H7N9 virus HA gene and NA gene, wherein comprise shown in table 16 primers for detection of HA gene and 6 primers for detection of NA gene totally 12 RT-LAMP combination of primers that primer forms.
In all test kits, can also comprise RT-LAMP reaction solution, in use, jointly form LAMP detection system with RT-LAMP combination of primers.
Described RT-LAMP reaction solution comprises: 20mM Tris-HCl (pH8.8), 10mM KCl, 10mM (NH
4)
2sO
4, 0.1%Tween20,0.8M trimethyl-glycine (betaine), 8mM MgSO
4, 1.4mM dNTPs (above material is the reaction mother liquor mixing), 8U Bst archaeal dna polymerase, 8U AMV reversed transcriptive enzyme.
RT-LAMP reaction solution and described combination of primers form LAMP detection system jointly, wherein contain 40pmol primers F IP and BIP, 20pmol primer LF and LB, 5pmol primers F 3 and B3.
In test kit, reagent code name is as follows:
Claims (10)
1. for detection of the specificity RT-LAMP combination of primers of H7N9 virus HA gene and NA gene, according to conservative target sequence, design, the preparation method of conservative target sequence is: first from U.S. gene database GenBank retrieval, obtain H7N9 virus HA gene (No. GenBank: KC885956.1) and NA gene (No. GenBank: KC885958.1) sequence, by BLAST software, carry out homology analysis again, find specific conservative target sequence, the nucleotide sequence of the conservative target sequence of HA gene specific is as shown in sequence in sequence table 13, the nucleotide sequence of the conservative target sequence of NA gene specific is as shown in sequence in sequence table 14.
2. the specificity RT-LAMP combination of primers for detection of H7N9 virus HA gene and NA gene according to claim 1, it is characterized in that: comprise 6 primers for detection of HA gene and 6 primers for detection of NA gene, the concrete sequence of each primer is as follows:
3. the specificity RT-LAMP combination of primers for detection of H7N9 virus HA gene and NA gene according to claim 2, it is characterized in that: wherein, for detection of the combination of primers of HA gene, comprise two outer primer H7-11F3 and H7-11B3, two inner primer H7-11FIP and H7-11BIP and two ring primer H7-11LF and H7-11LB; Outer primer: inner primer: ring primer in molar ratio 5:40:20 mixes.
4. the specificity RT-LAMP combination of primers for detection of H7N9 virus HA gene and NA gene according to claim 2, it is characterized in that: wherein, for detection of the combination of primers of NA gene, comprise two outer primer N9-37F3 and N9-37B3, two inner primer N9-37F3FIP and N9-37F3BIP and two ring primer N9-37F3LF and N9-37F3LB; Outer primer: inner primer: ring primer in molar ratio 5:40:20 mixes.
5. for detection of a RT-LAMP detection kit for H7N9 virus HA gene and/or NA gene, include described in claim 2 or 3 or 4 by 6 primers for detection of HA gene and/or 6 RT-LAMP combination of primers that form for detection of the primer of NA gene.
6. test kit according to claim 5, is characterized in that: described test kit also comprises RT-LAMP reaction solution, jointly forms LAMP detection system with RT-LAMP primer.
7. test kit according to claim 6, is characterized in that: the survey system of described test kit inspection comprises: 20mM Tris-HCl (pH8.8), 10mM KCl, 10mM (NH
4)
2sO
4, 0.1%Tween20,0.8M trimethyl-glycine (betaine), 8mM MgSO
4, 1.4mM dNTPs, 8U Bst archaeal dna polymerase, 8U AMV reversed transcriptive enzyme, 40pmol primers F IP and BIP, 20pmol primer LF and LB, 5pmol primers F 3 and B3.
8. with RT-LAMP test kit described in claim 5 or 6 or 7, detect a method for H7N9 virus HA and NA gene, comprise the following steps:
1) prepare 23 μ L RT-LAMP reaction solutions, while detecting HA gene, use outer primer H7-11F3& H7-11B3, inner primer H7-11FIP& H7-11BIP and ring primer H7-11LF& H7-11LB, uses outer primer N9-37F3& while detecting NA gene; N9-37B3, inner primer N9-37FIP& N9-37BIP and ring primer N9-37LF& N9-37LB;
2) extract according to a conventional method the nucleic acid in testing sample;
3) get 2 μ L nucleic acid extraction liquid and add step 1) preparation RT-LAMP reaction solution in, making end reaction volume is 25 μ L, mixes reaction solution;
4) put 60-65 ℃ of constant temperature 45min and carry out RT-LAMP amplification;
5) carry out result interpretation.
9. method according to claim 8, described step 5) result interpretation process is: amplification is gone to and in reaction solution, added 1 μ L fluorexon, according to the colour-change judged result of reaction solution: green, shows to exist in testing sample HA gene or the NA gene (result is positive) of H7N9 virus; Safran, represents not exist in testing sample HA gene or the NA gene (result is negative) of H7N9 virus.
10. method according to claim 8, described step 5) result interpretation process is: do not add fluorexon and directly with turbidimeter, according to turbidity, change judged result: turbidity (reduced turbidity >=0.3) rises, and shows to exist in testing sample HA gene or the NA gene (result is positive) of H7N9 virus; Turbidity is unchanged, shows not exist in testing sample HA gene or the NA gene (result is negative) of H7N9 virus.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104099430A (en) * | 2014-08-01 | 2014-10-15 | 中国检验检疫科学研究院 | Ring mediate isothermal amplification kit for diagnosing H7N9 hypotype avian influenza virus and application method thereof |
| CN104232622A (en) * | 2014-09-24 | 2014-12-24 | 中国人民解放军疾病预防控制所 | Nucleic acid isothermal amplification method and application thereof by polymerase spiral reaction |
| CN105779645A (en) * | 2014-12-22 | 2016-07-20 | 中国人民解放军疾病预防控制所 | LAMP kit for detecting Ebola virus and dedicated primer thereof |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110551850A (en) * | 2019-09-02 | 2019-12-10 | 拱北海关技术中心 | RT-LAMP primer and method for detecting highly pathogenic H7N9 avian influenza virus |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103290144A (en) * | 2013-05-29 | 2013-09-11 | 江苏省疾病预防控制中心 | Primer for detecting H7N9 subtype avian influenza virus infected by humans though RT-LAMP (reverse transcription loop-mediated isothermal amplification) and application of primer |
| 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 |
| CN103320532A (en) * | 2013-06-09 | 2013-09-25 | 中国人民解放军第四军医大学 | Rapid detection primer group of H7N7 subtype avian influenza virus and use thereof |
| CN103320544A (en) * | 2013-07-12 | 2013-09-25 | 中国人民解放军南京军区军事医学研究所 | Primer, kit and detection method for detecting avian influenza H7N9 virus by using RT-LAMP method |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6015664A (en) * | 1995-11-03 | 2000-01-18 | Mcw Research Foundation | Multiplex PCR assay using unequal primer concentrations to detect HPIV 1,2,3 and RSV A,B and influenza virus A, B |
-
2013
- 2013-10-15 CN CN201610437862.XA patent/CN105861758A/en active Pending
- 2013-10-15 CN CN201310481406.1A patent/CN103525949A/en active Pending
Patent Citations (4)
| 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 |
| CN103290144A (en) * | 2013-05-29 | 2013-09-11 | 江苏省疾病预防控制中心 | Primer for detecting H7N9 subtype avian influenza virus infected by humans though RT-LAMP (reverse transcription loop-mediated isothermal amplification) and application of primer |
| CN103320532A (en) * | 2013-06-09 | 2013-09-25 | 中国人民解放军第四军医大学 | Rapid detection primer group of H7N7 subtype avian influenza virus and use thereof |
| CN103320544A (en) * | 2013-07-12 | 2013-09-25 | 中国人民解放军南京军区军事医学研究所 | Primer, kit and detection method for detecting avian influenza H7N9 virus by using RT-LAMP method |
Non-Patent Citations (3)
| Title |
|---|
| JINHAI ZHANG等: "Rapid and Sensitive Detection of H7N9 Avian Influenza Virus by Use of Reverse Transcription–Loop-Mediated Isothermal Amplification", 《J. CLIN. MICROBIOL》 * |
| YIYUE GE等: "Rapid and Sensitive Detection of Novel Avian-OriginInfluenza A (H7N9) Virus by Reverse Transcription Loop- Mediated Isothermal Amplification Combined with a Lateral-Flow Device", 《PLOS ONE》 * |
| 国立感染症研究所インフルエンザウイルス研究センター: "鳥インフルエンザA(H7N9)ウイルス", 《検出マニュアル(第2 版)》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104099430A (en) * | 2014-08-01 | 2014-10-15 | 中国检验检疫科学研究院 | Ring mediate isothermal amplification kit for diagnosing H7N9 hypotype avian influenza virus and application method thereof |
| CN104232622A (en) * | 2014-09-24 | 2014-12-24 | 中国人民解放军疾病预防控制所 | Nucleic acid isothermal amplification method and application thereof by polymerase spiral reaction |
| CN105779645A (en) * | 2014-12-22 | 2016-07-20 | 中国人民解放军疾病预防控制所 | LAMP kit for detecting Ebola virus and dedicated primer thereof |
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