CN100378230C - H5, H7, H9 subtype auian flu virus real-time fluo rescent quantixative PCR detecting method - Google Patents

H5, H7, H9 subtype auian flu virus real-time fluo rescent quantixative PCR detecting method Download PDF

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CN100378230C
CN100378230C CNB2005101109905A CN200510110990A CN100378230C CN 100378230 C CN100378230 C CN 100378230C CN B2005101109905 A CNB2005101109905 A CN B2005101109905A CN 200510110990 A CN200510110990 A CN 200510110990A CN 100378230 C CN100378230 C CN 100378230C
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gene
influenza virus
avian influenza
quantitative pcr
virus
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CN1814805A (en
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张大兵
杨立桃
熊静静
潘爱虎
尹长松
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Shanghai Jiaotong University
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Abstract

The present invention relates to a real-time quantitative PCR detection method for subtype bird flu viruses H5, H7, H9, which belongs to the technical field of biological engineering. The present invention has the steps that standard molecules are constructed; virus specificity quantitative PCR primers and probes are designed; virus sample RNA is extracted; the extracted sample RNA carries out reverse transcription polymerase chain reaction to obtain sample cDNA; an M gene quantitative PCR detection method is optimized and established; a bird flu virus subtype specificity quantitative PCR detection method is optimized and established. The detection sensitivity of the present invention reaches 20 copies of virus molecules, the detection time on a single sample is 2 hours, the operation is simple, and the large-batch sample analysis can be simultaneously carried out. Compared with a virus fractional cultivation method by chicken embryo, the present invention has the advantages of high sensitivity and short detection time. The present invention is favorable for the monitoring and the identification of the bird flu viruses, and has good application prospects.

Description

The real-time fluorescence quantitative PCR detection method of H5, H7, H9 subtype avian influenza virus
Technical field
What the present invention relates to is a kind of detection method of technical field of bioengineering, specifically, is the real-time fluorescence quantitative PCR detection method of a kind of H5, H7, H9 subtype avian influenza virus.
Background technology
High pathogenic avian influenza (Highly Pathogenic Avian Influenza, be called for short HPAI), once claimed checken pest, cause by the specific hypotype of the A of orthomyxoviridae family type influenza virus, being generally H5 and H7 hypotype, secondly is some the highly pathogenic strains in the H9 hypotype; Its epidemiology characteristics are that morbidity is anxious, propagate soon, and the lethality rate height can cause pathogenic strike to aquaculture and human health.OIE (OIE) classifies it as category-A transmissible disease, and China is decided to be a class zoonosis with it.What cause that the people infects has H5N1, H7N7, three kinds of subtype avian influenza virus of H9N2, and wherein, H5N1 has caused global concern with its extremely strong pathogenic and too high lethality rate; And the strain of H9N2 subtype avian influenza virus also has the similar receptor-specific of human influenza virus, and host range is more extensive, has more danger.In monitoring and controlling unit to bird flu, the most important thing is and will diagnose fast, in time delimit epidemic place, epidemic-stricken area, and take measure of control such as blockade, prevent the diffusion of epidemic situation.Therefore, the on-the-spot quick diagnosis of bird flu epidemic situation, very crucial for taking resolutely effectively to put out with prevention and control measure.At present, the main diagnostic method for avian influenza virus is traditional influenza virus detection method, promptly adopt the chicken embryo to carry out the separation and Culture of virus, adopt blood clotting inhibition experiment and neuraminidase inhibition test to carry out the evaluation of hypotype then, during test fee, effort, can not make fast and timely diagnosis.Therefore, set up a kind of sensitivity, special, diagnostic method has important public hygienics meaning and biological diagnostics meaning widely fast.
The application of round pcr in virus detects makes and detects avian influenza virus fast and accurately and become possibility.What be applied to avian flu virus detection at present mainly is conventional qualitative PCR detection method, be to utilize the pcr amplification principle, according to known avian influenza virus gene order design Auele Specific Primer, viral DNA with extremely low concentration is that template amplification goes out special gene fragment, thereby reaches the purpose that detects virus; Have the high advantage of sensitivity and specificity, but qualitative PCR detection method because needing the aftertreatment of pcr amplification, it is easy to generate pollution, also false-positive result occurs than being easier to, and experimental period is also longer relatively.
Find through literature search prior art, the Chinese patent publication number is CN 1560597A, open day is on January 5th, 2005, patent name is: the avian influenza virus multiple PCR fast detecting method, this disclosure of the Invention a kind of identifying virus, particularly identify the detection method of bird flu H5, H9 subtype virus.Extract the sample gene RNA with Trizolkit, carry out the synthetic cDNA of reverse transcription again, carry out the specific a plurality of genes of disposable pcr amplification then, last electrophoresis is identified.Its weak point is: extend under the effect of Taq enzyme owing to obtain sample cDNA, thereby the non-specific amplification of primer can make detected result false positive occur; And the PCR product need carry out electrophoresis detection, and this link is more time-consuming, remolding sensitivity is lower and pollute easily, influences the accuracy and the sensitivity of detected result.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, the real-time fluorescence quantitative PCR detection method of a kind of H5, H7, H9 subtype avian influenza virus is provided, make its detection sensitivity reach the viruses molecule of 20 copies, for the single pattern detection time be 2 hours, easy and simple to handle, and can carry out large batch of sample analysis simultaneously, more traditional method of utilizing the chicken embryo to carry out viral separation and Culture was greatly improved aspect sensitivity and detection time, for the monitoring of avian influenza virus, identify that tool is very helpful and very high application prospect.
The present invention is achieved by the following technical solutions, the present invention is suitable for the specificity quantitative PCR detection of H5, H7 or H9 subtype avian influenza virus, comprising containing total conservative gene---the M gene of avian influenza virus, and H5, H7, H9 subtype avian influenza virus specific gene---the structure of the standard plasmid molecule of HA gene conservative sequence area, the quantitative PCR detection of avian influenza virus M gene, the quantitative PCR detection of H5, H7 or H9 subtype avian influenza virus specificity HA gene etc.
The present invention includes following steps:
(1) structure of standard molecule
Retrieval obtains the conservative gene of avian influenza virus in gene pool (Genbank), M gene and H5, H7, H9 hypospecificity gene, the sequence of HA gene, by online sequence alignment, determine the highly conserved sequence district of the total gene M of avian influenza virus and H5, H7, H9 hypospecificity gene HA, according to the conserved sequence district design primer of determining, utilize gene clone technology to make up to contain simultaneously the standard plasmid molecule in the highly conserved sequence district of the total gene M of avian influenza virus and H5, H7, H9 hypospecificity gene HA;
Described M gene refers in the avian influenza virus genome, that all contain, conservative gene.
Described HA gene refers to the gene that all contains in H5, H7, the H9 subtype avian influenza virus genome, but the HA gene order of three kinds of subtype virus is inconsistent.
The specific fragment of described HA gene refers at the peculiar one section HA gene order of each subtype virus genome, with the genomic HA gene order of other two kinds of subtype virus be different.
Described standard plasmid molecule refers to the artificial recombination plasmid that utilizes gene clone technology to make up, and it contains four specific sequences of external source, is respectively the specific sequence of M gene and H5, H7, three kinds of subtype avian influenza virus HA of H9 gene.
(2) virus-specific quantification PCR primer and probe design
Described virus-specific quantification PCR primer refers to: length is the oligonucleotide chain of 25 ± 5Nt, and the high conservative gene order of the total gene M of itself and avian influenza virus and H5, H7, H9 hypospecificity gene HA is identical or complementary;
Described virus-specific quantitative PCR probe refers to: length is the oligonucleotide chain of 30 ± 5Nt, and its 5 ' end mark FAM fluorescence excitation group, and 3 ' end or oligonucleotide chain central marker TAMRA fluorescent quenching group; Its high conservative gene order with the total gene M of avian influenza virus and H5, H7, H9 hypospecificity gene HA is complementary or identical fully; It combines with quantification PCR primer forms a cover quantitative PCR reaction system;
(3) extraction of Virus Sample RNA;
The extraction of described RNA refers to separation from sample, purifying acquisition RNA solution.
(4) sample rna that extracts is carried out inverse transcription polymerase chain reaction (RT-PCR) and obtain sample cDNA;
Described inverse transcription polymerase chain reaction is meant: according to the RevertAid of Promega company TMFirstStrand cDNA Synthesis Kit #K1622 carries out.
(5) optimization and the foundation of M gene quantification PCR detection method;
The optimization and the foundation of described M gene quantification PCR detection method refer to by the primer of each concentration in the 100-1000nM scope and the combination of probe, seek the reaction system of desired reaction efficiency and curve.
(6) optimization of avian influenza virus subtype specificity quantitative PCR detecting method and foundation;
The optimization and the foundation of described avian influenza virus subtype specificity quantitative PCR detecting method refer to by the primer of each concentration in the 100-1000nM scope and the combination of probe, seek the reaction system of desired reaction efficiency and curve.
The present invention adopts TaqMan probe real-time quantitative PCR to carry out avian influenza virus specific and detects, and have the following advantages: (1) specificity is good.Because TaqMan quantitatively uses hybridization that quantitative molecular is screened, and has very high accuracy.Simultaneously, target sequence is by primer and the dual control of probe, and specificity is good, and false positive is low; (2) highly sensitive.Detection technique of fluorescence is a very sensitive detection technique, so the sensitivity that TaqMan detects is very high; (3) linear relationship is good.Because the generation of fluorescent signal becomes relation one to one with each amplified production, can directly carry out quantitatively product by the detection of fluorescent signal; (4) simple to operate, level of automation height, anti-pollution.Use TaqMan quantitative amplification and detection in same pipe, to detect, do not need to uncap, be difficult for polluting.Increase simultaneously and detect one the step finish, simple to operate, easily be automated.
The fluorescent quantitative PCR technique that the present invention adopts has utilized dexterously that the DNA of round pcr efficiently increases, probe technique high specific and the susceptibility of spectroscopic techniques and the advantage of real-time quantitative analysis, the some shortcomings of conventional PCR qualitative detection have been overcome, the susceptibility and the specificity that detect have greatly been improved, shortened experimental period, simplified experiment and wiped work.And the result of fluoroscopic examination is by the computer for analysis reading, avoided the pollution due to the product postprocessing process, and more conventional PCR is more objective, sensitive, accurately.Simultaneously, the positive control that adopts among the present invention is the standard plasmid molecule according to the conservative gene sequences Design structure of avian influenza virus, with respect to the detection of adopting inactivation of viruses liquid as positive product in the past, more increased security, standard plasmid molecule can also duplicate in a large number, make that the source of positive criteria product is stable more and reliable, avoided the use of positive-virus strain in each experiment.
Embodiment
Conventional experimental technique in the following example, the molecular cloning of writing referring to Sambrook etc.: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989), the use of instrument is with reference to the instrumentation handbook.
Embodiment 1
The structure of standard molecule
One, experiment reagent
Restriction Enzyme Sac I, BamH I, Pst I, EcoR I, Hind III, Kpn I, and the corresponding damping fluid of restriction enzyme digestion (Buffer) (comprises 10 * M Buffer, 10 * H Buffer, 10 * L Buffer, 10 * K Buffer) (Shanghai white good development in science and technology company limited);
The PMD18-T carrier, T 4Dna ligase, T 4DNA connects damping fluid available from the white good development in science and technology in Shanghai company limited;
DNTPs, Taq archaeal dna polymerase and damping fluid thereof, DL2000 Marker are available from the precious biotechnology in Dalian company limited;
Primer is synthetic by the rich inferior biotechnology in Shanghai company limited;
Other biochemical reagents are import packing or homemade analytical pure.
Two, laboratory apparatus
PTC-100 type pcr amplification instrument (MJ Research Inc.);
DNA electrophoretic analysis system comprises camera bellows, digital camera, computer, scanner, ink-jet printer, photosensitive printer, Tanon UV-2000 uv analyzer, Gis gel images analysis software (sky, Shanghai energy company);
Constant incubator;
Three, experimental procedure
1, the sequence of the search total gene M of avian influenza virus and H5, H7, H9 hypospecificity gene HA in Genbank is determined the highly conserved sequence district of the total gene M of avian influenza virus and H5, H7, H9 hypospecificity gene HA by sequence alignment program (AlignX);
2, according to the gene conserved sequence design synthetic primer of determining (table 1), RT-PCR product (cDNA) with H5, H7, H9 subtype avian influenza virus RNA is a template, PCR (table 2) amplification obtains the conserved sequence of gene M and H5, H7, H9 hypospecificity gene HA, is called gene M-O, gene H5-HA, gene H7-HA, gene H9-HA;
3, the sequence of the gene M, gene H5, the gene H7 that obtain of amplification, gene H9 connect the PMD18-T carrier (10 μ l linked systems: 0.5 μ l PMD18-T carrier, 7.5 μ l gene amplification products, 1 μ l T4 dna ligase, 1 μ l T4 DNA connects damping fluid; 4 ℃ are spent the night), 42 ℃ of 90 seconds transformed into escherichia coli DH5 α obtain carrier molecule T-M, T-H5, T-H7 and T-H9;
4, the sequence of the gene M that obtains according to amplification, gene H5, gene H7, gene H9 designs the primer (table 3) that contains restriction enzyme site once more, with T-M, T-H5, T-H7 and T-H9 is template PCR (table 4) amplification gene M and H5, H7, H9 hypospecificity gene HA again, is called gene M, gene H5, gene H7, gene H9;
5, Kuo Zeng gene M, gene H5, gene H7, gene H9 obtain standard molecule in double digestion is connected to the BSK carrier of corresponding double digestion successively.
Gene H5 obtains carrier molecule BSK-H5 in Pst I, BamH I double digestion are connected to the BSK carrier of same double digestion (20 μ l enzymes are cut system: 2 μ l, 10 * K Buffer, 1 μ l Pst I, 1 μ l BamHI, 10 μ l gene H5 fragment or BSK carriers, 6 μ l ddH 2O; Placed 3 hours for 37 ℃);
Carrier molecule BSK-H5 and gene M are connected after cutting and obtain carrier molecule BSK-M-H5 (20 μ l enzymes are cut system: 2 μ l, 10 * M Buffer through Hind III, EcoR I are two, 1 μ l Hind III, 1 μ l EcoR I, 10 μ l gene M fragment or BSK-H5 carriers, 6 μ l ddH 2O; Placed 3 hours for 37 ℃);
Carrier molecule BSK-M-H5 and gene H7 are connected after cutting and obtain carrier molecule BSK-M-H5-H7 (20 μ l enzymes are cut system: 2 μ l, 10 * M Buffer through Kpn I, Hind III are two, 1 μ l Kpn I, 1 μ l HindIII, 10 μ l gene H7 fragment or BSK-M-H5 carriers, 6 μ l ddH 2O; Placed 3 hours for 37 ℃);
Carrier molecule BSK-M-H5-H7 and gene H9 are connected after cutting and obtain carrier molecule BSK-M-H5-H7-H9 (20 μ l enzymes are cut system: 1 μ l, 10 * K Buffer through Sac I, BamH I are two, 1 μ l Sac I, 1 μ lBamH I, 10 μ l gene H9 fragment or BSK-M-H5-H7 carriers, 7 μ l ddH 2O; Placed 3 hours for 37 ℃), promptly be to make up the standard molecule of finishing.
Table 1. avian influenza virus genes involved amplimer sequence
The gene title The primer title Sequence (5 '-3 ') Amplification length (bp)
Gene M-O M-O-F CAAAGCCGAAATCGCGCAGAGA 639
M-O-R CGTTCCGTTTCCTCGCTTGTACTG GAGCTAGGATGAGTCC
Gene H5-HA H5-HA-F GCTCCAGTACAAGCGAGGAAACG GAACGTATGACTATCCAC CATTTCCTTCTACGTAGGGTAGAT 282
H5-HA-R GCAAATTCTGCACTGCA
Gene H7-HA H7-HA-F TTTGCATCTACCCTACGTAGAAGG AAATGATGTTTGTTATC GTATGCCCCACGTCCGAATCCA 622
H7-HA-R CTATGATAGCAATCCCC
Gene H9-HA H9-HA-F ATCATAGTGGATTCGGACGTGG GGCATACATCACCCACCTA 385
H9-HA-R ATATTTACTGACATTGTGGAACG
Table 2-1. avian influenza virus genes involved pcr amplification system
Reaction reagent Volume (μ l) Final concentration
10×PCR Buffer 3 1×(50mM KCl，10mM Tris-HCl，PH8.3，1.5mM MgCl 2)
dNTPs 3 200μM
Primers F * 1 100nM
Primer R * 1 100nM
The Taq enzyme 1.5 1.5unit/reaction
The cDNA template 1
ddH2O Complement to 30 μ l
Attached: *The primer is corresponding with the primer in the table 1
Table 2-2. avian influenza virus genes involved pcr amplification condition
Cycle number Temperature (℃) Time
1 94 5min
30 94 30sec
58 40sec
72 30sec
1 72 2min
The primer sequence (containing restriction enzyme site) of table 3. avian influenza virus specific gene amplification
The gene title The primer title Sequence (5 '-3 ') Restriction enzyme site
Gene M M-F TTT AAGCTTGCATGCCTGCAGGTCGACGATT CAAAGCCGAAATCGCGCAGAGA Hind III
M-R AAA GAATTCTCTAGAGATTCGTTCCGTTTCC TCGCTTGTACTGGAGCTAGGATGAGTCC EcoR I
Gene H5 H5-F TTT CTGCAGGTCGACGATTGCTCCAGTACAA GCGAGGAAACGGAACGTATGACTATCCAC Pst I
H5-R AAA GGATCCTCTAGAGATTCATTTCCTTCTAC TAGGGTAGATGCAAATTCTGCACTGCA 3’ BamH I
Gene H7-F AAA GGTACCGAAGGAAATGATGTTTGTTAT CCTG Kpn I
H7 H7-R TTT AAGCTTCGTCCGAATCCACTATGATAGC Hind III
Gene H9 H9-F TTT GGATCCATATTTACTGACATTGTGGAACGG BamH I
H9-R AAA GAGCTCTACGTATGGGGCATACATCAC CCACCTAC Sac I
Table 4-1. avian influenza virus specific gene PCR amplification system
Reaction reagent Volume (μ l) Final concentration
10×PCR Buffer 3 1×(50mM KCl，10mM Tris-HCl，PH8.3，1.5 mM MgCl 2)
dNTPs 3 200μM
Primers F * 1 100nM
Primer R * 1 100nM
The Taq enzyme 1.5 1.5unit/reaction
Template 1
ddH2O Complement to 30 μ l
Attached: *The primer is corresponding with the primer in the table 3; Template is followed successively by T-M, T-H5, T-H7 and T-H9.
Table 4-2. avian influenza virus specific gene PCR amplification condition
Cycle number Temperature (℃) Time
1 94 5min
30 94 30sec
58 40sec
72 30sec
1 72 2min
Embodiment 2
The extraction of sample rna
According to Trizol kit extract avian influenza virus in the sample RNA (annotate: when RNA precipitate, to add glycogen 8-10 μ g, it can with the RNA co-precipitation, dissolve with the water of DEPC processing.)
One, experiment reagent
1.TRIZOL reagent;
2.DEPC the centrifuge tube of treated water, RNase-free and Tips:
3. new chloroform, Virahol and ethanol, 75% ethanol.
Two, laboratory apparatus
Desk centrifuge
Three, experimental procedure
1) after the detected liquid of .200 μ l adds 1ml Trizol liquid, acutely shake up, room temperature was placed 5-10 minute; 2). add 200 μ l chloroform/primary isoamyl alcohol (24: 1) or chloroforms, thermal agitation mixing 30 seconds, room temperature is placed
3-5 minute;
3). on the desk centrifuge, 12,000rpm, centrifugal 5 minutes of room temperature.
4). supernatant liquor is carefully transferred in the RNase-free 1.5ml centrifuge tube, added glycogen 8-10 μ g mixing, add isopyknic Virahol, placed 5 minutes under the room temperature;
5). on the desk centrifuge, 12,000rpm, centrifugal 5 minutes of room temperature; Carefully remove supernatant liquor, prevent that RNA precipitation from losing;
6). with 75% alcohol washed twice, each 700 μ l, 12,000rpm, centrifugal 2 minutes of room temperature.
7). siphon away supernatant as far as possible up hill and dale, prevent that RNA precipitation from losing; Dry 3-5 minute of traditional vacuum, or be placed under the room temperature alcohol is volatilized fully;
8). precipitation is dissolved with 30-50 μ l DEPC-H2O.As find to precipitate indissoluble, handled 10 minutes for 68 ℃.For pancreas, RNase content is very high in the tissue such as kidney, and precipitation is dissolved with 100% deionized formamide.
Embodiment 3
Reverse transcription (the RevertAid of sample rna TMFirst Strand cDNA Synthesis Kit #K1622)
One, experiment reagent
1. ThermoScript II (Reverse Transcriptase) (200u/ μ l)
Preserve damping fluid: 5mM Tris-HCl (pH 8.3), 0.1M NaCl, 1mM EDTA, 5mM DTT, 0.1%Triton X-100,50% glycerine;
2. nucleic acid inhibitor (Ribonuclease Inhibitor) (20u/ μ l)
Preserve damping fluid: 20mM HEPES-NaOH (pH 7.5), 50mM NaCl, 8mM DTT, 0.5mMELUGENT Detergent, 50% glycerine;
3.5 the reaction buffer of volume (5x Reaction Buffer) doubly
250mM Tris-HCl(pH 8.3，25)，250mM KCl，20mM MgCl 2，50mM DTT；
4.dNTP mixture (10mM dNTP Mix)
5. Oligonucleolide primers (Oligo (dT) 18Primer)
6.DEPC treated water (DEPC-treated Water)
Two, laboratory apparatus
PTC-100 type pcr amplification instrument (MJ Research Inc.)
Thermostat water bath etc.
Three, experimental procedure
1：RNA 1μl
Oligo(dT) 18 primer(0.5μg/μl) 1μl
+DEPC-treated water 10μl
Cumulative volume 12 μ l
Mixing gently, behind 70 ℃, 5min, ice bath 5min;
2：5x reaction buffer 4μl
RNasin(20u/μl) 1μl
10mM dNTP 2μl
Behind 37 ℃, 5min; Add
Reverse Transcriptase(200u/μl) 1μl
Final volume 20 μ l
42 ℃ then, 60min reverse transcription, 70 ℃, 10min deactivation reverse transcription obtain cDNA.
Embodiment 4
The optimization and the foundation of M gene quantification PCR detection method
One, experiment reagent
5 * PCR damping fluid, 1 * (50mM KCl, 10mM Tris-HCl, PH8.3)
DNTPs, Taq archaeal dna polymerase and damping fluid thereof, DL2000 Marker are available from the precious biotechnology in Dalian company limited.TaqMan probe, M-3 probe and primer M-1, M-2 are synthetic by the rich inferior biotechnology in Shanghai company limited;
Other biochemical reagents are import packing or homemade analytical pure.
Two, laboratory apparatus
Rotor-Gene 2000 quantitative PCR instrument (Corbett Research, Australia)
Three, experimentation
1, the primer of M gene specific quantitative PCR detection and probe design
M gene specific quantification PCR primer refers to: length is the oligonucleotide chain of 25 ± 5Nt, and the high conservative gene order of the total gene M of itself and avian influenza virus is identical or complementary;
M gene specific quantitative PCR probe refers to: length is the oligonucleotide chain of 30 ± 5Nt, and its 5 ' end mark FAM fluorescence excitation group, and 3 ' end or oligonucleotide chain central marker TAMRA fluorescent quenching group; Its high conservative gene order with the total gene M of avian influenza virus is complementary or identical fully.
According to the M gene conserved sequence district that obtains, utilize two pairs of TaqMan quantification PCR primers of primer-design software Primer Express2.0 design and probe.Concrete primer and probe sequence see Table 5.
The primer and the probe sequence of table 5.M gene specific quantitative PCR detection
Detection architecture Title Sequence (5 '-3 ') Amplification length (bp)
The M gene specific Primer M-1 AGGCTCTCATGGAATGGCTAAAG 131
Primer M-2 GGCATTTTGGACAAAGCGTCTAC
Probe M-3 FAM AGTCCTCGCTCACTGGGCACGGT TAMRA
2, M gene specific quantitative PCR detection system and reaction conditions
The optimization of M gene specific quantitative PCR detecting method refers to, and by the primer of each concentration in the 100-1000nM scope and the combination of probe, seeks the reaction system of desired M gene specific quantitative PCR reaction efficiency and curve.
Through optimizing, the system of M gene specific quantitative PCR detection sees Table 6; The pcr amplification condition sees Table 7.
Table 6.M gene specific quantitative PCR detection system
Reaction reagent Volume (μ l) Final concentration
5×PCR Buffer 5 1×(50mM KCl，10mM Tris-HCl，PH8.3)
MgCl 2 6 6mM
dNTPs 3 400μM dATP，dGTP，dCTP；800μM dUTP
Primer M-1 0.6 600nM
Primer M-2 0.6 600nM
Probe M-3 0.9 450nM
Hotstar Taq enzyme 1.5 1.5unit/reaction
The UNG enzyme 0.2 0.2unit/reaction
Dna profiling 1
ddH 2O Complement to 25 μ l
Table 7.M gene specific quantitative PCR detection amplification condition
Cycle number Temperature (℃) Time
1 50 2min
1 95 10min
35 94 30sec
56 40sec
72 30sec
Four, the result judges
1, interpretation of result condition enactment
Read detected result.The threshold setting principle is with the vertex of threshold line just above normal negative control product amplification curve, and result's demonstration is as the criterion.Or can adjust according to instrument noise situation.
2, quality control standard
(1) blank does not have the Ct value and does not have amplification curve.
(2) the Ct value of positive control answers＜28.0, and typical amplification curve occurs.Otherwise it is invalid that experiment this time is considered as.
3, the result describes and judges
(1) feminine gender
No Ct value and do not have amplification curve does not have avian influenza virus in the expression sample.
(2) positive
Ct value≤30.0, and typical amplification curve appears, there is avian influenza virus in the expression sample.
(3) effective principle
The sample of Ct value＞30.0 must be reformed.It is negative that the result that reforms does not have Ct value person, otherwise positive.
Embodiment 5
The optimization and the foundation of H5 subtype avian influenza virus specificity quantitative PCR detecting method
One, experiment reagent
5 * PCR damping fluid, 1 * (50mM KCl, 10mM Tris-HCl, PH8.3)
DNTPs, Taq archaeal dna polymerase and damping fluid thereof, DL2000 Marker are available from the precious biotechnology in Dalian company limited.TaqMan probe, H5-3 probe and primer H5-1, H5-2 are by synthetic by the rich inferior biotechnology in Shanghai company limited;
Other biochemical reagents are import packing or homemade analytical pure.
Two, laboratory apparatus
Rotor-Gene 2000 quantitative PCR instrument (Corbett Research, Australia)
Three, experimentation
1, the primer and the probe design of H5 subtype avian influenza virus specificity quantitative PCR detection
H5 subtype avian influenza virus specificity quantification PCR primer refers to: length is the oligonucleotide chain of 25 ± 5Nt, and the high conservative gene order of itself and H5 subtype avian influenza virus specific gene HA is identical or complementary;
H5 subtype avian influenza virus specificity quantitative PCR probe refers to: length is the oligonucleotide chain of 30 ± 5Nt, and its 5 ' end mark FAM fluorescence excitation group, and 3 ' end or oligonucleotide chain central marker TAMRA fluorescent quenching group; Its high conservative gene order with H5 subtype avian influenza virus specific gene HA is complementary or identical fully.
According to the H5 subtype avian influenza virus HA gene conserved sequence district that obtains, utilize two pairs of TaqMan quantification PCR primers of primer-design software Primer Express2.0 design and probe.Concrete primer and probe sequence see Table 8.
The primer and the probe sequence of table 8.H5 subtype avian influenza virus specificity quantitative PCR detection
Detection architecture Title Sequence (5 '-3 ') Amplification length (bp)
The H5 hypospecificity Primer H5-1 TGGAATCAATGGGAACTTACCAAA 101
Primer H5-2 GCACATCCATAAAGATAGACCAGC
Probe H5-3 FAM TGCCAGTGCTAGGGAACTCGCCAC TAMRA
2, H5 subtype avian influenza virus specificity quantitative PCR detection system and reaction conditions
The optimization of H5 subtype avian influenza virus hypospecificity quantitative PCR detecting method refers to, by the primer of each concentration in the 100-1000nM scope and the combination of probe, seek the reaction system of desired H5 subtype avian influenza virus specificity quantitative PCR reaction efficiency and curve.
Through optimizing, the system of H5 subtype avian influenza virus specificity quantitative PCR detection sees Table 9; The pcr amplification condition sees Table 7.
Table 9.H5 subtype avian influenza virus specificity quantitative PCR detection system
Reaction reagent Volume (μ l) Final concentration
5×PCR Buffer 5 1×(50mM KCl，10mM Tris-HCl，PH8.3)
MgCl 2 6 6mM
dNTPs 3 400μM dATP，dGTP，dCTP； 800μM dUTP
Primer H5-1 0.6 600nM
Primer H5-2 0.6 600nM
Probe H5-3 0.6 600nM
Hotstar Taq enzyme 1.5 1.5unit/reaction
The UNG enzyme 0.2 0.2unit/reaction
Dna profiling 1
ddH 2O Complement to 25 μ l
Four, experimental result is judged
1, interpretation of result condition enactment
Read detected result.The threshold setting principle is with the vertex of threshold line just above normal negative control product amplification curve, and result's demonstration is as the criterion.Or can adjust according to instrument noise situation.
2, quality control standard
(1) blank does not have the Ct value and does not have amplification curve.
(2) the Ct value of positive control answers＜28.0, and typical amplification curve occurs.Otherwise it is invalid that experiment this time is considered as.
3, the result describes and judges
(1) feminine gender
No Ct value and do not have amplification curve, no H5 subtype avian influenza virus in the expression sample.
(2) positive
Ct value≤30.0, and typical amplification curve appears, there is the H5 subtype avian influenza virus in the expression sample.
(3) effective principle
The sample of Ct value＞30.0 must be reformed.It is negative that the result that reforms does not have Ct value person, otherwise positive.
Embodiment 6
The optimization and the foundation of H7 subtype avian influenza virus quantitative PCR detecting method
One, experiment reagent
5 * PCR damping fluid, 1 * (50mM KCl, 10mM Tris-HCl, PH8.3)
DNTPs, Taq archaeal dna polymerase and damping fluid thereof, DL2000 Marker are available from the precious biotechnology in Dalian company limited.TaqMan probe, H7-3 probe and primer H7-1, H7-2 are synthetic by the rich inferior biotechnology in Shanghai company limited;
Other biochemical reagents are import packing or homemade analytical pure.
Two, laboratory apparatus
Rotor-Gene 2000 quantitative PCR instrument (Corbett Research, Australia)
Three, experimentation
1, the primer and the probe design of H7 subtype avian influenza virus specificity quantitative PCR detection
H7 subtype avian influenza virus specificity quantification PCR primer refers to: length is the oligonucleotide chain of 25 ± 5Nt, and the high conservative gene order of itself and H7 subtype avian influenza virus specific gene HA is identical or complementary;
H7 subtype avian influenza virus specificity quantitative PCR probe refers to: length is the oligonucleotide chain of 30 ± 5Nt, and its 5 ' end mark FAM fluorescence excitation group, and 3 ' end or oligonucleotide chain central marker TAMRA fluorescent quenching group; Its high conservative gene order with H7 subtype avian influenza virus specific gene HA is complementary or identical fully.
According to the H7 subtype avian influenza virus HA gene conserved sequence district that obtains, utilize two pairs of TaqMan quantification PCR primers of primer-design software Primer Express2.0 design and probe.Concrete primer and probe sequence see Table 10.
The primer and the probe sequence of table 10.H7 subtype avian influenza virus specificity quantitative PCR detection
Detection architecture Title Sequence (5 '-3 ') Amplification length (bp)
The H7 hypospecificity Primer H7-1 CCCAATGACACAGTCACTTTCAG 109
Primer H7-2 CATCAACCTGTACTCCACTCTGA
Probe H7-3 FAM ACTTCCCTCTCAGAAAACTTGC ACGGTCT TAMRA
2, H7 subtype avian influenza virus specificity quantitative PCR detection system and reaction conditions
The optimization of H7 subtype avian influenza virus hypospecificity quantitative PCR detecting method refers to, by the primer of each concentration in the 100-1000nM scope and the combination of probe, seek the reaction system of desired H7 subtype avian influenza virus specificity quantitative PCR reaction efficiency and curve.
Through optimizing, the system of H7 subtype avian influenza virus specificity quantitative PCR detection sees Table 11; The pcr amplification condition sees Table 7.
Table 11.H7 subtype avian influenza virus specificity quantitative PCR detection system
Reaction reagent Volume (μ l) Final concentration
5×PCR Buffer 5 1×(50mM KCl，10mM Tris-HCl，PH8.3)
MgCl 2 6 6mM
dNTPs 3 400μM dATP，dGTP，dCTP； 800μM dUTP
Primer H7-1 0.6 600nM
Primer H7-2 0.6 600nM
Probe H7-3 0.6 600nM
Hotstar Taq enzyme 1.5 1.5unit/reaction
The UNG enzyme 0.2 0.2unit/reaction
Dna profiling 1
ddH 2O Complement to 25 μ l
Four, the result judges
1, interpretation of result condition enactment
Read detected result.The threshold setting principle is with the vertex of threshold line just above normal negative control product amplification curve, and result's demonstration is as the criterion.Or can adjust according to instrument noise situation.
2, quality control standard
(1) blank does not have the Ct value and does not have amplification curve.
(2) the Ct value of positive control answers＜28.0, and typical amplification curve occurs.Otherwise it is invalid that experiment this time is considered as.
3, the result describes and judges
(1) feminine gender
No Ct value and do not have amplification curve, no H5 subtype avian influenza virus in the expression sample.
(2) positive
Ct value≤30.0, and typical amplification curve appears, there is the H5 subtype avian influenza virus in the expression sample.
(3) effective principle
The sample of Ct value＞30.0 must be reformed.It is negative that the result that reforms does not have Ct value person, otherwise positive.
Embodiment 7
The optimization and the foundation of H9 subtype avian influenza virus quantitative PCR detecting method
One, experiment reagent
5 * PCR damping fluid, 1 * (50mM KCl, 10mM Tris-HCl, PH8.3)
DNTPs, Taq archaeal dna polymerase and damping fluid thereof, DL2000 Marker are available from the precious biotechnology in Dalian company limited.TaqMan probe, H9-3 probe and primer H9-1, H9-2 are synthetic by the rich inferior biotechnology in Shanghai company limited;
Other biochemical reagents are import packing or homemade analytical pure.
Two, laboratory apparatus
Rotor-Gene 2000 quantitative PCR instrument (Corbett Research, Australia)
Three, experimentation
1, the primer and the probe design of H9 subtype avian influenza virus specificity quantitative PCR detection
H9 subtype avian influenza virus specificity quantification PCR primer refers to: length is the oligonucleotide chain of 25 ± 5Nt, and the high conservative gene order of itself and H9 subtype avian influenza virus specific gene HA is identical or complementary;
H9 subtype avian influenza virus specificity quantitative PCR probe refers to: length is the oligonucleotide chain of 30 ± 5Nt, and its 5 ' end mark FAM fluorescence excitation group, and 3 ' end or oligonucleotide chain central marker TAMRA fluorescent quenching group; Its high conservative gene order with H9 subtype avian influenza virus specific gene HA is complementary or identical fully.
According to the H9 subtype avian influenza virus HA gene conserved sequence district that obtains, utilize two pairs of TaqMan quantification PCR primers of primer-design software Primer Express2.0 design and probe.Concrete primer and probe sequence see Table 12.
The primer and the probe sequence of table 12.H9 subtype avian influenza virus specificity quantitative PCR detection
Detection architecture Title Sequence (5 '-3 ') Amplification length (bp)
H9 hypotype spy Primer H9-1 CTGACACAACAACAAGCGTTACA 138
The opposite sex Primer H9-2 GCAATGTTTGACCTGGCTTTAGT
Probe H9-3 FAM GATATCAATCGAACTTTCAAA CCAGTGATAGGGCC TAMRA
2, H9 subtype avian influenza virus specificity quantitative PCR detection system and reaction conditions
The optimization of H9 subtype avian influenza virus hypospecificity quantitative PCR detecting method refers to, by the primer of each concentration in the 100-1000nM scope and the combination of probe, seek the reaction system of desired H9 subtype avian influenza virus hypospecificity quantitative PCR reaction efficiency and curve.
Through optimizing, the system of H9 subtype avian influenza virus specificity quantitative PCR detection sees Table 13; The pcr amplification condition sees Table 7.
Table 13.H9 subtype avian influenza virus specificity quantitative PCR detection system
Reaction reagent Volume (μ l) Final concentration
5×PCR Buffer 5 1×(50mM KCl，10mM Tris-HCl，PH8.3)
MgCl 2 6 6mM
dNTPs 3 400μM dATP，dGTP，dCTP； 800μM dUTP
Primer H9-1 0.6 600nM
Primer H9-2 0.6 600nM
Probe H9-3 0.6 600nM
Hotstar Taq enzyme 1.5 1.5unit/reaction
The UNG enzyme 0.2 0.2unit/reaction
Dna profiling 1
ddH 2O Complement to 25 μ l
Four, the result judges
1, interpretation of result condition enactment
Read detected result.The threshold setting principle is with the vertex of threshold line just above normal negative control product amplification curve, and result's demonstration is as the criterion.Or can adjust according to instrument noise situation.
2, quality control standard
(1) blank does not have the Ct value and does not have amplification curve.
(2) the Ct value of positive control answers＜28.0, and typical amplification curve occurs.Otherwise it is invalid that experiment this time is considered as.
3, the result describes and judges
(1) feminine gender
No Ct value and do not have amplification curve, no H9 subtype avian influenza virus in the expression sample.
(2) positive
Ct value≤30.0, and typical amplification curve appears, there is the H9 subtype avian influenza virus in the expression sample.
(3) effective principle
The sample of Ct value＞30.0 must be reformed.It is negative that the result that reforms does not have Ct value person, otherwise positive.

Claims (6)

1. the real-time fluorescence quantitative PCR detection method of a H5, H7, H9 subtype avian influenza virus is characterized in that, comprises the steps:
(1) structure of standard molecule, retrieval obtains the conservative gene of avian influenza virus in gene pool, M gene and H5, H7, H9 hypospecificity gene, the sequence of HA gene, by online sequence alignment, determine the highly conserved sequence district of the total gene M of avian influenza virus and H5, H7, H9 hypospecificity gene HA, according to the conserved sequence district design primer of determining, utilize gene clone technology to make up to contain simultaneously the standard plasmid molecule in the highly conserved sequence district of the total gene M of avian influenza virus and H5, H7, H9 hypospecificity gene HA;
(2) virus-specific quantification PCR primer and probe design;
(3) extraction of Virus Sample RNA;
(4) sample rna that extracts is carried out inverse transcription polymerase chain reaction and obtain sample cDNA;
(5) optimization and the foundation of M gene quantification PCR detection method by the primer of each concentration in the 100-1000nM scope and the combination of probe, are sought the reaction system of desired reaction efficiency and curve;
(6) optimization of avian influenza virus subtype specificity quantitative PCR detecting method and foundation by the primer of each concentration in the 100-1000nM scope and the combination of probe, are sought the reaction system of desired reaction efficiency and curve.
2. the real-time fluorescence quantitative PCR detection method of H5 according to claim 1, H7, H9 subtype avian influenza virus is characterized in that, described HA gene refers to the gene that all contains in H5, H7, the H9 subtype avian influenza virus genome.
3. the real-time fluorescence quantitative PCR detection method of H5 according to claim 1, H7, H9 subtype avian influenza virus is characterized in that, the specific fragment of described HA gene refers at the peculiar one section HA gene order of each subtype virus genome.
4. the real-time fluorescence quantitative PCR detection method of H5 according to claim 1, H7, H9 subtype avian influenza virus, it is characterized in that, described standard plasmid molecule, refer to the artificial recombination plasmid that utilizes gene clone technology to make up, it contains four specific sequences of external source, is respectively the specific sequence of M gene and H5, H7, three kinds of subtype avian influenza virus HA of H9 gene.
5. the real-time fluorescence quantitative PCR detection method of H5 according to claim 1, H7, H9 subtype avian influenza virus, it is characterized in that, described virus-specific quantification PCR primer, refer to: length is the oligonucleotide chain of 25 ± 5Nt, and the high conservative gene order of the total gene M of itself and avian influenza virus and H5, H7, H9 hypospecificity gene HA is identical or complementary.
6. the real-time fluorescence quantitative PCR detection method of H5 according to claim 1, H7, H9 subtype avian influenza virus, it is characterized in that, described virus-specific quantitative PCR probe, refer to: length is the oligonucleotide chain of 30 ± 5Nt, its 5 ' end mark FAM fluorescence excitation group, and 3 ' end or oligonucleotide chain central marker TAMRA fluorescent quenching group; Its high conservative gene order with the total gene M of avian influenza virus and H5, H7, H9 hypospecificity gene HA is complementary or identical fully; It combines with quantification PCR primer forms a cover quantitative PCR reaction system.
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