CN103834747A - Method for detecting pathogenicity of influenza A (H1N1) virus based on pyrosequencing - Google Patents
Method for detecting pathogenicity of influenza A (H1N1) virus based on pyrosequencing Download PDFInfo
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Abstract
The invention relates to a method for detecting the pathogenicity of an influenza A (H1N1) virus based on pyrosequencing. The method comprises the steps of carrying out RT-PCR (Reverse Transcription-Polymerase Chain Reaction) amplification on a hemagglutinin (HA) gene of an H1N1 virus; carrying out pyrosequencing on a PCR amplification product to judge whether the cleavage site of the HA gene of the influenza A (H1N1) virus has a mutation, wherein the pyrosequencing is carried out in an SQA (Sequence Analysis) mode, and a nucleotide sampling sequence is shown as AGCT. The mutation of the cleavage site of the HA gene of the influenza A (H1N1) virus can be detected at rather high accuracy through comparing a detected result with the sequence of the cleavage site of the HA gene of a standard strain of the influenza A/H1N1 virus. The invention provides a simple and rapid experimental scheme for determining the virulence, pathogenicity and host range of the virus, the complex experiment steps related to complete genome sequencing are omitted, the variation direction of the virus can also be accurately mastered, the virus is conveniently monitored, an infection source is isolated, a transmission route is cut off, and the further development of an epidemic situation is stopped.
Description
Technical field
The present invention relates to a kind of method that detects H1N1virus virulence, be specifically related to a kind of method of the detection H1N1virus virulence based on tetra-sodium order-checking, belong to virus detection techniques field.
Background technology
The Influenza A H1N1 of in March, 2009 outburst is another great public health event after SARS, high pathogenic avian influenza, has caused huge fear to the people of other countries.Influenza A virus is the segmented RNA viruses of sub-thread minus strand, easily there is antigenic drift, antigen conversion and gene recombination, especially sudden change and the restructuring of the outer membrane protein hemagglutinin (HA) of virus, can form various mutant strains or recombinant strain, cause viral virulence and the variation of host range, thereby cause influenza pandemic in various degree.Can hemagglutinin (HA) be cut into HA1 and HA2 determines the pathogenic important factor of influenza virus, the variation of cracking site can cause the variation of pathogenicity, the sequence in analytical pyrolysis site, study its evolution laws can let us more in depth understand H1N1 pathogenic, virulence is big or small, host range.
The aminoacid sequence at cracking site place is one of factor determining influenza virus virulence.General highly pathogenic influenza virus is having continuous more than 4 basic aminoacids (RRRR) near cracking site, and the HA cracking site place of low pathogenicity influenza virus generally just only has single basic aminoacids (R).The multiple continuous basic aminoacidss in HA cracking site place of highly pathogenic influenza virus, can be by the cracking of multiple protein enzyme institute, thus virus can be in host's different tissues cell growth and breeding, cause host's systemic infection, exhaustion and cause death.And the HA protein cleavage site of low pathogenicity influenza virus can only be by the proteolytic enzyme institute cracking in the minority histocytes such as respiratory tract, thereby can only in host's respiratory tract, breed, cause temperate infection.The sequence signature of the cracking site of HA albumen is to viral host range, tissue tropism and pathogenicly have a critical impact, is the prerequisite that virus is propagated in body].Low pathogenicity influenza virus likely strengthens virulence by the transgenation of the multiple basic aminoacids in the connection peptides between HeHA2 district of HA1 district, becomes highly pathogenic virus strain, and therefore, cracking site sequential analysis and the monitoring of HA are extremely important
In order to strengthen the control to H1N1 fast propagation and to avoid its eruption and prevalence, developing quick, reliable high-throughout detection virus mutation technology is the focus of research,, there is shortcoming time-consuming, effort in the sudden change of detection virogene in traditional full-automatic DNA sequencing technology based on Sanger method.Tetra-sodium sequencing technologies is short segment DNA gene sequence analysis of new generation, can carry out fast, accurately and real-time short dna sequential analysis, detection flux is high, simple to operation, programmable, the research that utilizes tetra-sodium sequencing technologies to detect the cracking site of Influenza A H1N1 HA gene has no report.
Summary of the invention
The object of the invention is to provide a kind of method of the detection H1N1virus virulence based on tetra-sodium order-checking, it uses tetra-sodium sequence measurement to detect the sequence method of H1N1virus hemagglutinin (HA) cracking site, whether the cracking site that judges hemagglutinin (HA) undergos mutation, cause the variation of viral virulence, thereby cause influenza pandemic in various degree.
Can determine the pathogenic important factor of influenza virus owing to hemagglutinin (HA) be cut into HA1 and HA2, the variation of cracking site can cause the variation of pathogenicity, and therefore technical conceive of the present invention is to carry out tetra-sodium order-checking for H1N1virus cracking site to detect.The method that detection type H1N1 influenza virus virulence of the present invention changes, first adopt fragment (comprising cracking site sequence) in RT-PCR amplification H1N1 virus hemagglutinin (HA) gene, with tetra-sodium sequencing, amplified fragments is accurately checked order subsequently, thereby set up one cracking site sudden change fast and effectively detection method.For definite virus virulence, pathogenic and host range provides simply, experimental program efficiently, avoid the related loaded down with trivial details experimental procedure of full gene sequencing, can also accurately grasp viral variation direction, conveniently it is monitored, isolation contagium cuts off route of transmission, stops the further expansion of epidemic situation.
Cardinal principle of the present invention is:
Tetra-sodium sequencing technologies
1) tetra-sodium order-checking (Pyrosequencing) technology is brand-new a kind of DNA sequence analysis technology, can measure quickly and accurately one section of shorter target fragment.This technology need not be carried out electrophoresis, and DNA fragmentation also need not fluorescent mark, operates very easy.
2) tetra-sodium sequencing technologies is by the enzyme cascade chemiluminescence reaction in 4 kinds of enzymatic same reaction systems, ultimate principle is as follows: by 1 specific sequencing primer and the combination of single stranded DNA template, then add enzyme mixture (comprising DNA Polymerase, ATP Sulfurylase, Luciferase and Apyrase) and substrate mixture (comprising APS and Luciferin).In reaction system, add a kind of dNTP, if it can just with the next base pairing of DNA profiling, can be under the effect of archaeal dna polymerase, add 3 ' end of sequencing primer to, discharge the tetra-sodium group of mole number simultaneously.Under the effect of ATP sulfurylase, the PPi of generation can be with APS in conjunction with forming ATP; Under the catalysis of luciferase, the ATP of generation can, with fluorescein in conjunction with forming oxyluciferin, produce visible ray again simultaneously.Can obtain a special detected peaks by CCD optical system, the height of peak value is directly proportional with the base number matching.In reaction system, remaining dNTP and residual a small amount of ATP degrade under the effect of Apyrase.Add another kind of dNTP, 2nd～4 step reactions are repeated, can read DNA sequence dna information accurately according to the peak value figure obtaining.The height of each peak value with react in the Nucleotide number that mixes be directly proportional.Then add lower a kind of dNTP, continue the synthetic of DNA chain.
3) in tetra-sodium order-checking operating system, have 2 patterns and can carry out the detection of base mutation, i.e. sequential analysis pattern (Sequence Analysis, SQA) and single nucleotide polymorphism pattern (Single Nucleotide polymorphism, SNP).Wherein the Nucleotide application of sample of SQA pattern order is circular order application of sample method, by circulation repeatedly add A, G, C, tetra-kinds of Nucleotide of T to detect the sequence of sample to be checked, the once accurate base of about interpretation 40bp of this method, the interpretation situation of suddenling change voluntarily after measured sequence and known array are compared.The Nucleotide application of sample order of SNP pattern is particular order application of sample method, and system, according to the application of sample order of the site to be checked of setting and the site automatic product nucleus thuja acid of sequence around, only changes and detects the base of Single locus, whole sequence is not measured.For different detected objects, need to grope different tetra-sodium order-checking detection methods.
The method of the detection H1N1virus virulence based on tetra-sodium order-checking of the present invention, is characterized in that comprising the following steps:
Step 1, carries out RT-PCR amplification to H1N1 virus hemagglutinin (HA) gene;
Step 2, carries out tetra-sodium order-checking to pcr amplification product, judges whether the cracking site of Influenza A H1N1 HA gene has sudden change; Wherein tetra-sodium order-checking employing SQA pattern, Nucleotide application of sample order are AGCT.
In above-mentioned steps 1, H1N1 virus hemagglutinin (HA) gene is carried out to twice PCR amplification, wherein RT-PCR increases take sample H1N1virus RNA extract as template for the first time, increases for the second time take RT-PCR amplified production for the first time as template.
The above-mentioned for the first time PCR primer that amplification is used has sequence shown in H1F and H1R, primer amplification fragment length 950bp, and comprise cracking site sequence,
The one PCR primer sequence is as follows: H1F:ACGTGTTACCCAGGAGATTTC, H1R:TCTTTACCYACTRCTGTGAA
For the cracking site design tetra-sodium sequencing primer of Influenza A H1N1 HA gene;
The above-mentioned for the second time PCR primer that amplification is used has with biotin labeling, and this primer is high conservative and unique in H1N1virus hemagglutinin (HA) gene order; Used amplification is used for the second time PCR primer sequence and sequencing primer sequence refer to table 1
Table 1.HA gene cracking site sequencing primer and secondary amplification are used PCR primer
The concentration of the above-mentioned for the first time PCR primer that amplification is used and the PCR primer that amplification is used is for the second time 50nml/L.
In above-mentioned steps 1, RT-PCR amplified reaction process comprises the following steps:
Take sample H1N1virus RNA extract as template, adopt single stage method to carry out RT-PCR amplification, the HA1 gene fragment of amplification 950bp H1N1virus, RT-PCR reaction conditions is: 50 ℃ of reverse transcription 30min; 94 ℃ of denaturation 2min; 94 ℃ of 15s, 55 ℃ of 30s, 68 ℃ of 1min, 45 circulations; 68 ℃ of 5min.Get 5 μ l PCR products and carry out agarose gel electrophoresis identification and detection amplification.
By assay is compared with the HA gene cracking site sequence of H1N1virus type strain, the sudden change of the HA gene cracking site that detects H1N1virus that can pin-point accuracy can be conveniently applied among detection in clinical study.
The HA1 gene cracking site type strain sequence of H1N1virus type strain is in table 2
Composition and the position of table 2. H1N1virus type strain cracking site amino acid and gene
2. pair pcr amplification product carries out tetra-sodium order-checking, judges whether the cracking site of Influenza A H1N1 HA gene has sudden change; Wherein tetra-sodium order-checking adopts SQA pattern, Nucleotide application of sample order to be AGCT, specific as follows:
(1) take turns pcr amplification product by second and be fixed on magnetic bead,
(2) magnetic bead is adsorbed on vacuum sample translator, again vacuum sample translator is put into the slight vibration of 70% ethanol 5 seconds, then move on in sex change damping fluid and aspirate 5 seconds, make DNA sex change to obtain the single stranded DNA template of purifying, finally move on in lavation buffer solution and clean 5～10 seconds, wash loose single stranded DNA, thereby obtain the single stranded DNA as sequencing template
(3) primer hybridization: first add annealing buffer 50 μ L in the tetra-sodium order-checking each hole of microwell plate, add again sequence sequencing primer, the minimum requirements concentration of sequencing primer is 1 μ mol/L, maximum concentration is 0.2mmol/L, then vacuum sample translator is moved into tetra-sodium order-checking microwell plate from PCR plate, closes vacuum pump, by mixed to the single stranded DNA template that purifying is good and sequence order-checking thing primer, at 80 ℃, sex change 2 minutes, is then cooled to room temperature, makes primer and template annealing hybridization;
The sequence of sequencing primer: 5-GTTACTTTGGC CGTT-3
(4) tetra-sodium order-checking: utilize sequenator and test kit, add successively enzyme, substrate and 4 kinds of dNTPs in agent bin, select SQA detecting pattern, carry out sequencing reaction with the base addition sequence of A, G, C, T sequential loop application of sample 16 times; By CCD optical system, each application of sample after product is detected, to obtain special detected peaks, can read DNA sequence dna information accurately according to the peak value figure obtaining,
(5) tetra-sodium is checked order assay is compared with the HA1 gene cracking site sequence of H1N1virus type strain, and then judges whether the cracking site of Influenza A H1N1 HA gene has sudden change.
The HA1 gene cracking site that utilizes tetra-sodium sequencing technologies to carry out H1N1virus detects has unique advantage compared with other molecular detecting methods, for definite virus virulence, pathogenic and host range provides simply, experimental program efficiently, avoid the related loaded down with trivial details experimental procedure of full gene sequencing, can also accurately grasp viral variation direction, conveniently it is monitored, isolation contagium cuts off route of transmission, stops the further expansion of epidemic situation.
Accompanying drawing explanation
Fig. 1 is the HA gene fragment RT-PCR augmentation detection result of the present invention to H1N1virus.
Wherein, M swimming lane: DNA mark, other swimming lanes: HA gene fragment RT-PCR product
Fig. 2 is the HA gene cracking site tetra-sodium result with SQA pattern order-checking H1N1virus.
Embodiment
By the following examples content of the present invention is described in further detail.
1, RNA extracts
The template that the MDCK passage cell infecting with fresh propagative viruses extracts as RNA, adopt the HighPure Viral RNA Kit that Roche company produces to extract viral RNA, concrete operations are undertaken by test kit specification sheets, and the RNA of extraction increases for RT-PCR immediately or puts-80 ℃ of preservations.
2, RT-PCR reaction amplification HA1 gene fragment
With thering is the PCR primer of sequence shown in H1F and having with the PCR primer of sequence shown in H1R and carry out first round amplification, take the viral RNA that extracts as template.In 50 μ L reaction systems, the concentration of first primer the best is 50nmol/L.RT-PCR reaction conditions is: 50 ℃ of reverse transcription 30min; 94 ℃ of denaturation 2min; 94 ℃ of 15s, 55 ℃ of 30s, 68 ℃ of 1min, 45 circulations; 68 ℃ of 5min.Get 5 μ l PCR products and carry out agarose gel electrophoresis identification and detection amplification.
3, use and carry out second with the Reverse PCR primer of vitamin H (Biotin) mark and Forward PCR primer and take turns amplification, template is the PCR product of first round amplification.In 50 μ L reaction systems, the concentration of second primer the best is 50nmol/L.PCR reaction process is as follows:
First at 94 ℃, carry out preheating 10 minutes; At 94 ℃, keep carrying out for 45 seconds keeping annealing for 45 seconds at sex change, 64 ℃, keep increasing and extending for 90 seconds at 72 ℃, cycle annealing process 30 times; Finally at 72 ℃, keep 10 minutes.
The PCR product of getting 2 μ L carries out 2% agarose gel electrophoresis detection, electrophoresis 20 minutes under 12V/cm potential gradient, after under ultraviolet lamp, check expanding effect.Fig. 1 is the HA1 gene fragment RT-PCR product electrophorogram to H1N1virus, and wherein M is index zone, 1～8 HA gene fragment RT-PCR product that is H1N1virus.In figure, can see and have a band that single signal is stronger, and there is no remaining primer, also there is no primer dimer or other non-specific bands.
PCR second primer with sequence shown in the first primer of sequence shown in H1F and Forward primer is two forward primers.PCR first primer with sequence shown in H1R is two reverse primers with PCR second primer with sequence shown in Reverse primer, plays mark effect with biotin labeled primer in detection.
Tetra-sodium order-checking
1. detection reagent preparation
Binding buffer liquid: by 10mmol/L Tris-HCl, 2mol/L NaCl, 1mmol/L EDTA and 0.1%Tween20 deionized water dissolving, with 1mol/L HCl tune pH to 7.6.
Sex change damping fluid (denatureation buffer): 0.5mol/L NaOH.
Annealing buffer: 20mmol/L Tris-Acetate, 2mmol/L MgAc2 solution are mixed, with 4mol/L acetic acid tune pH to 7.6.
Lavation buffer solution (washing buffer): use 4mol/L acetic acid that 10mmol/L Tris-Acetate solution is adjusted to pH to 7.6.70% ethanol: add high purity water 20mL in 70mL dehydrated alcohol, be settled to 100mL after fully mixing.
2. detecting instrument and matched reagent
(1) the full-automatic tetra-sodium sequenator of PyroMark Q96ID, the strand of German QIAGEN company are prepared tool table (Vacuumprepworkstation), vacuum sample translator (Vacuum prep tool), vacuum pump, vortex vibrator, PCR instrument.
(2) the coated magnetic bead (Streptavidin Sepharose High Performance, Cat.No.17-5113-01) of streptavidin of the pyrosequencing test kit (PSQ96MA SQA Upgrade Kit) that German QIAGEN company produces and the production of GE Healthcare company of the U.S..
3. tetra-sodium order-checking detecting step
(1) PCR product is fixed: in PCR plate, put into the pcr amplification product that has of 40 μ L, then add respectively binding buffer liquid and the coated magnetic bead of 3 μ L streptavidin of 37 μ L.PCR plate is placed on to normal temperature on vortex vibrator and vibrates 20 minutes, PCR product is fixed on magnetic bead.
(2) purifying of single-stranded template: open vacuum pump, vacuum sample translator is moved on in PCR plate, capture the magnetic bead in conjunction with PCR product, check whether most of magnetic bead has all been attracted on vacuum sample translator.Again vacuum sample translator is put into the slight vibration of 70% ethanol 5 seconds, then move on in sex change damping fluid and aspirate 5 seconds, make DNA sex change to obtain the single stranded DNA template of purifying, finally move on in lavation buffer solution and clean 5～10 seconds, wash loose single stranded DNA, thereby obtain the single stranded DNA as sequencing template.
(3) primer hybridization: first add annealing buffer 50 μ L in the tetra-sodium order-checking each hole of microwell plate, then add and there is the sequencing primer of sequence shown in table 1.The concentration of sequencing primer is 20nmol/L.Again vacuum sample translator is moved into tetra-sodium order-checking microwell plate from PCR plate, close vacuum pump, by mixed to the single stranded DNA template that purifying is good and sequence order-checking thing primer, at 80 ℃, sex change 2 minutes, is then cooled to room temperature, makes primer and template annealing hybridization.
The sequence of sequencing primer: 5-GTTACTTTGGC CGTT-3
Tetra-sodium order-checking: utilize PyroMark Q96ID sequenator and test kit by German QIAGEN company, in agent bin, add successively enzyme, substrate and 4 kinds of dNTPs, select SQA detecting pattern, carry out sequencing reaction with the base addition sequence of A, G, C, T sequential loop application of sample 16 times.By CCD optical system, each application of sample after product is detected, to obtain special detected peaks, can read DNA sequence dna information accurately according to the peak value figure obtaining.
Fig. 2 is the HA gene cracking site tetra-sodium sequencing result that adopts SQA pattern H1N1virus, and detecting the sequence drawing is aggaatgttcc gtct attcaatctagaggc ctatttggg.It,, compared with the HA gene cracking site sequence of H1N1virus type strain, can be shown to whether cracking site gene order suddenlys change.
Claims (3)
1. the method for the detection H1N1virus virulence based on tetra-sodium order-checking, is characterized in that comprising the following steps:
1. pair H1N1 virus hemagglutinin (HA) gene carries out RT-PCR amplification;
1.1 use have the PCR primer of sequence shown in H1F and have and the PCR primer of sequence shown in H1R, and H1N1 virus hemagglutinin (HA) gene is carried out to first round amplification, take the viral RNA that extracts as template; RT-PCR reaction conditions is: 50 ℃ of reverse transcription 30min; 94 ℃ of denaturation 2min; 94 ℃ of 15s, 55 ℃ of 30s, 68 ℃ of 1min, 45 circulations; 68 ℃ of 5min;
First round amplification PCR primer sequence is as follows:
H1F:ACGTGTTACCCAGGAGATTTC
H1R:TCTTTACCYACTRCTGTGAA
1.2 use are carried out second with the Reverse PCR primer of vitamin H (Biotin) mark and Forward PCR primer and are taken turns amplification, and template is the PCR product of first round amplification; PCR reaction process is as follows:
First at 94 ℃, carry out preheating 10 minutes; At 94 ℃, keep carrying out for 45 seconds keeping annealing for 45 seconds at sex change, 64 ℃, keep increasing and extending for 90 seconds at 72 ℃, cycle annealing process 30 times; Finally at 72 ℃, keep 10 minutes.
Second takes turns amplification PCR primer
Reverse primer 5`-GGTTCCCAC GATATTTGTGGT-3
Forward primer 5 '-AAAAGGTCCACC AACTTGAGAAAT-3-
2. pair pcr amplification product carries out tetra-sodium order-checking, judges whether the cracking site of Influenza A H1N1 HA gene has sudden change; Wherein tetra-sodium order-checking adopts SQA pattern, Nucleotide application of sample order to be AGCT, specific as follows:
(1) take turns pcr amplification product by second and be fixed on magnetic bead,
(2) magnetic bead is adsorbed on vacuum sample translator, again vacuum sample translator is put into the slight vibration of 70% ethanol 5 seconds, then move on in sex change damping fluid and aspirate 5 seconds, make DNA sex change to obtain the single stranded DNA template of purifying, finally move on in lavation buffer solution and clean 5～10 seconds, wash loose single stranded DNA, thereby obtain the single stranded DNA as sequencing template
(3) primer hybridization: first add annealing buffer 50 μ L in the tetra-sodium order-checking each hole of microwell plate, add again sequence sequencing primer, the minimum requirements concentration of sequencing primer is 1 μ mol/L, maximum concentration is 0.2mmol/L, then vacuum sample translator is moved into tetra-sodium order-checking microwell plate from PCR plate, closes vacuum pump, by mixed to the single stranded DNA template that purifying is good and sequence order-checking thing primer, at 80 ℃, sex change 2 minutes, is then cooled to room temperature, makes primer and template annealing hybridization;
The sequence of sequencing primer: 5-GTTACTTTGGC CGTT-3
(4) tetra-sodium order-checking: utilize sequenator and test kit, add successively enzyme, substrate and 4 kinds of dNTPs in agent bin, select SQA detecting pattern, carry out sequencing reaction with the base addition sequence of A, G, C, T sequential loop application of sample 16 times; By CCD optical system, each application of sample after product is detected, to obtain special detected peaks, can read DNA sequence dna information accurately according to the peak value figure obtaining,
(5) tetra-sodium is checked order assay is compared with the HA1 gene cracking site sequence of H1N1virus type strain, and then judges whether the cracking site of Influenza A H1N1 HA gene has sudden change.
2. the method for the detection H1N1virus virulence based on tetra-sodium order-checking as claimed in claim 1, is characterized in that in above-mentioned steps 1, and in 50 μ L reaction systems, the concentration of first primer the best is 50nmol/L.
3. the method for the detection H1N1virus virulence based on tetra-sodium order-checking as claimed in claim 1, is characterized in that in above-mentioned steps 1, and in 50 μ L reaction systems, the concentration of second primer the best is 50nmol/L.
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| CN106282408A (en) * | 2016-08-18 | 2017-01-04 | 中国农业大学 | A kind of Manganic pyrophosphate complex initiation detects the method for clade2.1.3 branch H5N1 subtype avian influenza virus |
| CN107881224A (en) * | 2017-11-22 | 2018-04-06 | 凯杰（苏州）转化医学研究有限公司 | A kind of kit and its detection method of detection HER3 mutation |
| US11535901B2 (en) * | 2017-09-26 | 2022-12-27 | Yonsei University Industry Foundation (Yonsei Uif) | Probe for detecting and treating virus |
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| CN109266784B (en) * | 2018-07-27 | 2022-04-08 | 广东海洋大学 | Closed DNA fluorescent biosensor and application thereof in detection of influenza A H1N1 virus |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101845515A (en) * | 2009-12-04 | 2010-09-29 | 山东出入境检验检疫局检验检疫技术中心 | Method for detection of swine influenza A H1N1 virus based on pyrosequencing technology |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101845515A (en) * | 2009-12-04 | 2010-09-29 | 山东出入境检验检疫局检验检疫技术中心 | Method for detection of swine influenza A H1N1 virus based on pyrosequencing technology |
Non-Patent Citations (2)
| Title |
|---|
| ELISABETH NEUMEIER等: "Nucleotide sequence analysis of the HA1 coding portion of the hemagglutinin gene of swine H1N1 influenza viruses", 《VIRUS RESEARCH》, vol. 23, 31 December 1992 (1992-12-31), pages 107 - 117 * |
| 陈晓光等: "焦磷酸测序技术在检测甲型H1N1血凝素基因裂解位点突变中的应用", 《中国国境卫生检疫杂志》, vol. 37, no. 3, 30 June 2014 (2014-06-30) * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106282408A (en) * | 2016-08-18 | 2017-01-04 | 中国农业大学 | A kind of Manganic pyrophosphate complex initiation detects the method for clade2.1.3 branch H5N1 subtype avian influenza virus |
| CN106282408B (en) * | 2016-08-18 | 2019-12-24 | 中国农业大学 | Method for detecting clade2.1.3 branch H5N1 subtype avian influenza virus by pyrosequencing |
| US11535901B2 (en) * | 2017-09-26 | 2022-12-27 | Yonsei University Industry Foundation (Yonsei Uif) | Probe for detecting and treating virus |
| CN107881224A (en) * | 2017-11-22 | 2018-04-06 | 凯杰（苏州）转化医学研究有限公司 | A kind of kit and its detection method of detection HER3 mutation |
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| CN103834747B (en) | 2016-05-18 |
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