CN101886139A - Double-color fluorescence polymerase chain reaction (PCR) detection method of novel influenza A virus H1N1 subtype and kit thereof - Google Patents

Double-color fluorescence polymerase chain reaction (PCR) detection method of novel influenza A virus H1N1 subtype and kit thereof Download PDF

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CN101886139A
CN101886139A CN2009101366353A CN200910136635A CN101886139A CN 101886139 A CN101886139 A CN 101886139A CN 2009101366353 A CN2009101366353 A CN 2009101366353A CN 200910136635 A CN200910136635 A CN 200910136635A CN 101886139 A CN101886139 A CN 101886139A
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virus
influenza
hypotype
pcr
iav
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史成军
徐贵峰
罗宝正
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Inspection Technical Center Of Zhuhai Entry-Exit Inspection & Quarantine Bureau
BEIJING SUOAO BIOTECHNOLOGY Co Ltd
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Inspection Technical Center Of Zhuhai Entry-Exit Inspection & Quarantine Bureau
BEIJING SUOAO BIOTECHNOLOGY Co Ltd
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Abstract

The invention provides a method for rapidly and correctly detecting mRNA of novel influenza A virus H1N1 subtype (IAV-H1N1) and various influenza A virus subtypes (IAV-U) by a double-color fluorescence quantitive polymerase chain reaction (PCR) technique (a PCR-double-color fluorescence probe method). The method comprises the following steps of: (1) collecting and conveying infected or suspected patient samples; (2) preprocessing the samples and extracting RNA; (3) detecting the samples by a one-step PCR-fluorescence probe in vitro enlargement method; and (4) after the amplification reactions are finished, analyzing the corresponding sample according to the fluorescence intensity of each amplification reaction so as to judge that the IAV-H1N1 subtype and/or the various IAV-U subtypes exist in collected samples, correctly quantify the IAV-H1N1 subtype and/or the various IAV-U subtypes (figure 3), and fulfill the aim of rapidly and accurately detecting the IAV-H1N1 subtype and/or the various IAV-U subtypes in real time.

Description

A kind of novel influenza A virus H1N1 hypotype two-color fluorescence PCR detection method and test kit thereof
One, technical field
This examination invention relates to the method that detects novel influenza A virus H1N1 hypotype (IAV-H1N1) and each hypotype of influenza A virus (IAV-U) mRNA, particularly relates to using Two Colour Fluorescence probe quantitative round pcr (PCR-Two Colour Fluorescence probe method) to detect the method for novel influenza A virus H1N1 hypotype in sample samples such as (comprise throat swab sample, serum, secretory product and) the animal lungs tissues of dying of illness and each hypotype mRNA of influenza A virus fast and accurately.The invention further relates to the test kit that is used for this virus clinical detection.
Two, background technology
Novel influenza A virus H1N1 hypotype is to cause that (Swine Influenza, main pathogens SI), this kind disease are a kind of acute, infecting both domestic animals and human respiratory infectious diseases to porcine influenza.Separated and identified the first strain influenza A virus H1N1 hypotype in 1931.1976, classical influenza A virus H1N1 hypotype was separated to from the North of Italy pig farm first, these virus isolated strains be popular in the porcine influenza of classics of the U.S. at that time very near dependency arranged.Infected influenza A virus H1N1 hypotype about 500 people of the U.S. the same year, this virus with at that time in the pig body isolating virus identical, confirmed first that under field conditions (factors) swine influenza virus can be propagated to the people from pig.Domestic report mostly is H1N1 and H3N2 causes porcine influenza.In recent years, the particularly report of H1N1 hypotype of the sick different strains of people infected pigs influenza virus is arranged all over the world, but unpopular on a large scale.
But countries and regions such as the Mexico and the U.S. have broken out the epidemic situation that the people infects influenza A virus since in April, 2009.Subsequently, this epidemic situation worldwide spreads rapidly, in short period of time, the generation of influenza A virus epidemic situation is infected in countries and regions such as Mexico, the U.S., Canada, Switzerland, Austria, Peru, Spain, Britain, Australia, Korea S successively, in one month, whole world confirmed cases and doubtful accumulative total several thousand examples, dead hundreds of example, break out fast, develop rapid, make common people shock and fear, the World Health Organization on April 29th, 2009 guarded against influenza and transfers to level V on the rank, and showing is very popular is imminent.To this epidemic situation, China Ministry of Health and relevant departments pay much attention to, and have taked corresponding counter-measure, comprise that epidemic situation prevention and control and cause of disease detect.The whole nation each big disease prevention and control center, each provincial disease prevention and control center, hospital, Entry-Exit Inspection and Quarantine Bureau at different levels and animal epidemic at different levels control center pay much attention to this.
Studies confirm that according to the World Health Organization, the people infected pigs influenza virus that area such as the Mexico and the U.S. takes place is novel influenza A virus H1N1 hypotype strain, this strain includes the gene segment of porcine influenza, bird flu and three kinds of influenza viruses of human influenza, is a kind of new type influenza virus.Metainfective clinical early symptom of people and influenza are similar, and fever, cough, fatigue, poor appetite etc. are arranged, and symptoms such as diarrhoea or vomiting can also occur.The state of an illness can be made progress rapidly, high suddenly heat, pneumonia, and respiratory insufficiency, multiple organ injury can appear in weight person, cause death.
Novel influenza A virus H1N1 hypotype belongs in orthomyxoviridae family (0rthomyxoviridae), and influenza A virus belongs to (Influenza virusA).It is spherical that virion is, and diameter is 80nm~120nm, and cyst membrane is arranged.Virus is made up of 8 segmented sub-thread strand RNAs, and virus protein contains 5 peptide species, i.e. hemagglutinin, neuraminidase, stromatin, nucleoprotein and polymerase.
At present the laboratory examination for this kind novel influenza A virus H1N1 hypotype and influenza A virus mainly comprises following aspect: 1. peripheral hemogram: total white blood cells is generally not high or reduce.The patient with severe symptoms has total white blood cells and lymphopenia more, and blood platelet reduction is arranged; 2. serodiagnosis: can use indirect ELISA, antigen to catch ELISA, fluorescent immune method etc.; 3. reverse transcription-polymerase chain reaction (RT-PCR); 3. virus is separated: (throat swab, oral cavity gargle, nasopharynx or tracheae aspirate, phlegm or lung tissue etc.) are separated and are cultivated this virus from patient respiratory road sample.But make a definite diagnosis to depend on from respiratory tract sample or serum and be separated to specific virus; RT-PCR detects above-mentioned sample, has viral RNA to exist, and will confirm through order-checking.Below mainly be divided into two class methods: immunological detection method and nucleic acid detection method.Immunological method mainly is to detect the specific antibody that whether has novel influenza A virus H1N1 subtype virus in the serum, specifically be that method with ELISA detects, yet this method must be to be based upon virus infection takes place and to produce on the basis of antibody, and in view of the influenza disease the height variability and the intercrossing of learning with other serum virus, its specificity is difficult to guarantee, occurs false positive and false negative easily.From sample, separate swine influenza virus, carry out egg inoculation and cell cultures and may compare sensitivity, but this method needs the time in 2-3 week, more loaded down with trivial details on the schedule of operation, should not promote such as hospital, disease prevention and control center, inspection and quarantine center and animal epidemic control center in all test experience some basic unit laboratories particularly.Other has regular-PCR to carry out the method for novel influenza A virus H1N1 hypotype detection of nucleic acids such as reverse transcription-polymerase chain reaction (RT-PCR), though tool remolding sensitivity aforesaid method slightly improves, but this method only is based on a pair of nucleic acid primer and increases, the result is easy to generate non-specific amplification, even can be because false positive appears in the reason of operation, and its result's of regular-PCR TRAP judgement need be carried out gel electrophoresis analysis, complex operation to product.
Its principle of fluorescent quantitative PCR technique be in conventional pcr amplification system, add one section with the special complementary fluorescence labeling probe of target gene.5 of probe ' end is marked with reporter group, and as FAM, VIC etc., 3 ' end is marked with the fluorescent quenching group, as TAMRA etc.When probe was complete, reporter group institute emitted fluorescence energy was absorbed by quenching group, and instrument detecting is less than fluorescent signal.Along with the carrying out of pcr amplification, the Taq enzyme runs in the chain extension process with template bonded probe will cut off probe the generation fluorescent signal.With the variation of fluorescence detector detection fluorescent value, can accurately judge the amount of amplified production.Fluorescence quantifying PCR method is exactly according to this principle.In the PCR process, the continuously variation of fluorescent signal in the detection reaction system.When signal is strengthened to a certain threshold value, the cycle index (Ct represents with cycle threshold) of this moment just goes on record.Strict linear relationship is arranged between the logarithm of starting template in CT value and the PCR system, utilize the Ct and the quantitative templates number of the positive quantitative criterion product amplification of each gradient to make typical curve, just can accurately make the quantity of starting template nucleic acid again according to the Ct value of testing sample through logarithm match mapping.
Double-colored or multicolor fluorescence quantitative PCR technique is meant two or more goal gene that increase simultaneously in same fluorescent quantitative PCR test, and the fluorescent probe without wavelength that each goal gene adopts detects.Fluorescently-labeled probe has multiple, such as FAM, VIC, JOE, NED, HEX etc., every kind of fluorescent signal difference that fluorescently-labeled probe is produced in the pcr amplification process.The test kit that utilizes this principle invention is in the detection of same sample, not only can detect novel influenza A virus H1N1 hypotype (IAV-H1N1), can also detect each hypotype of influenza virus (IAV-U), realized detecting simultaneously in the sample function of novel influenza A virus H1N1 hypotype and each hypotype of influenza A virus, reach and detect in real time accurately and virus is carried out the purpose of somatotype, it is very convenient to use.Because this method introduced specificity amplification primer and fluorescent probe, make that the sensitivity and the specificity that detect are strengthened significantly, thereby avoided the not high problem of failing to pinpoint a disease in diagnosis easily with mistaken diagnosis of other detection method specificitys.
Three, summary of the invention
The invention provides the method that detects novel influenza A virus H1N1 hypotype and each hypotype mRNA of influenza A virus, particularly provide a kind of use double color fluorescent quantitative round pcr (PCR-Two Colour Fluorescence probe method) to detect the method for novel influenza A virus H1N1 hypotype in sample samples such as (comprise throat swab sample, serum, secretory product and) the animal lungs of dying of illness or each hypotype mRNA of influenza A virus fast and accurately.The present invention further provides and be used for the clinical test kit that quick and precisely detects of this viroid patient suspected.
At present comprise mainly that at the novel influenza A virus H1N1 of this kind hypotype associated diseases laboratory examination indirect ELISA, antigen in the serodiagnosis catches ELISA, fluorescent immune method etc., nucleic acid detection method such as reverse transcription-polymerase chain reaction (RT-PCR). virus separation and inoculation method etc.But this several method has sensitivity and poor specificity is arranged respectively, occurs false positive and false-negative deficiency easily, even some detection method process is loaded down with trivial details consuming time longer, is difficult to reach the purpose that quick and precisely detects.
In order to overcome defective and the deficiency in the existing detection technique, the invention provides the method that a kind of use double color fluorescent quantitative round pcr (PCR-Two Colour Fluorescence probe method) detects the mRNA of novel influenza A virus H1N1 hypotype in sample samples such as (comprise throat swab sample, serum, secretory product and) the animal lungs of dying of illness and each hypotype of influenza A virus fast and accurately.This method comprises: (1) is gathered and is transported and infect or patient suspected's sample (comprising throat swab sample, serum, secretory product and the animal lungs etc. of dying of illness); (2) sample pre-treatment and extraction RNA; (3) one step PCR-Two Colour Fluorescence probe amplification in vitro methods detect sample: synthetic specific primer and fluorescent probe, also carry out augmentation detection with novel influenza A virus H1N1 hypotype PCR reaction solution (PCR MIX) to sample with double color fluorescent quantitative PCR reaction technology; (4) amplified reaction finishes the back and according to the fluorescence intensity of each amplified reaction respective sample is analyzed, thereby judges the existence of novel influenza A virus H1N1 hypotype in the sample of being gathered and each hypotype of influenza A virus and according to positive quality control product the virus in its sample is carried out detection by quantitative.
Detect the specific oligonucleotide primers of novel influenza A virus H1N1 hypotype and the region of variability that fluorescent probe is positioned at this each hypotype of C-type virus C, there is not homology with other subtype gene sequences, Genbank is gone up all influenza A virus H1N1 hypotypes of delivering (comprising novel influenza A virus H1N1 hypotype) virus gene sequence, through biological new informatics analysis, the variability maximum of hemagglutinin (HA) in eight gene fragments of influenza A virus, some zone of the HA gene of novel influenza A virus H1N1 hypotype is obviously different with other influenza A virus, and wherein a lot of gene orders morph and recombinate.Through comparison, select as lower area as the novel influenza A virus H1N1 hypospecificity target area of increasing:
GGTACGGTTA?TCACCATCA?AAATGAGCAGG?GGTCA?GG ATA
GCAGCCGACC?TGAAGAGCAC?ACAGAATGCC?ATTGACGAG?A?TTACTAAC(SEQ?ID?NO.1)
Primer and probe sequence are as follows
(1) novel influenza A virus H1N1 hypotype upstream primer:
5’-GGTACGGTTATCACCATCAAAA-3’(SEQ?ID?NO.2)
(2) novel influenza A virus H1N1 hypotype downstream primer:
5’-GTTAGTAATCTCGTCAATGGCATT-3’(SEQ?ID?NO.3)
(3) novel influenza A virus H1N1 hypotype fluorescent probe:
5’-FAM-ATATGCAGCCGACCTGAAGAGCACACA-TAMRA-3’(SEQ?ID?NO.4)
Through bioinformatic analysis, the variability minimum of eight gene fragment mesostromas (M) gene of influenza A virus.Through comparison, selecting as lower area also is influenza A virus universal (IAV-U) specificity target area as each hypotype of amplification influenza A virus:
TGTGCCACTT?GTGAACAGAT?TGC TGATTCA?CAGCATCGGT
CTCACAGACA?GATGGCTACT?AC CACCAATC?CACTAATCAG?GC(SEQID?NO.5)
(4) the universal upstream primer of influenza A virus:
5’-TGTGCCACTTGTGAACAGATTG-3’(SEQ?ID?NO.6)
(5) the universal downstream primer of influenza A virus:
5’-GCCTGATTAGTGGATTGGTG-3’(SEQ?ID?NO.7)
(6) the universal fluorescent probe of influenza A virus:
5’-VIC-TGATTCACAGCATCGGTCTCACAGAC-TAMRA-3’(SEQ?ID?NO.8)
According to a preferred embodiment of the invention, the viral a kind of novel influenza A virus H1N1 hypotype that causes many countries and regions influenza epidemic situation that is the World Health Organization's in April, 2009 announcement of wherein said detection.This strain includes the gene segment of porcine influenza, bird flu and three kinds of influenza viruses of human influenza, is a kind of novel influenza A virus that has made a variation.
According to a preferred embodiment of the invention, wherein said sample is from examinee's throat swab sample, serum, secretory product and the animal lungs etc. of dying of illness.
According to a preferred embodiment of the invention, thus wherein said sample process process use be a kind of viral concentrated solution reagent and can obtain to concentrate through high speed centrifugation after virion.
According to a preferred embodiment of the invention, wherein said extraction RNA use is that a kind of Trizol reagents RNA extracting solution is fully handled sample.
According to a preferred embodiment of the invention, the specific gene of the novel influenza A virus H1N1 of wherein said detection hypotype is hemagglutinin (HA) gene, and the specific gene that detects each hypotype of influenza A virus (influenza A virus universal (IAV-U)) is stromatin (M) gene.
According to a preferred embodiment of the invention, wherein said Two Colour Fluorescence mark is meant that the fluorescent mark of the probe that detects novel influenza A virus H1N1 hypotype (IAV-H1N1) hemagglutinin (HA) gene is FAM, the fluorescent mark that detects the probe of each hypotype of influenza A virus (influenza A virus universal (IAV-U)) stromatin (M) gene is VIC, and quenching group is TAMRA.
According to a preferred embodiment of the invention, wherein said novel influenza A virus H1N1 hypotype double color fluorescent quantitative PCR reaction system (PCR MIX) is by novel influenza A virus H1N1 hypotype (A-H1N1) specificity upstream and downstream primer,, a specificity fluorescent probe (Fam fluorescent mark), the specificity upstream and downstream primer of influenza A virus universal (IAV-U),, a specificity fluorescent probe (VIC fluorescent mark), (FQ-Buffer includes magnesium ion to the quantitative fluorescent PCR reaction buffer, Tris-HCl etc.), four kinds of nucleotide monomers (dNTPs), the reaction system that compositions such as pcr amplification toughener and deionized water constitute.
According to a preferred embodiment of the invention, wherein said amplification procedure be at a kind of hyperchannel and detect in real time fluorescent signal the fluorescent quantitation amplification device on carry out.
Another object of the present invention provides a kind of test kit that is used for the novel influenza A virus H1N1 hypotype (IAV-H1NI) of rapid detection, and this test kit comprises that (1) viral nucleic acid extracts reagent; (2) reversed transcriptive enzyme system and Taq enzyme system; (3) novel influenza A virus H1N1 hypotype two-color fluorescence PCR reaction system (PCRMIX); (4) the positive and negative quality control product of novel influenza A virus H1N1 hypotype.
According to a preferred embodiment of the invention, wherein said novel influenza A virus H1N1 hypotype positive quality control product is meant that the amplified fragments of 89bp is connected to the recombinant plasmid that constitutes on the T carrier through the clone, and through quantitatively strict, its concentration is 10 7Copies/ml.The amplification of positive quality control, novel influenza A virus H1N1 hypotype (IAV-H1N1), the common qualitative PCR of influenza A virus is seen Fig. 1.Positive quality control product is carried out gradient dilution, the typical curve of its amplification and kinetic curve such as Fig. 2 and 3.
According to a preferred embodiment of the invention, wherein said test kit is a Two Colour Fluorescence probe PCR technology owing to what adopt, not only can detect novel influenza A virus H1N1 hypotype, can also detect the detection of promptly so-called influenza A virus universal (IAV-U) to each hypotype of influenza A virus.The every part of reagent one-time detection that is to say this test kit just has the ability that detects two kinds of viruses simultaneously.
According to a preferred embodiment of the invention, it is simultaneously novel influenza A virus H1N1 hypotype (IAV-H1NI) and influenza A virus universal (IAV-U) RNA to be increased and carry out detection by quantitative by a step PCR-Two Colour Fluorescence probe amplification in vitro method that wherein said quick real-time quantitative detects, need not special reverse transcription reaction process, simplified testing process and saved detection time.
According to a preferred embodiment of the invention, wherein said fluorescent quantitation amplification device can be the instrument of ABIPRISM 7700, ABI PRISM5700, ABI GeneAmp 7000 (need cut the reaction lid), use thin-walled tubes such as ABI RISM7300/7500 (using 8 pipes), MJ Opticon (using 8 pipes), also can be use instruments capillaceous such as LightCycler
According to a preferred embodiment of the invention, wherein said result judges that the Ct value that will satisfy the positive quality control product amplification all should be less than 30, and is standard S type amplification curve.Otherwise it is invalid that test this time is considered as, and Total Test should carry out again.
According to a preferred embodiment of the invention, need use the manual setting threshold value to make negative quality control product Ct value more than 40 during wherein said interpretation of result, the Ct value is positive less than 35 round-robin; The Ct value is gray area between 35-40 circulation, need carry out revision test.After the revision test, if the Ct value still between 35-40 circulation, is judged as the positive, there do not have fluorescent value to increase to be negative.
In order to finish method of the present invention, at first collect specimen is placed in the centrifuge tube, airtight censorship.Getting 500 μ l sample liquid then adds 500 μ l virus concentrated solution and fully shakes 4 ℃ of refrigerated centrifuges 13, the centrifugal 10min of 000rpm, remove supernatant, keep the sample liquids about 50 μ l, add fully concussion of 500 μ l Trizol reagents (RNA extracting solution), room temperature is placed 5min.For serum or blood plasma etc., getting 500 μ l serum adds 500 μ l virus concentrated solution and fully shakes, 4 ℃ of refrigerated centrifuges 13, the centrifugal 10min of 000rpm, remove supernatant, keep the sample liquids about 50 μ l, add fully concussion of 500ul Trizol reagents (RNA extracting solution), room temperature is placed 5min.For tissue such as lungs etc., get that tissue shreds with glass homogenizer homogenate or scissors about 50mg, add 500 μ l Trizol reagents (RNA extracting solution), grind or vibration, transfer in the EP pipe of 1.5mL room temperature placement 5min.Add the 100ul chloroform then, firmly shake 15s, room temperature leaves standstill 5min, and 4 ℃ 13, the centrifugal 10min of 000rpm.Carefully with the upper water phase transition in clean centrifuge tube, add the equal-volume Virahol, abundant mixing, 13, the centrifugal 10min of 000rpm.Abandon supernatant, add the DEPC ethanol of 500 μ l 75%, abundant mixing, 13, the centrifugal 10min of 000rpm, the careful suction removed most of ethanol.With extraction tube uncovered in air at room temperature dry 5min treat that ethanol volatilization is clean, with 20 μ l DEPC H2O dissolution precipitations.
According to another preferred embodiment according to the present invention, take out novel influenza A virus H1N1 hypotype-PCR reaction solution (PCRMIX contains amplification primer probe of usefulness and ion etc.), Taq enzyme system, reversed transcriptive enzyme system, room temperature melt and the vibration mixing after, 10, the centrifugal 10s of 000rpm.Each test reaction system is formulated as follows table:
Reagent ??PCRMIX Taq enzyme system Reversed transcriptive enzyme system
Consumption ??20μl ??1μl ??1μl
Calculate the usage quantity of good each reagent, add in the clean 0.2ml centrifuge tube of a proper volume, abundant mixing, 10, the centrifugal 10s of 000rpm adds 22 μ l respectively in setting each PCR reaction tubes, add to handle back sample (RNA of extraction), positive quality control product and negative quality control product 3 μ l in every pipe, 10, instantaneous centrifugal 10 seconds of 000rpm.Each reaction tubes is put into the reactive tank of quantitative PCR instrument, by correspondence positive quality control product, negative quality control product and key sample not are set in proper order, and sample title and mark fluorescent radical species (reporter group is set to FAM and VIC, quenching group TAMRA) are set.
According to another preferred embodiment of the present invention, the condition that is used to increase is: ABI PRISM 7700, ABI PRISM5700, ABI GeneAmp7000 (need cut the reaction lid), ABI PRISM 7300/7500 (using 8 pipes), MJ Opticon (using 8 pipes) etc. use the instrument of thin-walled tube, cycling condition: 42 ℃ → 20 minutes, 93 ℃ then → 2 minutes, then 93 ℃ 30 seconds → 55 ℃ 45 seconds, detect fluorescent signal, 40 circulations.LightCycler etc. use instrument capillaceous: cycling condition: 42 ℃ → 20 minutes, 93 ℃ → 2 minutes, then 93 ℃ 5 seconds → 58 ℃ 45 seconds, detect fluorescent signal, totally 40 circulations.After reaction finishes, use the manual setting threshold value to make negative quality control product Ct value more than 40, the Ct value is positive less than 35 round-robin; The Ct value is gray area between 35-40 circulation, need carry out revision test.After the revision test, if the Ct value still between 35-40 circulation, is judged as the positive, there do not have fluorescent value to increase to be negative.Will note following problem during judged result: negative quality control product should be total negative; Novel influenza A virus H1N1 hypotype-positive quality control product, the Ct value of positive quality control product all should be less than 30, and are standard S type amplification curve.Above condition should satisfy simultaneously, should carry out again otherwise test this time is considered as invalid Total Test.
Test kit of the present invention is mainly used in and detects a kind of novel influenza A virus H1N1 hypotype and each hypotype of influenza A virus, the former is a kind of new variant virus, it has integrated swine influenza virus, avian influenza virus and human influenza virus's gene fragment, be a kind of virus of new reorganization, it has caused the outburst of influenza epidemic situation in April, 2009 world wide.The whole testing process of test kit of the present invention comprises that influenza A virus H1N1 hypotype mRNA, single stage method double color fluorescent quantitative PCR detect virus mRNA in the extraction sample, and result's steps such as analysis.In order to finish these steps, test kit of the present invention can be divided into viral nucleic acid again and extract reagent and two parts of double color fluorescent quantitative PCR reaction reagent.Wherein, the former mainly comprises viral concentrated solution and RNA extracting solution etc., and the latter mainly comprises PCR MIX (1 each primer of μ M and probe, 200 μ MdNTP, 50mM Tris-HCl (pH8.3), 3.5mM MgCl2,50mM KCl, 25mM (NH4) 2SO4 etc.), reversed transcriptive enzyme, heat-resisting Taq enzyme, positive quality control product, negative quality control product and DEPC treated water etc.
Test kit of the present invention confirms that through repeatedly different repeated experiments checkings this test kit has the performance and the advantage of better repeatability, specificity and stability.
In the method for the present invention, owing to used at novel influenza A virus H1N1 hypotype and designed primer and the Two Colour Fluorescence probe amplification specific gene fragment of each hypotype nucleotide sequence of influenza A virus, not only can detect novel influenza A virus H1N1 hypotype, can also detect simultaneously each hypotype of the susceptible poison of stream first type, thereby realized in the sample simultaneously purpose that novel influenza A virus H1N1 hypotype and each hypotype of influenza A virus are accurately detected simultaneously in real time, to use very convenient.Because this method has been introduced specificity amplification primer and Two Colour Fluorescence probe, make to detect to have better sensitivity and specificity, thereby avoided the not high problem of other detection method specificitys.The present invention has simultaneously introduced contrast and the quantitative criterion of positive criteria product as experiment, and the RNA of novel influenza A virus H1N1 hypotype and each hypotype of influenza A virus is carried out rapid detection and quantitative.The another one aspect, the present invention has used single stage method fluorescent quantitative PCR technology, has simplified process of the test and has improved detection efficiency.In general, the judgement from the processing sample to the result only needed about 2 hours.So compare with traditional method, tool of the present invention also has simple to operate, advantage is quickly and easily arranged.Therefore, test kit of the present invention provides new method for the rapid detection of clinical samples and large sample generaI investigation, and uses for patient suspected's examination reliable methodology foundation is provided.
Four, description of drawings
Fig. 1 shows conventional pcr amplification specific fragment, 2% agarose gel electrophoresis, behind pcr amplification, its amplified production is observed under ultraviolet lamp through 2% agarose gel electrophoresis, can see the electrophoretic band consistent with the purpose clip size, from left to right be molecular weight Marker, positive control, IAV-H1N1 specific amplification fragment, IAV-U specific amplification fragment, negative control successively.Fig. 2 shows positive quantitative criterion template pcr amplification electrophorogram, and the right side swimming lane is dna molecular amount Marker; From 0 to 6 is corresponding successively: 10 0, 10 1, 10 2, 10 3, 10 4, 10 5, 10 6The positive quality control product amplified production of dilution; Fig. 3 shows positive quality control product standard form amplification dynamic curve, gradient dilution; The positive quality control product typical curve of Fig. 4.
Five, embodiment:
Embodiment 1: collection of specimens
Suitable sample type comprises throat swab sample, serum, secretory product and the animal lungs etc. of dying of illness.At first, for the throat swab sample, need to gather fall ill throat swab sample in 3 days of patient, with special-purpose sampling cotton swab, appropriateness firmly swabs pharynx rear wall and tonsilla position, both sides, should avoid touching tongue; Rapidly cotton swab is put into the sampling tube that 1~2ml preserves liquid (keeping liquid or physiological saline) is housed,, screw pipe lid and airtight censorship at the cotton swab bar that fractures near top end; For serum, get patient suspected's peripheric venous blood 3ml under the aseptic condition, go in the 5ml drying tube, room temperature leaves standstill 30min, and the centrifugal 5min of 3000g gets in the centrifuge tube that 1ml serum goes to the 1.5ml cleaning airtight censorship.For the preservation of sample, sample can be stored in-20 ℃ in a short time, and prolonged preservation can be put-70 ℃, but can not surpass 6 months, and sample transports and should adopt 0 ℃ of curling stone, and collect specimen (in the 12h) is immediately sent to detection.
Embodiment 2: the extraction of influenza A virus mRNA
For samples such as throat swabs, getting 500 μ l sample liquid adds 500 μ l virus concentrated solution and fully shakes, 4 ℃ of refrigerated centrifuges 13, the centrifugal 10min of 000rpm, remove supernatant, keep the sample liquids about 50 μ l, add fully concussion of 500 μ lTrizol reagents (RNA extracting solution), room temperature is placed 5min.For serum or blood plasma etc., getting 500 μ l serum adds 500 μ l virus concentrated solution and fully shakes, 4 ℃ of refrigerated centrifuges 13, the centrifugal 10min of 000rpm, remove supernatant, keep the sample liquids about 50 μ l, add fully concussion of 500ul Trizol reagents (RNA extracting solution), room temperature is placed 5min.For tissue such as lungs etc., get that tissue shreds with glass homogenizer homogenate or scissors about 50mg, add 500 μ l Trizol reagents (RNA extracting solution), grind or vibration, transfer in the EP pipe of 1.5mL room temperature placement 5min.Add the 100ul chloroform then, firmly shake 15s, room temperature leaves standstill 5min, and 4 ℃ 13, the centrifugal 10min of 000rpm.Carefully with the upper water phase transition in clean centrifuge tube, add the equal-volume Virahol, abundant mixing, 13, the centrifugal 10min of 000rpm.Abandon supernatant, add the DEPC ethanol of 500 μ l 75%, abundant mixing, 13, the centrifugal 10min of 000rpm, the careful suction removed most of ethanol.With extraction tube uncovered in air at room temperature dry 5min treat that ethanol volatilization is clean, with 20 μ l DEPC H2O dissolution precipitations.
Embodiment 3: detect novel influenza A virus H1N1 hypotype (IAV-H1N1) and each hypotype of influenza A virus (IAV-U) with this test kit
Take out influenza A virus H1N1 hypotype two-color fluorescence PCR MIX, Taq enzyme system, reversed transcriptive enzyme system, behind room temperature thawing and the vibration mixing, 10, the centrifugal 10s of 000rpm.Each test reaction system is formulated as follows, PCRMIX 20 μ l, Taq enzyme system and each 1ul of reversed transcriptive enzyme calculate the usage quantity of each reagent well, add in the clean 0.2ml centrifuge tube of a proper volume, abundant mixing, 10, the centrifugal 10s of 000rpm, in the PCR reaction tubes of setting, add 22 μ l respectively, in every pipe, add and handle back sample (extracting RNA) or IAV-positive quality control product and negative quality control product 3 μ l, 10, instantaneous centrifugal 10 seconds of 000rpm.
Each reaction tubes is put into the reactive tank of quantitative PCR instrument, by correspondence negative quality control product is set in proper order, positive quality control product and key sample not, and the sample title is set, mark fluorescent radical species (reporter group FAM and VIC quencher group TAMRA) and cycling condition: ABI PRISM 7700, ABI PRISM5700, ABIGeneAmp 7000 (need cut the reaction lid), ABI PRISM7300/7500 (using 8 pipes), MJOpticon (using 8 pipes) etc. use the instrument cycling condition of thin-walled tube: 42 ℃ → 20 minutes, back 93 ℃ → 2 minutes, the back 93 ℃ 30 seconds → 55 ℃ 45 seconds, 40 circulations.LightCycler etc. use instrument capillaceous, cycling condition: 42 ℃ → 20 minutes, 93 ℃ → 2 minutes, back 93 ℃ 5 seconds → 58 ℃ 45 seconds, totally 40 circulations.
Embodiment 4: interpretation of result and judgement
After reaction finishes, use the manual setting threshold value to make negative quality control product Ct value more than 40, the Ct value is positive less than 35 round-robin; The Ct value is gray area between 35-40 circulation, need carry out revision test.After the revision test, if the Ct value still between 35-40 circulation, is judged as the positive, there do not have fluorescent value to increase to be negative.Need to satisfy: negative quality control product should be total negative; The Ct value of positive quality control product all should be less than 30, and are standard S type amplification curve.Above condition should satisfy simultaneously, otherwise that test this time is considered as is invalid, and Total Test should carry out again.
Embodiment 5: the clinical application of the inventive method
Use method of the present invention and test kit, pick up from the breadboard sample of Zhuhai, Guangdong Province Entry-Exit Inspection and Quarantine Bureau to 600 parts and carry out single stage method double color fluorescent quantitative PCR and detect, and in the experiment of comparing of other like products, finally through the experimental verification of checking order.The result shows: the test kit of the inventive method, sensitivity are 10 2Copies, accuracy and specificity reach 100%, and repeatability and stability are better, and preservation condition of this test kit and validity period are :-20 ℃, 12 months.The formulation that the invention of present method can be the prevention and control scheme of the detection of novel influenza A virus H1N1 hypotype and each hypotype mRNA of influenza A virus and epidemic situation provides molecular biology experiment data science, individuation, for patient's diagnosis provides reliable Molecular Virology evidence.Exist if occur influenza A virus H1N1 hypotype mRNA virus in patient's throat swab or the serum, then will take corresponding measure immediately, also avoid producing unnecessary consequence.Test kit result's of the present invention judgement has following several situation, sees Table 1:
The novel influenza A virus H1N1 of table 1. hypotype double color fluorescent quantitative PCR detection kit result judges
Positive quality control Negative Quality Control FAM fluorescence VIC fluorescence The result judges
??+ ??- ?+ ?+ The novel I AV-H1N1 positive; The IAV-U positive
??+ ??- ?+ ?- The novel I AV-H1N1 positive; The IAV-U feminine gender
??+ ??- ?- ?+ Novel I AV-H1N1 feminine gender; The IAV-U positive
??+ ??- ?- ?- Novel I AV-H1N1 feminine gender; The IAV-U feminine gender
Six, sequence table
SEQUENCE?LISTING
<110〉Beijing Suo Ao Bioisystech Co., Ltd
<120〉a kind of novel influenza A virus H1N1 hypotype two-color fluorescence PCR detection method and test kit thereof
<130>
<160>8
<170>PatentIn?version?3.3
<210>1
<211>88
<212>DNA
<213〉IAV-H1N1 HA gene
<400>1
ggtacggtta?tcaccatcaa?aatgagcagg?ggtcaggata?gcagccgacc?tgaagagcac????60
acagaatgcc?attgacgaga?ttactaac???????????????????????????????????????88
<210>2
<211>22
<212>DNA
<213〉artificial sequence (primer)
<400>2
ggtacggtta?tcaccatcaa?aa?????????????????????????????????????????????22
<210>3
<211>24
<212>DNA
<213〉artificial sequence (primer)
<400>3
gttagtaatc?tcgtcaatgg?catt???????????????????????????????????????????24
<210>4
<211>27
<212>DNA
<213〉artificial sequence (probe)
<400>4
atatgcagcc?gacctgaaga?gcacaca????????????????????????????????????????27
<210>5
<211>82
<212>DNA
<213〉IAV-U M gene
<400>5
tgtgccactt?gtgaacagat?tgctgattca?cagcatcggt?ctcacagaca?gatggctact????60
accaccaatc?cactaatcag?gc?????????????????????????????????????????????82
<210>6
<211>22
<212>DNA
<213〉artificial sequence (primer)
<400>6
tgtgccactt?gtgaacagat?tg????????22
<210>7
<211>20
<212>DNA
<213〉artificial sequence (primer)
<400>7
gcctgattag?tggattggtg???????????20
<210>8
<211>26
<212>DNA
<213〉artificial sequence (probe)
<400>8
tgattcacag?catcggtctc?acagac????26

Claims (4)

1. the present invention is a kind of novel influenza A virus H1N1 (A (H 1 N 1) virus) hypotype two-color fluorescence PCR detection method and test kit thereof, from sample, extract novel influenza A virus H1N1 hypotype (IAV-H1N1) RNA, by a step PCR-Two Colour Fluorescence probe amplification in vitro method novel influenza A virus H1N1 hypotype and each hypotype RNA of first C-type virus C are increased, reach the purpose of quick real-time quantitative detection.
2. a kind of novel influenza A virus H1N1 according to claim 1 (A (H 1 N 1) virus) hypotype two-color fluorescence PCR detection method and test kit thereof, be characterised in that what be used to detect novel influenza A virus H1N1 hypotype (IAV-H1N1) is the stronger hemagglutinin of this C-type virus C variability (Hemagglutinin) gene, Auele Specific Primer and fluorescent probe sequence are as follows: (1) novel influenza A virus H1N1 hypotype upstream primer: 5 '-GGTACGGTTATCACCATCAAAA-3 '; (2) novel influenza A virus H1N1 hypotype downstream primer: 5 '-GTTAGTAATCTCGTCAATGGCATT-3 '; (3) novel influenza A virus H1N1 hypotype fluorescent probe: 5 '-FAM-ATATGCAGCCGACCTGAAGAGCACACA-TAMRA-3 '.
Be used to detect each hypotype of influenza A virus (IAV-U) be this C-type virus C conservative stromatin (Matrix Protein) gene, Auele Specific Primer and probe sequence are as follows: the universal upstream primer of (4) influenza A virus: 5 '-TGTGCCACTTGTGAACAGATTG-3 '; (5) the universal downstream primer of influenza A virus: 5 '-GCCTGATTAGTGGATTGGTG-3 '; (6) the universal fluorescent probe of influenza A virus: 5 '-VIC-TGATTCACAGCATCGGTCTCACAGAC-TAMRA-3 '.
3. according to the described a kind of novel influenza A virus H1N1 hypotype double color fluorescent quantitative PCR detection kit of claim 1, it is characterized in that, prepare reaction system in the following manner: PCR reaction solution (PCR MIX) 20 μ l, Taq enzyme system and each 1 μ l of reversed transcriptive enzyme totally 22 μ l add in the clean 0.2ml centrifuge tube of a proper volume, abundant mixing, 10, the centrifugal 10s of 000rpm adds processing back sample (i.e. the RNA of Ti Quing), positive quality control product and negative quality control product and carries out augmentation detection in every pipe.
4. be mainly following composition according to the described PCR reaction solution of claim 3 (PCR MIX): 1 each primer of μ M and probe, 200uMdNTP, 50mM Tris-HCl (pH8.3), 5mM MgCl 2, 50mMKCl, 25mM (NH 4) 2SO 4Form, the one step amplification program is: ABI PRISM 7700, ABIPRISM5700, ABIGeneAmp7000, ABI PRISM7300/7500, MJ Opticon etc., 42 ℃ → 20 minutes, 93 ℃ then → 2 minutes, 93 ℃ 30 seconds → 55 ℃ 45 seconds (detection fluorescence) then, 40 circulations; Amplification programs such as LightCycler are: 42 ℃ → 20 minutes, and 93 ℃ then → 2 minutes, 93 ℃ of 5 seconds → 58 ℃ 45 seconds (detection fluorescence), totally 40 circulations then.
CN2009101366353A 2009-05-11 2009-05-11 Double-color fluorescence polymerase chain reaction (PCR) detection method of novel influenza A virus H1N1 subtype and kit thereof Pending CN101886139A (en)

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* Cited by examiner, † Cited by third party
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CN102286639A (en) * 2011-08-05 2011-12-21 江苏硕世生物科技有限公司 Type A H1N1/influenza A virus nucleic acid dual fluorescent polymerase chain reaction (PCR) detection kit
CN102676691A (en) * 2011-10-31 2012-09-19 武汉大学 H1N1, PRRSV and O-FMDV quadruple detection kit
CN102816865A (en) * 2011-10-31 2012-12-12 武汉大学 H1N1, PRRSV and CSFV quadruple detection kit
CN103131794A (en) * 2011-12-05 2013-06-05 中国人民解放军军事医学科学院卫生学环境医学研究所 Suspension chip method capable of quickly diagnosing A-type H1N1 influenza viruses
CN108192996A (en) * 2018-03-13 2018-06-22 中国人民解放军南京军区南京总医院 A kind of multiple RT-RPA primers for influenza A virus detection and H1 and H3 partings combine and its application
CN109212207A (en) * 2018-10-12 2019-01-15 北京理工大学 For detecting fluorescent reagent of influenza virus hemagglutinin albumen and preparation method thereof and detection method in oral cavity

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102286639A (en) * 2011-08-05 2011-12-21 江苏硕世生物科技有限公司 Type A H1N1/influenza A virus nucleic acid dual fluorescent polymerase chain reaction (PCR) detection kit
CN102676691A (en) * 2011-10-31 2012-09-19 武汉大学 H1N1, PRRSV and O-FMDV quadruple detection kit
CN102816865A (en) * 2011-10-31 2012-12-12 武汉大学 H1N1, PRRSV and CSFV quadruple detection kit
CN102816865B (en) * 2011-10-31 2014-04-02 武汉大学 H1N1, PRRSV and CSFV quadruple detection kit
CN102676691B (en) * 2011-10-31 2014-07-16 武汉大学 H1N1, PRRSV and O-FMDV quadruple detection kit
CN103131794A (en) * 2011-12-05 2013-06-05 中国人民解放军军事医学科学院卫生学环境医学研究所 Suspension chip method capable of quickly diagnosing A-type H1N1 influenza viruses
CN108192996A (en) * 2018-03-13 2018-06-22 中国人民解放军南京军区南京总医院 A kind of multiple RT-RPA primers for influenza A virus detection and H1 and H3 partings combine and its application
CN108192996B (en) * 2018-03-13 2020-04-21 中国人民解放军南京军区南京总医院 Multiple RT-RPA primer combination for detecting influenza A virus and parting H1 and H3 and application thereof
CN109212207A (en) * 2018-10-12 2019-01-15 北京理工大学 For detecting fluorescent reagent of influenza virus hemagglutinin albumen and preparation method thereof and detection method in oral cavity
CN109212207B (en) * 2018-10-12 2019-08-20 北京理工大学 For detecting fluorescent reagent of influenza virus hemagglutinin albumen and preparation method thereof and detection method in oral cavity

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Application publication date: 20101117