CN101845515A - Method for detection of swine influenza A H1N1 virus based on pyrosequencing technology - Google Patents
Method for detection of swine influenza A H1N1 virus based on pyrosequencing technology Download PDFInfo
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Abstract
The invention discloses a method for the detection of swine influenza A H1N1 virus based on pyrosequencing technology, belonging to the technical field of animal pathogen detection. The method comprises the steps: firstly designing specific primers and pyrosequencing primers of the swine influenza A H1N1 virus; extracting RNA of a sample to be detected and then amplifying respectively by H1 and N1 primers of the swine influenza A H1N1 virus; detecting the amplified product by agarose gel electrophoresis preparing a pyrosequencing single-chain template in case that the length of H1 primer-amplified fragment is 102bp and the length of N1 primer-amplified fragment is 249bp, implementing pyrosequencing reaction, and finally judging whether the swine influenza A H1N1 virus is contained in the sample according to the amplification result and the pyrosequencing result. The method can realize short DNA sequence analysis accurately and fast by using the pyrosequencing technology and has the characteristics of high flux, low cost, direct use of the product for sequencing, no need of secondary treatment, extremely simple and convenient operation and small number of required sample.
Description
Technical field:
The invention belongs to the detection technique field of animal pathogenic, relate to a kind of method of employing tetra-sodium order-checking (Pyrosequencing) technology for detection swine influenza A H 1 N 1 virus.
Background technology:
Swine influenza A H 1 N 1 virus is the pig acute respiratory disease that is caused by the variable A type influenza virus of a series of antigenic structures.In nearly 90 years of influenza virus invasion and attack swinery, porcine influenza has brought catastrophic loss for global pig industry, and its antigenic variation that constantly takes place makes porcine influenza be difficult to thoroughly be removed.Because pig can serve as the intermediate host of bird flu and human influenza, swine influenza virus can be propagated to the people, thereby has caused the potential threat to human health again.The pig Influenza A H1N1 epidemic situation of outburst in 2009 has not only proposed acid test to world's public health, and world economy has been produced great effect.After epidemic situation was broken out, some countries import prohibition in succession brought financial loss for two places meat product processing and exporter from the pork and the relevant meat product of epidemic situation severely afflicated areas such as Mexico, the U.S..Therefore, deeply reinforcement is very urgent to the research of porcine influenza.
At present, be used for methods such as the main virulent isolation identification of method that the porcine influenza cause of disease detects, serological diagnostic method, diagnosis of molecular biology.The isolation identification time and effort consuming of virus, the test conditions of having relatively high expectations can not satisfy the anti-system of the urgent epidemic situation of acute infectious disease requirements of one's work; Serological diagnostic method mainly comprises blood clotting and hemagglutination-inhibition test, neuraminidase inhibition test, neutralization test, agar gel diffusion test, immunofluorescence technique, enzyme linked immunosorbent assay, its method has high specificity, advantage that susceptibility is high, but method is quite loaded down with trivial details, is not suitable for a large amount of samples are detected; The diagnosis of molecular biology method mainly comprises polymerase chain reaction (PCR), real time fluorescent quantitative poly chain reaction, amplification of nucleic acid sequences experiment (NASBA) method, these methods compare faster, sensitivity, but accuracy is not enough, and cross-contamination issue is more serious.
The tetra-sodium sequencing technologies is the new technology that developed recently gets up, it is present unique detection technique that obtains quantitative sequence results, has high accuracy, and circulation ratio is splendid, it is a kind of universal technology platform, diverse in function, Application Areas is extensive, has high-throughput, characteristics cheaply, the polymerase chain reaction product can be directly used in order-checking, need not carry out secondary treatments such as product purification, operates very easy, required sample size is little, meet the requirement of inspection and quarantining for import/export,, strengthen the monitoring of influenza epidemic situation in view of the seriousness of swine influenza virus harm, improve the accuracy and the detection efficiency of detection method, effective propagation of controlling influenza virus is had important and practical meanings in present stage.
Summary of the invention:
The objective of the invention is to overcome the shortcoming that prior art exists, seek to provide a kind of tetra-sodium sequencing technologies detection method of swine influenza A H 1 N 1 virus, to overcome the deficiency of existing detection technique, for pig aquaculture, meat product processing and aspects such as exporter and international meat-based food trade provide a kind of quick, easy, efficient, practical detection method.
To achieve these goals, cardinal principle of the present invention is to utilize the tetra-sodium sequencing technologies, by sequencing primer and the hybridization of polymerase chain reaction (PCR) amplification single stranded deoxyribonucleic acid template, hatch with deoxyribonucleic acid polymerase, adenosine triphyosphate (ATP) sulfurylase, luciferase, apyrase and substrate (APS), fluorescein, add four kinds of triphosphoric acid base deoxynucleotides (dNTPs) one by one: (triphosphoric acid adenyl-deoxyribonucleotide dATP; Triphosphoric acid thymidylic acid dTTP; Triphosphoric acid deoxycytidylic acid dCTP; Triphosphoric acid guanine deoxyribonucleoside acid dGTP), as matching with template, the end of this triphosphoric acid base deoxynucleotide and primer forms covalent linkage, discharges tetra-sodium group (PPi); Adenosine triphyosphate (ATP) sulfurylase catalysis tetra-sodium under the situation that substrate (APS) exists forms adenosine triphyosphate, the fluorescein that adenosine triphyosphate drives the luciferase mediation transforms to oxyluciferin, and oxyluciferin sends the visible light signal that is directly proportional with the adenosine triphyosphate amount; The optical signals charge-coupled device (CCD) is collected and is converted into the peak by software; The peak height of each optical signal is directly proportional with the Nucleotide number that mixes in the reaction, adenosine triphyosphate and uncorporated triphosphoric acid base deoxynucleotide are degraded by apyrase, the cancellation optical signal, and regeneration reaction system, utilize optical signal to transform the peak figure that obtains, the swine influenza A H 1 N 1 virus in the sample is detected accurately.
Realization of the present invention at first designs swine influenza A H 1 N 1 virus Auele Specific Primer and tetra-sodium sequencing primer; Extract the Yeast Nucleic Acid (RNA) of testing sample again, carry out reverse transcription-polymerase chain reaction (RT-PCR) amplification with swine influenza A H 1 N 1 virus H1, N1 primer respectively then; With carrying out agarose gel electrophoresis amplified production is detected, if H1 primer amplification fragment length is 102bp, N1 primer amplification fragment length is 249bp, then prepare tetra-sodium order-checking single-stranded template, carry out the tetra-sodium sequencing reaction again, judge whether contain swine influenza A H 1 N 1 virus in the sample according to RT-PCR amplification and tetra-sodium sequencing result at last.
Design swine influenza A H 1 N 1 virus Auele Specific Primer of the present invention and tetra-sodium sequencing primer are the gene orders according to swine influenza virus, choose conserved sequence, by Assay Design SW software design swine influenza A H 1 N 1 virus specific primer sequence, to amplify the single band of specificity, design the tetra-sodium sequencing primer to determine virus subtype according to the specific nucleotide sequence that comprises in the amplification region (target sequence) again; Swine influenza A H 1 N 1 virus RT-PCR and sequencing primer are:
H1 upstream region of gene primer 5 '-GCCGGATTCATTGAAGGAG-3 ',
H1 gene downstream primer 5 '-GCTTTTCTGATCTGCTGCGTAAC-3 ', 5 ' carries out biotin labeling
H1 gene sequencing primer 5 '-TGAAGGAGGATGGACA-3 ',
N1 upstream region of gene primer 5 '-AATTGGCATGGCTCAAATCG-3 ',
N1 gene downstream primer 5 '-TGGATCCCAAATCATCTCAAAA-3 ', 5 ' carries out biotin labeling
N1 gene sequencing primer 5 '-CGTTTAAATACGGCAAT-3 '.
Swine influenza A H 1 N 1 virus H1 target sequence: ATGATAGATGG; Swine influenza A H 1 N 1 virus N1 target sequence: AATGGAGCAAATGG; H1 hypotype expanding fragment length is 102bp, and N1 hypotype expanding fragment length is 249bp.
The RNA of extraction testing sample of the present invention utilizes the TRIZOL lysate to handle the swine influenza A H 1 N 1 virus testing sample, trichloromethane extracting protein, and isopropanol precipitating obtains RNA; Also available business-like RNA extracts test kit and extracts.
Of the present invention respectively with influenza virus A porcine H1, the N1 primer carries out the RT-PCR amplification: wherein, the H1 primer amplification is that the testing sample RNA that will extract carries out reverse transcription, the reverse transcription reaction system is for adding 2 μ L 25mmol/L magnesium chlorides (MgCl2) respectively in 3 μ LRNA templates, 1 μ L, 10 * RNA PCR damping fluid, 1 μ L 2.5mmol/L triphosphoric acid base deoxynucleotide (dNTP), 0.25 μ L 40U/ μ L RNA enzyme inhibitors, 0.5 μ L 5U/ μ L AMV RNA ThermoScript II, 0.5 μ L 20pmol/ μ L H1 upstream primer and H1 downstream primer, with diethylpyrocarbonate (diethypyrocarbonate, DEPC) water is supplied volume to 10 μ L, instantaneous centrifugal mixing, the reverse transcription condition is 42 ℃ of 30min, 98 ℃ of 1min, 4 ℃, add 10 times of polymerase chain reaction damping fluids (10 * PCR buffer), 5 μ L in 10 μ L cDNA (complementary DNA) solution after reverse transcription, 5U/ μ L Taq archaeal dna polymerase 0.25 μ L, 20pmol/ μ L H1 upstream primer and H1 downstream primer 0.5 μ L, DEPC water is supplied volume to 50 μ L, the PCR cycling condition is: behind 94 ℃ of pre-sex change 5min, enter 94 ℃ of sex change 30s, 54 ℃ of annealing, 72 ℃ are extended 30s, circulate altogether 50 times; 72 ℃ are extended 10min more then; The N1 primer amplification is that the testing sample RNA that will extract carries out reverse transcription, the reverse transcription reaction system is for adding 2 μ L 25mmol/L MgCl2 respectively in 3 μ L RNA templates, 1 μ L, 10 * RNA PCRbuffer, 1 μ L 2.5mmol/L dNTP, 0.25 μ L 40U/ μ L RNA enzyme inhibitors, 0.5 μ L 5U/ μ LAMV RNA ThermoScript II, 0.5 μ L 20pmol/ μ L N1 upstream primer and N1 downstream primer, supply volume to 10 μ L with DEPC water, instantaneous centrifugal mixing, reverse transcription condition are 42 ℃ of 30min, 98 ℃ of 1min, 4 ℃, add 10 * PCR buffer, 5 μ L in the 10 μ L cDNA solution after reverse transcription, 5U/ μ L Taq archaeal dna polymerase 0.25 μ L, 20pmol/ μ L N1 upstream primer and N1 downstream primer 0.5 μ L, DEPC water is supplied volume to 50 μ L, the PCR cycling condition is: behind 94 ℃ of pre-sex change 5min, enter 94 ℃ of sex change 30s, 59 ℃ of annealing, 72 ℃ are extended 30s, circulate altogether 50 times; 72 ℃ are extended 10min more then.
Pcr amplification product electrophoresis detection of the present invention: get the 2g agarose, in the 100mL electrophoretic buffer, heat, fully dissolve, adding ethidium bromide stock solution to final concentration is 0.5 μ g/mL, glue adds electrophoretic buffer in electrophoresis chamber, make liquid level just not have gel; 3 μ L～6 μ L pcr amplification products are mixed point sample with an amount of sample loading buffer respectively; 9V/cm constant voltage electrophoresis migrates to the gel middle part until the tetrabromophenol sulfonphthalein indicator; Ultraviolet Detector is observed electrophoresis result and record down.
Preparation tetra-sodium order-checking single-stranded template of the present invention: use 50 μ l to be marked with the PCR product of vitamin H and the magnetic bead of 200 μ g Streptavidin bag quilts, under 25 ℃ of room temperatures, hatched 20 minutes, to pick up with the PCR product after magnetic bead combines with vacuum prep tool, in 70% ethanol, clean 5s then; Sex change damping fluid (denatureation buffer) is washed 5s; Move on at last and clean 10s in the dcq buffer liquid (washing buffer); Vaccum prep tool puts into the plate that contains sequencing primer, shakes, and discharges magnetic bead.Sample was put into 80 ℃ of baking ovens 2 minutes, again cool to room temperature.
Tetra-sodium sequencing reaction of the present invention: sequencing reaction is detecting on the tetra-sodium sequenator under 28 ℃, application of sample uses 600 millibars/8 milliseconds application of sample pressure and time, the every wheel 65 seconds reaction times, primer strand extends along with the adding of different dNTP, combination along with nucleic acid, ccd video camera detects the optical signal that sends, and reads dna sequence dna.
Of the present inventionly whether contain swine influenza A H 1 N 1 virus: H1 in the sample and the N1RT-PCR reaction is male about judging according to RT-PCR amplification and tetra-sodium sequencing result, carry out the tetra-sodium sequencing analysis, order-checking fragment and H1, N1 target fragment are identical, are defined as the swine influenza A H 1 N 1 virus hypotype; H1 and N1RT-PCR reaction are male, and it is non-swine influenza A H 1 N 1 virus that H1 and N1 order-checking fragment and target fragment have an above base different decision; H1, N1RT-PCR reaction all or have one negative to be judged to be non-swine influenza A H 1 N 1 virus.
The present invention is applicable to swine influenza A H 1 N 1 virus carried out rapid detection, can be widely used in the detection of meat swine influenza A H 1 N 1 virus in the eqpidemic disease monitoring of meat packing plant, pig plant and the foreign trade; Compared with prior art, it uses the tetra-sodium sequencing technologies can carry out the short dna sequential analysis quickly and accurately, is convenient to make up the normalizing operation flow process; Have high-throughput, characteristics cheaply, the PCR product can be directly used in order-checking, need not carry out secondary treatments such as product purification, operates very easyly, and required sample size is little.
Description of drawings:
Fig. 1 for the present invention to the HA gene amplification collection of illustrative plates of pork sample, M:DL2000 wherein, 1: the pork sample.
Fig. 2 for the present invention to the NA gene amplification collection of illustrative plates of pork sample, M:DL2000 wherein, 1: the pork sample.
Fig. 3 is that the present invention is to HA gene tetra-sodium sequencing result in the pork sample.
Fig. 4 is that the present invention is to NA gene tetra-sodium sequencing result in the pork sample.
Embodiment:
Also further set forth the present invention in conjunction with the accompanying drawings below by specific embodiment.
Embodiment 1: detect in the pork product that exports whether contain swine influenza A H 1 N 1 virus
1, design specific primer sequence
Land the U.S. state-run biology information technology center (NCBI) by Internet, porcine influenza H1N1 virus HA that query and search has been announced and the nucleotide sequence of NA, do not comprise not clear base components series, the same time that homology is nearer is only chosen a strain with the strain in place, with the Editseq in DNAStar 7.1 software packages and MegAlign software HA to selected sample, the NA gene nucleotide series is edited, comparison one by one, with Clustal W Method (software) default parameters to selected HA, the NA nucleotide sequence carries out homology relatively, find the conserved sequence region of representing each hypotype, and characterize this regional specific nucleotide sequence (target detect sequence).
Design of pcr amplification primer and sequencing primer design all can be undertaken by Assay Design SW software, use one group of higher primer of score to experimentize, homology analysis result according to DNASTAR, choose sequence area conservative in the sample gene, design of amplification primers, so that amplify the single band of specificity, for follow-up order-checking lays the foundation.For the specific nucleotide sequence (target sequence) that comprises in each amplification region designs sequencing primer, H1 hypotype expanding fragment length is 102bp simultaneously, and N1 hypotype expanding fragment length is 249bp.
Swine influenza A H 1 N 1 virus RT-PCR and sequencing primer are:
H1 upstream region of gene primer 5 '-GCCGGATTCATTGAAGGAG-3 ',
H1 gene downstream primer 5 '-GCTTTTCTGATCTGCTGCGTAAC-3 ', 5 ' carries out biotin labeling
H1 gene sequencing primer 5 '-TGAAGGAGGATGGACA-3 ',
N1 upstream region of gene primer 5 '-AATTGGCATGGCTCAAATCG-3 ',
N1 gene downstream primer 5 '-TGGATCCCAAATCATCTCAAAA-3 ', 5 ' carries out biotin labeling
N1 gene sequencing primer 5 '-CGTTTAAATACGGCAAT-3 '.
2, extract the RNA of testing sample
Utilize TRIZOL lysate method to extract the RNA of testing sample, concrete steps are to get the 1.5mL centrifuge tube of sterilization, add 600 μ L TRIZOL lysates, add each 200mg of pork sample to be measured, add 200 μ L chloroforms again, grind, vibration mixing 30s on the vortex mixer in 4 ℃ of centrifugal 15min of 12000r/min, draws supernatant liquor 500 μ L and transfers in the new 1.5mL centrifuge tube, add-20 ℃ of precooling 500 μ L Virahols again, behind the precipitation at room temperature 30min,, carefully remove supernatant in 4 ℃ of centrifugal 15min of 12000r/min, add 600 μ L, 70% ethanol, put upside down washing,, remove supernatant again in 4 ℃ of centrifugal 10min of 12000r/min, centrifuge tube is inverted on the thieving paper drying at room temperature.Add 10 μ L ultrapure waters at last, mixing gently, the RNA on the dissolving tube wall, the centrifugal 5s of 2000r/min promptly obtains the RNA of sample.The RNA that extracts must carry out pcr amplification in 2 hours; Must place refrigerator if need prolonged preservation.
3, carry out the RT-PCR amplification with swine influenza A H 1 N 1 virus H1, N1 primer
(1) H1 primer amplification:
The testing sample RNA that extracts is carried out reverse transcription, the reverse transcription reaction system is for adding 2 μ L 25mmol/L MgCl2 respectively in 3 μ L RNA templates, 1 μ L, 10 * RNA PCR buffer, 1 μ L 2.5mmol/LdNTP, 0.25 μ L 40U/ μ L RNA enzyme inhibitors, 0.5 μ L 5U/ μ L AMV RNA ThermoScript II, 0.5 μ L 20pmol/ μ L H1 upstream primer and H1 downstream primer, supply volume to 10 μ L with DEPC water, instantaneous centrifugal mixing, reverse transcription condition are 42 ℃ of 30min, 98 ℃ of 1min, 4 ℃, add 10 * PCR buffer, 5 μ L in the 10 μ L cDNA solution after reverse transcription, 5U/ μ L Taq archaeal dna polymerase 0.25 μ L, 20pmol/ μ L H1 upstream primer and H1 downstream primer 0.5 μ L, DEPC water is supplied volume to 50 μ L, the PCR cycling condition is: behind 94 ℃ of pre-sex change 5min, enter 94 ℃ of sex change 30s, 54 ℃ of annealing, 72 ℃ are extended 30s, circulate altogether 50 times; 72 ℃ are extended 10min more then.
(2) N1 primer amplification:
The testing sample RNA that extracts is carried out reverse transcription, the reverse transcription reaction system is for adding 2 μ L 25mmol/L MgCl2 respectively in 3 μ L RNA templates, 1 μ L, 10 * RNA PCR buffer, 1 μ L 2.5mmol/LdNTP, 0.25 μ L 40U/ μ L RNA enzyme inhibitors, 0.5 μ L 5U/ μ L AMV RNA ThermoScript II, 0.5 μ L 20pmol/ μ L N1 upstream primer and N1 downstream primer, supply volume to 10 μ L with DEPC water, instantaneous centrifugal mixing, the reverse transcription condition is 42 ℃ of 30min, 98 ℃ of 1min, 4 ℃, add 10 * PCR buffer, 5 μ L in the 10 μ L cDNA solution after reverse transcription, 5U/ μ L Taq archaeal dna polymerase 0.25 μ L, 20pmol/ μ L N1 upstream primer and N1 downstream primer 0.5 μ L, DEPC water is supplied volume to 50 μ L; The PCR cycling condition is: behind 94 ℃ of pre-sex change 5min, enter 94 ℃ of sex change 30s, and 59 ℃ of annealing, 72 ℃ are extended 30s, circulate altogether 50 times; 72 ℃ are extended 10min more then.
4, carry out agarose gel electrophoresis
The PCR product is carried out agarose gel electrophoresis, get the 2g agarose, heat in the 100mL electrophoretic buffer, fully dissolve, adding the ethidium bromide stock solution is 0.5 μ g/mL to final concentration, glue.In electrophoresis chamber, add electrophoretic buffer, make liquid level just not have gel; 3 μ L～6 μ L pcr amplification products are mixed point sample with an amount of sample loading buffer respectively; 9V/cm constant voltage electrophoresis migrates to the gel middle part until the tetrabromophenol sulfonphthalein indicator; Ultraviolet Detector is observed electrophoresis result down, and pork sample to be checked can amplify specific band, and the H1 hypotype is 102bp, and the N1 hypotype is 249bp, and electrophoresis result is seen Fig. 1, Fig. 2.
5, preparation tetra-sodium order-checking single-stranded template
Use 50 μ l to be marked with the PCR product of vitamin H and the magnetic bead of 200 μ g Streptavidin bag quilts, under 25 ℃ of room temperatures, hatched 20 minutes, will pick up with the PCR product after magnetic bead combines, in 70% ethanol, clean 5s then with vacuum prep tool; Denatureation buffer washes 5s; Move on at last among the washing buffer and clean 10s; Vaccum prep tool puts into the plate that contains H1 gene, N1 gene sequencing primer, shakes, and discharges magnetic bead.Sample was put into 80 ℃ of baking ovens 2 minutes, again cool to room temperature.
6, tetra-sodium order-checking
React on tetra-sodium sequenator (PYROMARK ID), under 28 ℃ of conditions, application of sample uses 600 millibars/8 milliseconds application of sample pressure and time, the every wheel 65 seconds reaction times, and primer strand extends along with the adding of different dNTP; Along with the combination of nucleic acid, ccd video camera detects the optical signal that sends, H1 gene, N1 tetra-sodium sequencing result and see Fig. 3, Fig. 4 respectively, and the sequence that reads is respectively ATGATAGATGG, AATGGAGCAAATGG.
7, the result judges
The H1 gene amplification fragment length of pork sample is 102bp, and N1 gene amplification fragment length is 249bp, with conformance to standard; Through the tetra-sodium order-checking, the H1 sequence of mensuration is consistent with swine influenza A H 1 N 1 virus H1 target sequence, for: ATGATAGATGG; The N1 sequence of measuring is consistent with swine influenza A H 1 N 1 virus N1 target sequence, for: AATGGAGCAAATGG, therefore judge in the pork sample and contain swine influenza A H 1 N 1 virus.
Embodiment 2: detect certain and whether culture in swinery infected pigs's H1N1virus, be sampled as hog snout chamber swab
1, design specific primer sequence
With embodiment 1.
2, extract the RNA of testing sample:
Hog snout chamber swab sample is behind thorough mixing on the mixing tank, with the autoclaving tweezers liquid in the swab is extruded, room temperature is placed 30min, get supernatant liquor, adopt RNA to extract test kit (TaKaRa MiniBEST ViralRNA/DNA Extraction Kit Ver 3.0), carry out the extraction of RNA to specifications.
3, carry out the RT-PCR amplification with swine influenza A H 1 N 1 virus H1, N1 primer
With embodiment 1.
4, carry out agarose gel electrophoresis:
The PCR product is carried out agarose gel electrophoresis, get the 2g agarose, in the 100mL electrophoretic buffer, heat, fully dissolve, adding the ethidium bromide stock solution is 0.5 μ g/mL to final concentration, glue, in electrophoresis chamber, add electrophoretic buffer, make liquid level just not have gel; 3 μ L～6 μ L pcr amplification products are mixed point sample with an amount of sample loading buffer respectively; 9V/cm constant voltage electrophoresis migrates to the gel middle part until the tetrabromophenol sulfonphthalein indicator; Ultraviolet Detector is observed electrophoresis result down.
Therefore sample RT-PCR to be checked does not amplify specific band, judges in the swinery sample of certain plant not contain swine influenza A H 1 N 1 virus.
The nucleotides sequence tabulation
＜110〉Inspection and Quarantine Technology Center, Shandong Inspection and Quarantine
＜120〉method of detection of swine influenza A H 1 N 1 virus based on pyrosequencing technology
<160>8
<170>PatentIn?version?3.5
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<211>19
<212>DNA
＜213〉artificial sequence
<220>
<221>primer_bind
<222>(1)..(19)
＜223〉be used to the to increase upstream primer of swine influenza A H 1 N 1 virus H1 gene
<400>1
gccggattca?ttgaaggag 19
<210>2
<211>23
<212>DNA
＜213〉artificial sequence
<220>
<221>primer_bind
<222>(1)..(23)
＜223〉be used to the to increase downstream primer of swine influenza A H 1 N 1 virus H1 gene
<400>2
gcttttctga?tctgctgcgt?aac 23
<210>3
<211>16
<212>DNA
＜213〉artificial sequence
<220>
<221>primer_bind
<222>(1)..(16)
＜223〉be used for the primer that swine influenza A H 1 N 1 virus H1 gene tetra-sodium checks order
<400>3
tgaaggagga?tggaca 16
<210>4
<211>20
<212>DNA
＜213〉artificial sequence
<220>
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<222>(1)..(20)
＜223〉be used to the to increase upstream primer of swine influenza A H 1 N 1 virus N1 gene
<400>4
<210>5
<211>22
<212>DNA
＜213〉artificial sequence
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<221>primer_bind
<222>(1)..(22)
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<400>5
tggatcccaa?atcatctcaa?aa 22
<210>6
<211>17
<212>DNA
＜213〉artificial sequence
<220>
<221>primer_bind
<222>(1)..(17)
＜223〉be used for the primer that swine influenza A H 1 N 1 virus N1 gene tetra-sodium checks order
<400>6
cgtttaaata?cggcaat 17
<210>7
<211>11
<212>DNA
<213>swine?influenza?virus
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<222>(1)..(11)
＜223〉target sequence of swine influenza A H 1 N 1 virus H1 gene tetra-sodium order-checking
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atgatagatg?g 11
<210>8
<211>14
<212>DNA
<213>swine?influenza?virus
<220>
<221>gene
<222>(1)..(14)
＜223〉target sequence of swine influenza A H 1 N 1 virus N1 gene tetra-sodium order-checking
<400>8
aatggagcaa?atgg 14
Claims (8)
1. the method for a detection of swine influenza A H 1 N 1 virus based on pyrosequencing technology is characterized in that design swine influenza A H 1 N 1 virus Auele Specific Primer and tetra-sodium sequencing primer earlier; Extract the RNA of testing sample again, carry out the RT-PCR amplification with swine influenza A H 1 N 1 virus H1, N1 primer respectively then; With carrying out agarose gel electrophoresis amplified production is detected, if H1 primer amplification fragment length is 102bp, N1 primer amplification fragment length is 249bp, then prepare tetra-sodium order-checking single-stranded template, carry out the tetra-sodium sequencing reaction again, judge in the sample whether contain swine influenza A H 1 N 1 virus according to RT-PCR amplification and tetra-sodium sequencing result at last.
2. the method for detection of swine influenza A H 1 N 1 virus based on pyrosequencing technology according to claim 1, it is characterized in that described design swine influenza A H 1 N 1 virus Auele Specific Primer and tetra-sodium sequencing primer are the gene orders according to swine influenza virus, choose conserved sequence, by Assay Design SW software design swine influenza A H 1 N 1 virus specific primer sequence, to amplify the single band of specificity, design the tetra-sodium sequencing primer to determine virus subtype according to the specific nucleotide sequence that comprises in the amplification region again; Swine influenza A H 1 N 1 virus RT-PCR and sequencing primer are:
H1 upstream region of gene primer 5 '-GCCGGATTCATTGAAGGAG-3 '
H1 gene downstream primer 5 '-GCTTTTCTGATCTGCTGCGTAAC-3 ', 5 ' carries out biotin labeling
H1 gene sequencing primer 5 '-TGAAGGAGGATGGACA-3 '
N1 upstream region of gene primer 5 '-AATTGGCATGGCTCAAATCG-3 '
N1 gene downstream primer 5 '-TGGATCCCAAATCATCTCAAAA-3 ', 5 ' carries out biotin labeling
N1 gene sequencing primer 5 '-CGTTTAAATACGGCAAT-3 ';
Swine influenza A H 1 N 1 virus H1 target sequence: ATGATAGATGG; Swine influenza A H 1 N 1 virus N1 target sequence: AATGGAGCAAATGG; H1 hypotype expanding fragment length is 102bp, and N1 hypotype expanding fragment length is 249bp.
3. the method for detection of swine influenza A H 1 N 1 virus based on pyrosequencing technology according to claim 1, the RNA that it is characterized in that described extraction testing sample utilizes the TRIZOL lysate to handle the swine influenza A H 1 N 1 virus testing sample, trichloromethane extracting protein, isopropanol precipitating obtains RNA, or extracts test kit with business-like RNA and extract.
4. the method for detection of swine influenza A H 1 N 1 virus based on pyrosequencing technology according to claim 1, it is characterized in that described respectively with influenza virus A porcine H1, the N1 primer carries out the RT-PCR amplification: wherein, the H1 primer amplification is that the testing sample RNA that will extract carries out reverse transcription, the reverse transcription reaction system is for adding 2 μ L 25mmol/L magnesium chlorides respectively in 3 μ L RNA templates, 1 μ L, 10 * RNA PCR damping fluid, 1 μ L 2.5mmol/L triphosphoric acid base deoxynucleotide, 0.25 μ L 40U/ μ L RNA enzyme inhibitors, 0.5 μ L 5U/ μ LAMV RNA ThermoScript II, 0.5 μ L 20pmol/ μ L H1 upstream primer and H1 downstream primer, supply volume to 10 μ L with diethylpyrocarbonate water, instantaneous centrifugal mixing, the reverse transcription condition is 42 ℃ of 30min, 98 ℃ of 1min, 4 ℃, add 10 times of polymerase chain reaction damping fluid 5 μ L in the 10 μ L cDNA solution after reverse transcription, 5U/ μ L Taq archaeal dna polymerase 0.25 μ L, 20pmol/ μ L H1 upstream primer and H1 downstream primer 0.5 μ L, DEPC water is supplied volume to 50 μ L, the PCR cycling condition is: behind 94 ℃ of pre-sex change 5min, enter 94 ℃ of sex change 30s, 54 ℃ of annealing, 72 ℃ are extended 30s, circulate altogether 50 times; 72 ℃ are extended 10min more then; The N1 primer amplification is that the testing sample RNA that will extract carries out reverse transcription, the reverse transcription reaction system is for adding 2 μ L 25mmol/L MgCl2 respectively in 3 μ L RNA templates, 1 μ L, 10 * RNA PCR buffer, 1 μ L 2.5mmol/L dNTP, 0.25 μ L 40U/ μ L RNA enzyme inhibitors, 0.5 μ L 5U/ μ L AMVRNA ThermoScript II, 0.5 μ L 20pmol/ μ L N1 upstream primer and N1 downstream primer, supply volume to 10 μ L with DEPC water, instantaneous centrifugal mixing, reverse transcription condition are 42 ℃ of 30min, 98 ℃ of 1min, 4 ℃, add 10 * PCR buffer, 5 μ L in the 10 μ L cDNA solution after reverse transcription, 5U/ μ L Taq archaeal dna polymerase 0.25 μ L, 20pmol/ μ L N1 upstream primer and N1 downstream primer 0.5 μ L, DEPC water is supplied volume to 50 μ L, the PCR cycling condition is: behind 94 ℃ of pre-sex change 5min, enter 94 ℃ of sex change 30s, 59 ℃ of annealing, 72 ℃ are extended 30s, circulate altogether 50 times; 72 ℃ are extended 10min more then.
5. the method for detection of swine influenza A H 1 N 1 virus based on pyrosequencing technology according to claim 1, it is characterized in that described pcr amplification product electrophoresis detection: get the 2g agarose, in the 100mL electrophoretic buffer, heat, fully dissolve, adding ethidium bromide stock solution to final concentration is 0.5 μ g/mL, glue adds electrophoretic buffer in electrophoresis chamber, make liquid level just not have gel; 3 μ L～6 μ L pcr amplification products are mixed point sample with an amount of sample loading buffer respectively; 9V/cm constant voltage electrophoresis migrates to the gel middle part until the tetrabromophenol sulfonphthalein indicator; Ultraviolet Detector is observed electrophoresis result and record down.
6. the method for detection of swine influenza A H 1 N 1 virus based on pyrosequencing technology according to claim 1, it is characterized in that described preparation tetra-sodium order-checking single-stranded template: use 50 μ l to be marked with the PCR product of vitamin H and the magnetic bead of 200 μ g Streptavidin bag quilts, under 25 ℃ of room temperatures, hatched 20 minutes, to pick up with the PCR product after magnetic bead combines with vacuum prep tool, in 70% ethanol, clean 5s then; The sex change damping fluid is washed 5s; Move on at last in the dcq buffer liquid and clean 10s; Vaccum prep tool puts into the plate that contains sequencing primer, shakes, and discharges magnetic bead, sample is put into 80 ℃ of baking ovens 2 minutes, again cool to room temperature.
7. the method for detection of swine influenza A H 1 N 1 virus based on pyrosequencing technology according to claim 1, it is characterized in that described tetra-sodium sequencing reaction: sequencing reaction is detecting on the tetra-sodium sequenator under 28 ℃, application of sample uses application of sample pressure and the time of 600mbar/8msec, the every wheel 65 seconds reaction times, primer strand extends along with the adding of different dNTP, along with the combination of nucleic acid, ccd video camera detects the optical signal that sends, and reads dna sequence dna.
8. the method for detection of swine influenza A H 1 N 1 virus based on pyrosequencing technology according to claim 1, it is characterized in that describedly whether containing swine influenza A H 1 N 1 virus: H1 in the sample and the N1RT-PCR reaction is male about judging according to RT-PCR amplification and tetra-sodium sequencing result, carry out the tetra-sodium sequencing analysis, order-checking fragment and H1, N1 target fragment are identical, are defined as the swine influenza A H 1 N 1 virus hypotype; H1 and N1RT-PCR reaction are male, and it is non-swine influenza A H 1 N 1 virus that H1 and N1 order-checking fragment and target fragment have an above base different decision; H1, N1RT-PCR reaction all or have one negative to be judged to be non-swine influenza A H 1 N 1 virus.
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| CN103834747A (en) * | 2014-03-16 | 2014-06-04 | 山东国际旅行卫生保健中心 | Method for detecting pathogenicity of influenza A (H1N1) virus based on pyrosequencing |
| 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 |
| CN106868221A (en) * | 2017-04-07 | 2017-06-20 | 中华人民共和国济南出入境检验检疫局 | Porcine pseudorabies pyrosequencing detection method and primer special |
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| CN104195269B (en) * | 2014-09-12 | 2016-04-13 | 山东出入境检验检疫局检验检疫技术中心 | A kind of method detecting tomato spotted wilf virus |
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| WO2009008921A2 (en) * | 2007-03-28 | 2009-01-15 | Yoshihiro Kawaoka | Influenza b viruses with reduced sensitivity to neuraminidase inhibitors |
| CN101338345A (en) * | 2008-08-12 | 2009-01-07 | 广州华峰生物科技有限公司 | H5 avian influenza viral gene rapid diagnosis kit based on ring mediating isothermal amplification technology and detecting process thereof |
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| CN103834747A (en) * | 2014-03-16 | 2014-06-04 | 山东国际旅行卫生保健中心 | Method for detecting pathogenicity of influenza A (H1N1) virus based on pyrosequencing |
| CN103834747B (en) * | 2014-03-16 | 2016-05-18 | 山东国际旅行卫生保健中心 | The method of the detection H1N1virus pathogenicity based on pyrophosphoric acid order-checking |
| 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 |
| CN106868221A (en) * | 2017-04-07 | 2017-06-20 | 中华人民共和国济南出入境检验检疫局 | Porcine pseudorabies pyrosequencing detection method and primer special |
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