CN101649356A - Fluorescent quantitative detection kit of H1N1 influenza virus A and detection method thereof - Google Patents
Fluorescent quantitative detection kit of H1N1 influenza virus A and detection method thereof Download PDFInfo
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Abstract
The invention provides a nucleic-acid fluorescent quantitative RT-PCR detection kit of H1N1 influenza virus A and a detection method thereof. The detection kit mainly comprises a specific primer, a fluorescent probe, a PCR buffer solution, a deoxidized nucleoside triphosphate compound, a DNA polymerase, a reverse transcriptase and an RNA enzyme inhibitor, wherein the specific primer and the fluorescent probe have the following sequences: an upstream primer H1N1 A-FP:5'-AGGTTTGAGATATTCCCCAAGACA-3'; a downstream primer H1N1 A-RP:5'-AATTTTTGTAGAAGCTTTTTGCTCC-3'; and a specific probe H1N1 A-P:5'-F-CATGGCCCAATCATGACTCGAACA-Q-3', wherein F is a fluorescent reporter group; and Q is a fluorescent quenching group. The invention has the advantages that the nucleic-acid fluorescent quantitative RT-PCR detection kit of the H1N1 influenza virus A and the detection method thereof can be applied to the emergent detection and the monitoring of a lab where the H1N1 influenza virus A causes an outbreakepidemic.
Description
(1) technical field
The present invention relates to H1N1virus nucleic acid fluorescent quantitative RT-PCR detection kit and detection method, can be applicable to H1N1virus and cause the emergent detection in the laboratory of breaking out epidemic situation.
(2) background technology
Since 18 days March in 2009, the people has been taken place and has been infected H1N1virus and break out epidemic situation in Mexico.Since Mexico appearance on April 13rd, 2009 the 1st routine people infects the death of H1N1virus, detect affirmation through the laboratory to existing 21 countries in the whole world on May 5 and have the Influenza A H1N1 case, total number of the infected rises to 1124 examples, wherein death toll 26 examples.Except that the Mexico and the U.S., the affirmation case of H1N1 has all been reported by many countries.For this reason, The World Health Organization (WHO) rises to 4 grades in the warning level with global flu outbreak, April 29 was further brought up to 5 grades again, this means in of six compasses of competency of World Health Organization, have at least two countries viral interpersonal communication to occur,, announce that entering level V is an intensive signal though can not be affected in this stage most countries, show and be very popular extremely urgently, demonstrated fully the World Health Organization current new type influenza popular attention degree.
At nature, influenza virus especially continues popular animals such as people and pig and horses in the middle of the birds.At present, caused worldwide popular swine influenza virus serotype that three kinds of H1N1, H1N2, H3N2 are only arranged in the swinery in the world.Porcine influenza is one of modal in the world pig transmissible disease, often causes the acute respiratory disease outburst of pig, and its cause of disease swine influenza virus also is the member of influenza A virus.The acceptor of existing avian influenza virus on the porcine respiratory epithelial cell, sialic acid a-2, (SA a 2 3Ga1) has human influenza virus's acceptor sialic acid a-2 again to the 3-galactoside, and (SAa 2,6Ga1) for the 6-galactoside.Therefore, swinery has played vital role on the link that stores influenza virus, induces the influenza virus new subtype to occur.
Have the expert to nearly three during the last ten years the reprovision of swine influenza virus study, think from 1970, in the swinery of the U.S., just be separated to the reprovision swine influenza virus that contains classic swine influenza virus and human influenza virus, the also recombined strain of separation of human influenza virus and avian influenza virus in the Europe and the swinery in Asia, and the lasting existence in the swinery in Europe and Asia of these reprovision viruses.1984 in the extensive porcine influenza of Europe and Asia appearance, also is people source H3N2 and fowl source H1N1 virus gene fragment are carried out reprovision in the pig body result probably.In addition, from 1998, the H3N2 virus of three source reprovisions was widely current in the U.S. in the swinery, and this virus contains human influenza virus's HA, NA and PB1 gene, the PB2 of North America avian viruses and PA gene, and the NP of classic swine disease poison, M and NS simultaneously.
Porcine influenza is except the influence to pig industry, the most outstanding epidemiology characteristics are abilities that it has while infected person and fowl, therefore the meaning of swine influenza virus on having the livestock industry transmissible disease, in the mankind's influenza pandemic, also has important public health meaning.The influenza virus in the swinery of being popular in has has an ability of duplicating on human body, this has just increased new influenza virus and has propagated behind the pig reprovision and give human possibility.Therefore, many scholars think, the intravital influenza virus of pig is the potential source of human influenza virus, in the epidemic strain gene of human flu outbreak next time, the influenza virus fragment that is popular in now in the swinery will be had, to the monitoring of the people in the swinery, pig, avian influenza virus gene, might predict the strain of mankind's flu outbreak next time.In case this class reprovision virus has stronger infectivity, pathogenic, and can infect the mankind, just might cause new worldwide flu outbreak, novel first type (H1N1) influenza that current Mexico takes place also has this characteristics.
Between the popular later stage, the activity of influenza may return to the seasonal influenza level of seeing usually.This time H1N1virus popular might then show as seasonal A type influenza virus.In this stage, importantly continue to monitor, this is also to the improvement of detection method with improve and have higher requirement.
Behind the fluorescence quantitative detecting method of announcement on April 30, there are many countries and regions to announce the gene order of H1N1virus within the next few days again at this H1N1virus.But well-known, influenza virus is a kind of very fast RNA viruses of speed that makes a variation, and occurring in nature makes a variation as influenza A virus frequently, and the biology that variation amplitude is big is rare, and its variation can be changed these two kinds of mechanism by antigenic drift and antigen and realize.In 8 genes of influenza virus, the HA variation is the fastest, secondly is NA.Probability as annual each nucleotide diversity of the HA1 gene of influenza A virus is 3～4 * 10
-3, and the mutation rate of cell chromosome DNA only is 10
-8-10
-10And HA and the NA gene important goal gene that detects of H1N1virus just.Though the method for WHO can detect this popular influenza virus, because the height variability of influenza virus, also should consider the monitoring at the pig source influenza virus of China simultaneously, therefore the detection of going to improve influenza virus from methodological angle also needs to carry out a lot of exploration work.
In view of comprising that people's seasonal influenza, bird flu and the Influenza A H1N1 importance in public health grows with each passing day,, waste time and energy and waste clinical samples and reagent so in the influenza monitoring, will carry out the detection of multiple influenza virus simultaneously.On the basis of popular H1N1virus sequential analysis at present, the molecular biology of setting up multiple fluorescence quantitative PCR is quick, special, accurate, sensitive gene diagnosis method and diagnostic kit are particularly important.
According to relevant regulations, this time the cell cultures of H1N1virus needs to carry out in the BSL-3 laboratory, and this is positive in having increased the Biosafety risk to the detection of producing each influenza network laboratories.Therefore it is positive in having very significant meaning to improving influenza test to carry out safe and stable RNA.
(3) summary of the invention
The object of the invention provides and comes into vogue in the fluorescence quantitative RT-PCR detecting kit and the detection method of the H1N1virus in the whole world from Mexico a kind of in March, 2009.
The technical solution used in the present invention is:
A kind of H1N1virus fluorescence quantitative RT-PCR detecting kit, mainly comprise Auele Specific Primer and fluorescent probe, PCR damping fluid, deoxidation nucleoside triphosphate mixture and archaeal dna polymerase, reversed transcriptive enzyme, RNA enzyme inhibitors, these components belong to common practise to those skilled in the art, can select for use as required; Described Auele Specific Primer and fluorescent probe sequence are as follows:
Upstream primer H1N1-FP:5 '-AGGTTTGAGATATTCCCCAAGACA-3 '
Downstream primer H1N1-RP:5 '-AATTTTTGTAGAAGCTTTTTGCTCC-3 '
Specific probe H1N1-P:5 '-F-CATGGCCCAATCATGACTCGAACA-Q-3 '; Wherein F is the fluorescence report group, and Q is the fluorescent quenching group, in the described specific probe F and Q be chosen as one of following: 1. F is FAM, and Q is BHQ1; 2. F is VIC, and Q is BHQ1; 3. F is TAMRA, and Q is BHQ2; 4. F is ROX, and Q is BHQ2; 5. F is CY5, and Q is BHQ3.Serial probe of the present invention entrusts the super generation bio tech ltd in Shanghai synthetic.
The H 1 N 1 influenza virus gene DNA standard substance that test kit of the present invention also comprises, described standard substance sequence is as follows: AGGTTTGAGATATTCCCCAAGACAAGTTCATGGCCCAATCATGACTCGAACAAAGG TGTAACGGCAGCATGTCCTCATGCTGGAGCAAAAAGCTTCTACAAAAATT.
Reversed transcriptive enzyme described in the test kit of the present invention is the MMLV reversed transcriptive enzyme.
Test kit of the present invention, archaeal dna polymerase described in the test kit, reversed transcriptive enzyme, RNA enzyme inhibitors recommend to come from their mixed preparation Enzyme Mix, described Enzyme Mix is an archaeal dna polymerase, and the ratio mixed preparing that reversed transcriptive enzyme, RNA enzyme suppress to make by 1: 50: 5 unit forms.
Test kit of the present invention is to the H1N1virus fluorescence quantitative RT-PCR detecting method, and described method comprises:
(1) extracts testing sample RNA; If concentration is on the low side, need be concentrated into 5ng/ul; The described sample RNA of this step extracts and can carry out according to a conventional method, as adopts RNeasyMini Kit or other test kit of German QIAGEN company;
(2) be template with testing sample RNA, be hybridly prepared into reaction system with single stage method and following material and carry out the PCR reaction: Auele Specific Primer and fluorescent probe, PCR damping fluid, deoxidation nucleoside triphosphate mixture and archaeal dna polymerase, reversed transcriptive enzyme, RNA enzyme inhibitors; Reaction product places the quantitative PCR instrument to carry out fluoroscopic examination; Described Auele Specific Primer and fluorescent probe sequence are as follows: upstream primer H1N1-FP:5 '-AGGTTTGAGATATTCCCCAAGACA-3 ' downstream primer H1N1-RP:5 '-AATTTTTGTAGAAGCTTTTTGCTCC-3 ' specific probe H1N1-P:5 '-F-CATGGCCCAATCATGACTCGAACA-Q-3 '; Wherein F is the fluorescence report group, and Q is the fluorescent quenching group, and described F is that FAM, VIC, TAMRA, ROX, CY5Q are BHQ1, BHQ2, BHQ3.
(3) select fluoroscopic examination model F AM fluorescence, baseline adjustment is got 3～15 round-robin fluorescent signals, with the vertex setting threshold line of threshold line just above normal negative control; Testing sample fluorescence growth curve surpasses threshold line, and is good logarithmic growth, then is judged as the positive, and promptly then testing sample contains first type N1H1 influenza virus; If no typical amplification curve is judged as feminine gender.
Above-mentioned steps (1) sample RNA extracting method adopts the RNeasyMini Kit of German QIAGEN company, extracts according to the test kit specification sheets.
It is as follows that PCR reaction system final concentration is formed recommendation in the described step (2):
PCR damping fluid final concentration is 1 *
Deoxidation nucleoside triphosphate mixture dATP, dCTP, dGTP, each 0.2mmol/L of dTTP, dUTP
MgCl
2 5mmol/L
Upstream primer 0.4 μ mol/L
Downstream primer 0.4 μ mol/L
Probe 0.2 μ mol/L
Archaeal dna polymerase 4U/ reaction
MMLV reversed transcriptive enzyme 200U/ reaction
RNA enzyme inhibitors 20U/ reaction
Template ribonucleic acid 5ng/ μ L
Solvent is ddH
2O.
The PCR damping fluid is phosphoric acid buffer and deoxidation nucleoside triphosphate mixture One Step RT-PCR Reaction Buffer in the described PCR reaction system.Described PCR reaction system archaeal dna polymerase, MMLV reversed transcriptive enzyme, their mixed preparation Enzyme Mix of RNA enzyme inhibitors system, among the described Enzyme Mix, archaeal dna polymerase, the ratio of reversed transcriptive enzyme, RNA enzyme inhibitors is 1: 50: 5.One Step RT-PCR Reaction Buffer can mix earlier with Enzyme Mix and be called onestep RT-PCR Master Mix, again to treat that test sample RNA is a template, the damping fluid final concentration is 1 *, be meant that the final concentration of each component of damping fluid in reaction system is identical with each component concentrations among 1 * one stepRT-PCRMaster Mix.Usually adopt 2 * one stepRT-PCRMaster Mix of reaction system 1/2 volume.1 * one step RT-PCR Master Mix damping fluid of the present invention (the super generation bio tech ltd in Shanghai) Code:Q40101.
The PCR reaction conditions is as follows in the described step (2): 50 ℃ of 30,95 ℃ of 5min carry out reverse transcription, 95 ℃ of 10s then, and 60 ℃ of 30～35s carry out the single-point fluoroscopic examination at 60 ℃, carry out 45～50 circulations altogether.
The present invention's beneficial effect of the present invention is mainly reflected in: the inventive method detects the specificity that height is arranged to H1N1virus, the sensitivity that present method detects, be special, the responsive method of a kind of rapid detection H1N1virus, be highly suitable for the laboratory early diagnosis that H 1 N 1 influenza A virus infection causes the burst epidemic situation.
(4) description of drawings
X-coordinate is represented the CT value of quantitative fluorescent PCR among Fig. 1, Fig. 2, Fig. 3, and ordinate zou is represented fluorescence intensity.
Fig. 1 is the sensitivity test of Influenza A H1N1 probe: the fluorescence RT-PCR method detects the sensitivity of H1N1virus; From 1 to 4 be followed successively by 1,0.1,0.01,0.001TCID
50
Fig. 2 is for using the detection picture of this system to the clinical sample of the H1N1 of Zhejiang Province's discovery, and on behalf of strong positive sample, the weak positive of b representative, c, a1, a2, a3 represent negative sample
Fig. 3 for the fluorescence RT-PCR method detect H1N1virus specificity (5, HKH1N1 is for making a definite diagnosis positive sample, 6 be that seasonal H1N1,7 is seasonal H3N1,8 is the malicious pearl of SWH1N1 for Zhejiang Province's disease control preservation, the negative contrast of NTC.)
(5) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1:
1 materials and methods
Virus strain and clinical samples:
H1N1, SWH1N1, seasonal H1N1, seasonal H3N1 virus strain derive from Hong Kong and Zhejiang Center For Disease Control and Prevention's strain isolated.Clinical sample derives from the throat swab of recent Zhejiang Province A (H 1 N 1) virus outburst epidemic situation suspected patient, and band ice is transported to the laboratory after the sample collection.
1.2 primer and probe
H1N1 all over the world, seasonal H1N1, pig H1N1 influenza virus strain have been downloaded from the NCBI gene pool of the U.S..It has been carried out homology relatively, and at all influenza virus gene group HA gene regions design H1N1 Auele Specific Primer and Taqman probe, sequence is as follows:
Upstream primer H1N1-FP:5 '-AGGTTTGAGATATTCCCCAAGACA-3 '
Downstream primer H1N1-RP:5 '-AATTTTTGTAGAAGCTTTTTGCTCC-3 '
Specific probe H1N1-P:5 '-FAM-CATGGCCCAATCATGACTCGAACA-BHQ1-3 '; Primer that the present invention uses and probe entrust the super generation bio tech ltd in Shanghai synthetic.
1.3 the extraction of viral quantitative criterion and viral RNA:
With the isolating A (H 1 N 1) virus of conclusive evidence patient's sample is standard strain, carries out virus titer titration (1 * 10 with dog kidney passage cell (MDCK)
3TCID
50/ ml) back is as with reference to strain.With its be diluted to 1,0.1,0.01,0.001TCID
50Each reaction tubes.The Reansy Mini Kit of German QIAGEN company is adopted in the extraction of viral RNA, presses the test kit specification sheets and extracts, and obtaining viral RNA is that template ribonucleic acid is standby.
1.4 the optimization of fluorescence RT-PCR reaction system and condition:
Test kit: the one step RT-PCR Master Mix that selects the super generation bio tech ltd in Shanghai for use, Code:Q40101, the by specification operation, (wherein the PCR damping fluid is 10X buffer2.5ul to contain 2 * One Step RT-PCR Reaction Buffer 7.5ul among the one step RT-PCR Master Mix, 5X RT buffer 4ul dNTP (10mM) 1ul) and Enzyme Mix 5ul (contain archaeal dna polymerase 4U among the 5ulEnzyme Mix, reversed transcriptive enzyme 200U, RNA enzyme 20U), get each 1 μ l of upstream and downstream primer (10 μ mol/L) again, probe (10 μ mol/L) 0.5 μ l, it is 25 μ l that template ribonucleic acid 2 μ l, DEPC water supply 25 μ l reaction systems.Reaction conditions is 50 ℃ of 30min, and 95 ℃ of 5min carry out reverse transcription, 95 ℃ of 10s then, and 60 ℃ of 30s carry out the single-point fluoroscopic examination at 60 ℃, carry out 45 circulations altogether.
The result judges: select fluoroscopic examination model F AM, the fluorescence baseline adjustment is got 3-15 round-robin fluorescent signal mean value, and threshold setting is with the vertex of threshold line just above normal negative control product, and sample is typical amplification curve, and be good logarithmic growth, be judged as the positive.Do not have typical amplification curve, be judged as feminine gender.The optimization Test of system, be in the reaction system of template with the positive nucleic acid of same concentrations, primer concentration is from 0.10～1.00 μ M, concentration and probe concentration is from 0.10～0.50 μ M, adopt the optimum concn of preferred primer of matrix method and probe, according to minimum Ct value and high fluorescent increased value (Δ Rn) best primer of selection and concentration and probe concentration.The preferred primer 400nmol of the present invention, probe 200nmol.
1.5 fluorescence RT-PCR specificity, susceptibility and replica test
Select H1N1, seasonal H1N1, pig H1N1 influenza virus strain and Zhejiang Province's outburst epidemic situation patient diagnosed's in 2009 throat swab clinical sample, above-mentioned virus strain and sample are extracted nucleic acid respectively, detect the specificity of verification method with H1N1virus fluorescence RT-PCR method; To the influenza A virus (1 * 10 of demarcating TCID50
3TCID
50/ ml) extract RNA respectively after the dilution, parallelly carry out fluorescence RT-PCR and RT-PCR reaction, its sensitivity of comparison.In addition, the viral dilution liquid of each concentration is made 5 duplicate detection, the Ct value base of calculation that obtains is poor, and the repeatability of verification method the results are shown in Figure 3, and the result shows that primer of the present invention and probe have specificity to H1N1virus.
Be that probe replaces constant test of other condition of H1N1-P:5 '-FAM-CATGGCCCAATCATGACTCGAACA-BHQ1-3 ' in 1.2 with 5 '-VIC-CATGGCCCAATCATGACTCGAACA-BHQ 1-3 ', 5 '-TAMRA-CATGGCCCAATCATGACTCGAACA-BHQ2-3 ', 5 '-ROX-CATGGCCCAATCATGACTCGAACA-BHQ2-3 ', 5 '-CY5-CATGGCCCAATCATGACTCGAACA-BHQ3-3 ' F respectively, the result is also identical.
2 results
2.1 fluorescence RT-PCR reaction system and condition
Select the one step RT-PCR Master Mix of the super generation bio tech ltd in Shanghai for use, Code:Q40101, the by specification operation, 2 * One StepRT-PCR Reaction Buffer 7.5ul among the one step RT-PCR Master Mix, archaeal dna polymerase, MMLV reversed transcriptive enzyme, their mixed preparation Enzyme Mix 5ul of RNA enzyme inhibitors system, get each 1 μ l of upstream and downstream primer (10 μ mol/L) again, probe (10 μ mol/L) 0.5 μ l, template ribonucleic acid 2-3 μ l, DEPC water is supplied 25 μ l, making reaction system is 25 μ l altogether, wherein.Detect with the Rotor-Gene6000 fluorescence detecting system, reaction parameter is: reaction conditions is 50 ℃ of 30min, and 95 ℃ of 5min carry out reverse transcription, 95 ℃ of 10s then, 60 ℃ of 30s carry out the single-point fluoroscopic examination at 60 ℃, carry out 45 circulations altogether, can obtain minimum Ct value and high fluorescent.
2.2 specificity test
The fluorescence RT-PCR method that the present invention sets up has specificity preferably to H1N1virus, and popular Influenza A H1N1 patient's throat swab also shows positive reaction in the recent period.And with equal no cross reaction (see figure 3) such as other influenza viruses such as seasonal H1N1, pig H1N1, H3N1, H5N1, H7N1, H9N1 influenza virus.
2.3 sensitivity test
To the H1N1virus strain, adopt mdck cell to carry out virus titer and measure (100TCID50/ml), with its be diluted to 1,0.1,0.01,0.001TCID
50The Reansy Mini Kit of German QIAGEN company is adopted in the extraction of viral RNA, presses the test kit specification sheets and extracts, and obtains viral RNA.Detect with the fluorescence RT-PCR method, fluorescence RT-PCR method detection sensitivity reaches 0.001TCID as a result
50(see figure 1).
2.4 replica test
The H1N1virus strain becomes 3 different concentration by 10 times of gradient dilutions, reaction system is described in 1.4, sample to each concentration is made 5 duplicate detection, and different IPs acid concentration detection Ct value standard deviation separately has better repeatability (table 1) between 0.10～0.31 as a result.
Table 1 fluorescence RT-PCR method detects the replica test of enterovirus
2.5 the detection of clinical sample
The throat swab, the gargarism that break out the epidemic situation suspected patient from the Influenza A H1N1 of various places, recent Zhejiang Province report are directly extracted viral RNA totally 40 parts of clinical samples, detect enterovirus simultaneously with A (H 1 N 1) virus fluorescence RT-PCR method of the present invention and WHO method, the inventive method detects positive 40 parts of enterovirus as a result, the WHO method detects 35 parts, and fluorescence RT-PCR method of the present invention detects the positive rate height of enterovirus than WHO method.Fluorescence RT-PCR method of the present invention is used for the checking of clinical sample detection carries out 4 parallel laboratory test chambers, has all obtained satisfied result, and what Fig. 2 showed is the detection collection of illustrative plates of part clinical sample.
Sequence table _ ST25.txt
SEQUENCE?LISTING
＜110〉Zhejiang Center For Disease Control and Prevention
＜120〉first type HIN1 influenza virus fluorescence quantitative detection kit and detection method
<130>
<160>4
<170>PatentIn?version?3.4
<210>1
<211>24
<212>DNA
<213>Unknown
<220>
＜223〉artificial sequence
<400>1
aggtttgaga?tattccccaa?gaca 24
<210>2
<211>25
<212>DNA
<213>Unknown
<220>
＜223〉artificial sequence
<400>2
aatttttgta?gaagcttttt?gctcc 25
<210>3
<211>24
<212>DNA
<213>Unknown
<220>
＜223〉artificial sequence
<400>3
catggcccaa?tcatgactcg?aaca 24
<210>4
<211>106
<212>DNA
<213>H1N1swine?influenza?virus
<400>4
aggtttgaga?tattccccaa?gacaagttca?tggcccaatc?atgactcgaa?caaaggtgta 60
acggcagcat?gtcctcatgc?tggagcaaaa?agcttctaca?aaaatt 106
Claims (10)
1. H1N1virus fluorescence quantitative RT-PCR detecting kit, mainly comprise Auele Specific Primer and fluorescent probe, PCR damping fluid, deoxidation nucleoside triphosphate mixture and archaeal dna polymerase, reversed transcriptive enzyme, RNA enzyme inhibitors, described Auele Specific Primer and fluorescent probe sequence are as follows:
Upstream primer: 5 '-AGGTTTGAGATATTCCCCAAGACA-3 '
Downstream primer: 5 '-AATTTTTGTAGAAGCTTTTTGCTCC-3 '
Specific probe: 5 '-F-CATGGCCCAATCATGACTCGAACA-Q-3 '; F is the fluorescence report group, and Q is the fluorescent quenching group.
2. test kit as claimed in claim 1 is characterized in that the H 1 N 1 influenza virus gene DNA standard substance that described test kit also comprises, described standard substance sequence is as follows:
AGGTTTGAGATATTCCCCAAGACAAGTTCATGGCCCAATCATGACTCGAACAAAGGTGTAACGGCAGCATGTCCTCATGCTGGAGCAAAAAGCTTCTACAAAAATT。
3. test kit as claimed in claim 1 is characterized in that the reversed transcriptive enzyme described in the described test kit is the MMLV reversed transcriptive enzyme.
4. test kit as claimed in claim 1, it is characterized in that the archaeal dna polymerase described in the described test kit, reversed transcriptive enzyme, RNA enzyme inhibitors come from their mixed preparation Enzyme Mix, described Enzyme Mix is an archaeal dna polymerase, and the ratio mixed preparing that reversed transcriptive enzyme, RNA enzyme suppress to make by 1: 50: 5 unit forms.
5. as the described test kit of one of claim 1～4, it is characterized in that being chosen as of F and Q in the described specific probe is one of following: 1. F is FAM, and Q is BHQ1; 2. F is VIC, and Q is BHQ1; 3. F is TAMRA, and Q is BHQ2; 4. F is ROX, and Q is BHQ2; 5. F is CY5, and Q is BHQ3.
6. one kind is utilized test kit as claimed in claim 1 to the H1N1virus fluorescence quantitative RT-PCR detecting method, and described method comprises:
(1) extracts testing sample RNA;
(2) to treat that test sample RNA is a template, be hybridly prepared into reaction system with single stage method and following material and carry out the PCR reaction: Auele Specific Primer and fluorescent probe, PCR damping fluid, deoxidation nucleoside triphosphate mixture and archaeal dna polymerase, reversed transcriptive enzyme, RNA enzyme inhibitors; Reaction product places the quantitative PCR instrument to carry out fluoroscopic examination; Described Auele Specific Primer and fluorescent probe sequence are as follows:
Upstream primer: 5 '-AGGTTTGAGATATTCCCCAAGACA-3 '
Downstream primer: 5 '-AATTTTTGTAGAAGCTTTTTGCTCC-3 '
Specific probe: 5 '-F-CATGGCCCAATCATGACTCGAACA-Q-3 '; Wherein F is the fluorescence report group, and Q is the fluorescent quenching group;
(3) select fluoroscopic examination model F AM fluorescence, baseline adjustment is got 3～15 round-robin fluorescent signals, with the vertex setting threshold line of threshold line just above normal negative control; Testing sample fluorescence growth curve surpasses threshold line, and is good logarithmic growth, then is judged as the positive, and promptly then testing sample contains first type N1H1 influenza virus; If no typical amplification curve is judged as feminine gender.
7. method as claimed in claim 3 is characterized in that described step (1) sample RNA extracting method adopts the RNeasy Mini Kit of German QIAGEN company, extracts according to the test kit specification sheets.
8. method as claimed in claim 3 is characterized in that PCR reaction system final concentration is composed as follows in the described step (2):
PCR damping fluid final concentration is 1 *
Deoxidation nucleoside triphosphate mixture dATP, dCTP, dGTP, each 0.2mmol/L of dTTP, dUTP
MgCl
2 5mmol/L
Upstream primer 0.4 μ mol/L
Downstream primer 0.4 μ mol/L
Probe 0.2 μ mol/L
Archaeal dna polymerase 4U/ reaction
MMLV reversed transcriptive enzyme 200U/ reaction
RNA enzyme inhibitors 20U/ reaction
Template ribonucleic acid 5ng/ μ L
Solvent is ddH
2O.
9. method as claimed in claim 8 is characterized in that the PCR reaction system is prepared as follows in the described step (2), (a) PCR damping fluid and deoxidation nucleoside triphosphate mixture is made mixed preparation One Step RT-PCR Reaction Buffer; (b) with archaeal dna polymerase, reversed transcriptive enzyme, RNA enzyme are made by the ratio mixed preparing of 1: 50: 5 unit, make among every 5ul Enzyme Mix to contain archaeal dna polymerase 4U reversed transcriptive enzyme 200U, RNA enzyme 20U; (c) with One StepRT-PCR Reaction Buffer and Enzyme Mix by the upstream primer, downstream primer, probe and the template ribonucleic acid that add proportional quantity behind the proportioning mixing, supply DEPC water again.
10. method as claimed in claim 3 is characterized in that the PCR reaction conditions is as follows in the described step (2): 50 ℃ of 30min, and 95 ℃ of 5min carry out reverse transcription, 95 ℃ of 10s then, 60 ℃ of 30～35s carry out the single-point fluoroscopic examination at 60 ℃, carry out 45～50 circulations altogether.
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| CN101875933A (en) * | 2010-06-22 | 2010-11-03 | 中国人民解放军军事医学科学院微生物流行病研究所 | Kit for identifying subtypes of influenza A virus |
| CN101928787A (en) * | 2010-08-20 | 2010-12-29 | 浙江省疾病预防控制中心 | Primer, probe and method for detecting resistance mutation of influenza A H1N1 viruses |
| CN101948934A (en) * | 2010-09-13 | 2011-01-19 | 中国医学科学院医学实验动物研究所 | Kit for detecting seasonal influenza virus H1N1 through real-time PCR |
| CN102154514A (en) * | 2011-03-18 | 2011-08-17 | 武汉大学 | One-step real-time fluorescence quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) kit for influenza A virus H1N1 |
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