CN102154511B - Preparation method and application of reagent for detecting novel type A H1N1 2009 influenza virus with one-step method - Google Patents
Preparation method and application of reagent for detecting novel type A H1N1 2009 influenza virus with one-step method Download PDFInfo
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Abstract
The invention relates to a one-step-method fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection kit for detecting a type A H1N1 2009 influenza virus (also called pH1N1-2009 influenza virus) and a detection method and application thereof. The one-step-method fluorescent RT-PCR detection kit provided by the invention comprises a pair of specific primers and a fluorescent probe, wherein the specific primers and the fluorescent probe are designed by taking a NA (neuraminidase) gene conservative segment of the pH1N1-2009 influenza virus as a target; and the detection principle is that: real-time quantitative differential detection is performed on a sample by using a fluorescent probe in combination with the specificity of target RNA (Ribonucleic Acid). By adopting the one-step-method fluorescent RT-PCR detection kit and the detection method provided by the invention, differential detection can be quickly performed on the pH1N1-2009 influenza virus. The detection kit and the detection method are easy and convenient to operate, and have high detection specificity and wide application range.
Description
Technical field
The present invention relates to a kind of virus detection kit, relate in particular to a kind of pH1N1-2009 influenza virus detection kit.
Background technology
Influenza Virus orthomyxovirus section consists of genome by 8 sub-thread strand RNAs.Influenza virus is different according to the antigenicity of core protein (NP) and matrix (M), can be divided into A, B, C three types, wherein pathogenic the strongest, in the world popular the widest be A type influenza virus.A type influenza virus can be divided into 15 hypotypes according to the difference of surface glycoprotein hemoglutinin (HA), and H1-H15 can be divided into 9 hypotype N1-N9 according to the difference of surface glycoprotein neuraminidase (NA).
Wherein, the H1N1 influenza virus constantly breaks through the kind boundary in mutation process, human life's health is brought serious threat.1918-1919 Europe during influenza pandemic causes more than 5,000 ten thousand people dead, and cause of disease is H1N1 subtype influenza virus.Shope in 1931 first in the pig body isolation identification H1N1 subtype influenza virus.Occur in so-called " New Jersey " event in the U.S. in 1976, about 500 people infect H1N1 subtype influenza virus, this virus be separated in the pig body at that time viral identical.In April, 2009, run initially in Mexico, propagating into very soon the U.S. is to be caused by new H1N1virus in the influenza of Global prevalence afterwards.This time epidemic situation caused hundred people to infect, and had caused a large amount of epidemic situations to propagate into thereafter the whole world, had caused personnel's death and a large amount of financial losses.Cause this time extensive popular virus more in the past the virus of isolation identification obvious variation has all occured, velocity of propagation is unusually rapid, brings global fear.On June 2nd, 2009, the pig on one pig farm, Canada West Alberta detects H1N1virus with it, and this is to have found first that in the world pig is infected by this new virus.Gene test finds, these pigs virus with it is consistent with this virus in the current popular of Mexico, the U.S. and other countries' discovery, this new H1N1virus pH1N1-2009 influenza virus that is otherwise known as.
The H1N1 influenza virus possesses to stride between group's propagation and group propagates two kinds of circulation ways.H1N1 new type influenza virus is former to be a kind of disease that infects in pig, belongs to influenza A virus.Belong to the high-incidence season autumn and winter, can propagate the whole year.The porcine influenza epidemic situation of Mexico in 2009 and U.S.'s outburst, it is caused to be H1N1 virus.Mexico outburst H1N1 epidemic disease tide caused hundred people to infect, and had caused a large amount of epidemic situations to propagate into thereafter the whole world, had caused personnel's death and a large amount of financial losses to April in 2009 3.
Because variation has occured virus, without any meaning, the mode that the most effective control epidemic situation enlarges is to set up fast diagnostic method to diagnostic techniques in the past to the control pathophoresis.The most frequently used quick detection means is real-time fluorescence detection method at present, and the method for therefore setting up a kind of real-time fluorescence detection H1N1 virus is significant.Because but H1N1 virus direct infection people causes the mankind ill or dead, its public health meaning is day by day remarkable.Reinforcement aspect reducing the world economy loss and improving human hygiene and health, all has profound significance to the research of H1N1 virus method for quick.
As everyone knows, based on the advantage on ageing and the accuracy, the real-time fluorescence RT-PCR technology has progressively replaced general RT-PCR method on a lot of projects that epidemic disease detects, become the most frequently used molecular biology method of epidemic disease detection field, but up to the present, not yet there is the report of differentiating simultaneously the RT-PCR method for quick research of detection pH1N1-2009 influenza virus single stage method and correlation detection reagent in China.
Summary of the invention
The purpose of this invention is to provide a kind of single stage method fluorescent RT-PCR detection reagent box for detection of the pH1N1-2009 influenza virus.Inventive point is that described test kit comprises a pair of Auele Specific Primer and a fluorescent probe, realizes the quick discriminating of pH1N1-2009 influenza virus is detected by the specific binding of this Auele Specific Primer, fluorescent probe and H1N1 Influenza Virus RNA.
Another object of the present invention provides the detection method of above-mentioned single stage method fluorescent RT-PCR detection reagent box.
Another object of the present invention provides above-mentioned single stage method fluorescent RT-PCR detection reagent box and is differentiating the application that detects in the pH1N1-2009 influenza virus.
The preparation method of single stage method fluorescent RT-PCR detection reagent box provided by the invention is as follows:
At first, synthesize Auele Specific Primer and fluorescent probe sequence.
It is target that the present invention selects the conserved sequence fragment of pH1N1-2009 influenza virus NA gene, is 121bp for the amplification target fragment length of pH1N1-2009 influenza virus NA gene test, and the sequence of this amplification target fragment is such as sequence table＜400〉shown in 4.According to the characteristics of pH1N1-2009 influenza virus NA gene conserved sequence, use the PrimerSeleCT software design primer sequence to be measured among primer Express 3.0 softwares and the DNAStar; Adopt β-acetonitrile phosphorous acid amination synthesis method, use full-automatic dna synthesizer to carry out primer synthetic to be measured and the probe of OligoDNA, at probe 5 ' end mark fluorescent reporter group FAM (6-Fluoresceincarboxylic acid), 3 ' holds mark quenching group BHQ simultaneously.
Secondly, screening Auele Specific Primer and fluorescent probe sequence.
Synthetic primer and probe are passed through respectively specificity comparative experiments, sensitivity test experience, repeatability and accuracy test experience; Final definite more excellent a pair of Auele Specific Primer and fluorescent probe of properties, primer and probe sequence are:
Primer: F:5 '-CAACACCAACTT TGC TGC-3 ' (such as sequence table＜400〉1 shown in)
R:5 '-GGAACCGATTCT TCACTG TTGTC-3 ' (such as sequence table＜400〉2 shown in)
Probe: probe:5 '-FAM-CAGTCAGTGGTTCCGTGGAAATTAGC-BHQ-3 ' (such as sequence table＜400〉3 shown in)
At last, determine that reaction system and test kit form.
Reaction system is set as 25 μ L, screens within the specific limits above-mentioned Auele Specific Primer, fluorescent probe optimum concn.Screening experiment all adopts the equivalent template at every turn and only establishes a variable, and take factors such as CT value, fluorescence increment and plateaus as investigating foundation, repeated experiments 3 times if the result is stable, can be defined as optimum concentration value.
The common practise that consists of this area of fluorescent RT-PCR detection reagent box, the technician can select according to actual needs.
As preferably, single stage method fluorescent RT-PCR detection reagent box component provided by the invention is as shown in the table:
| Solution A (2 * RT-PCR mixed solution contains primer and probe) | 1mL |
| Solution B (Taq archaeal dna polymerase; The AMV ThermoScript II) | 30μL |
| Solution C (DEPC-H 2O) | 1mL |
| Positive control | 1mL |
| Negative control | 1mL |
Mentioned solution A comprises and takes from AgPath-ID
TM2 * RT-PCR mixed solution and Auele Specific Primer and fluorescent probe in the One-Step RT-PCR Kit test kit, as preferably, the corresponding 1 μ l Auele Specific Primer (F) of 2 * RT-PCR mixed solution of 12.5 μ l, 1 μ l Auele Specific Primer (R) and 0.5 μ l fluorescent probe in the solution A, namely wherein volume proportion is 12.5: 1: 1: 0.5.
Mentioned solution B takes from AgPath-ID
TMOne-Step RT-PCR Kit test kit comprises Taq archaeal dna polymerase and AMV ThermoScript II.Above-mentioned AgPath-ID
TMOne-Step RT-PCR Kit test kit is available from ABI company.
Solution C is DEPC-H
2O.
Positive control is influenza split vaccine diluent, available from the Beijing Kexing Biotech Products Co., Ltd.
Negative control is the SPF chick embryo allantoic liquid, available from Beijing Cimmeria Wei Tong company.
The present invention also provides the detection method of above-mentioned single stage method fluorescent RT-PCR detection reagent box, and concrete steps are:
(1) extracts sample rna
(2) preparation of reaction component: the reaction cumulative volume is 25 μ l, adds following component in the reaction tubes:
(3) fluorescence RT-PCR response procedures
By following parameter the fluorescence RT-PCR response procedures is set:
In the detection method of above-mentioned single stage method fluorescent RT-PCR detection reagent box, the setting of fluorescence RT-PCR response procedures parameter is to obtain through comparative experiments repeatedly by optimizing good reaction system.Experimental principle is certain reaction conditions (temperature, reaction times and cycle number) scope of setting in advance, repeatedly test in the scope, each test is adopted with pipe equivalent template, each reaction conditions revision test 3 times, take CT value, fluorescence increment, plateau and the factor such as consuming time as investigating foundation, determine optimum reaction condition.
(4) interpretation of result: directly read detected result, with the setting threshold line that is as the criterion of the vertex just above normal negative control amplification curve; The CT value is judged as feminine gender greater than 38.0 or without amplification curve; The amplification curve CT value that represents FAM is less than or equal to 35.0, and typical amplification curve occurs, is judged as the positive.The sample that the CT value of amplification curve is between 35.0～38.0 is considered as suspicious specimen, and suggestion is reformed.Reform the CT value as a result greater than 38.0 or negative without the amplification curve person, otherwise positive.
The beneficial effect of single stage method fluorescent RT-PCR detection reagent box provided by the invention is:
1, can be to Nasopharyngeal swabs, organize the sample of the various ways such as ground sample suspension, chick embryo allantoic liquid, cerebrospinal fluid to carry out rapid detection, applied widely;
2, single step reaction is finished detecting step, receives behind the sample can draw detected result, weak point consuming time easy and simple to handle in 110 minutes;
3, increased the restricted condition of probe specificity binding purposes amplified production, specificity, susceptibility and accuracy are high;
4, the purpose expanding fragment length only has 121bp, can detect the small segment purpose sample that often is missed in conventional RT-PCR detects.
Embodiment
Below among each embodiment, as being commercially available standard specimen without the reagent, the material that clearly indicate concrete source.The pH1N1-2009 Influenza Virus RNA of using among each embodiment and positive control are influenza split vaccine diluent, available from the Beijing Kexing Biotech Products Co., Ltd.
The preparation and determination methods method of embodiment 1 one-step method real-time fluorescent RT-PCR stdn test kit
One, reaction principle
The reaction principle of one-step method real-time fluorescent RT-PCR stdn test kit provided by the invention is: selecting the conserved sequence fragment of pH1N1-2009 influenza virus NA gene is target, the specific probe that design is complementary with it and supporting primer form test kit, by real time fluorescence quantifying PCR method, realize the detection to the pH1N1-2009 influenza virus.
Two, preparation method
1, design synthesising probing needle and primer
Primer, probe in the one-step method real-time fluorescent RT-PCR test kit provided by the invention is based on the conserved sequence fragment design of pH1N1-2009 influenza virus NA gene, and amplification target fragment length scale is 121bp.Amplification target nucleotides sequence is classified as: 5 '-CAACACCAACTTTGCTGCTGGACAGTCAGTGGTTTCCGTGAAATTAGCGGGCAATT CCTCTCTCTGCCCTGTTAGTGGATGGGCTATATACAGTAAAGACAACAGTATAAGA ATCGGTTCC-3 '.
Design procedure is: according to the characteristics of pH1N1-2009 influenza virus NA gene conserved sequence, use the PrimerSeleCT software among primer Express 3.0 softwares and the DNAStar, the synthetic primer to be measured of design and probe; The synthetic employing β of primer and probe-acetonitrile phosphorous acid amination synthesis method uses full-automatic dna synthesizer to carry out the synthetic of OligoDNA; The synthetic two ends mark that carries out simultaneously of probe, the fluorescence report group of probe 5 ' end mark is FAM (6-Fluoresceincarboxylic acid), the group of 3 ' end mark is BHQ; , simultaneous test preferred through a large amount of reaction conditions and proof test are selected the good primer of best performance and probe (concrete preferred method is seen embodiment 2).Final a pair of Auele Specific Primer and specificity fluorescent probe of determining is 121bp for the amplification target fragment length of pH1N1-2009 influenza virus NA gene test.The sequence of primer and probe is:
Primer: F:5 '-CAACACCAACTT TGC TGC-3 '
R：5’-GGAACCGATTCT TCACTG TTGTC-3’
Probe: probe:5 '-FAM-CAGTCAGTGGTTCCGTGGAAATTAGC-BHQ-3 '
Detecting primer sequence is (dna sequence dna).
2, determine reaction system
Reaction system is set as 25 μ L, screening primer, probe optimum concn.Primer concentration is increased progressively with 0.05 μ mol/L by 0.25 μ mol/L～0.6 μ mol/L, concentration and probe concentration is increased progressively with 0.05 μ mol/L by 0.1 μ mol/L～0.45 μ mol/L, each test is adopted the equivalent template and is only established a variable, take factors such as CT value, fluorescence increment and plateaus as investigating foundation, repeated experiments 3 times, if the result is stable, can be defined as optimum concentration value.
Grope the RT-PCR optimum reaction condition to optimize good reaction system, in the scope of " 95 ℃ of 1～12min; 95 ℃ of 35～45 circulations, 15～30s and 50～60 ℃, 35～60s ", repeatedly test, each test is adopted with pipe equivalent template, each reaction conditions revision test 3 times, take CT value, fluorescence increment, plateau and the factor such as consuming time as investigating foundation, determine respectively optimum reaction condition.
Through optimizing, pH1N1-2009 influenza virus one-step method real-time fluorescent RT-PCR adopts 25 μ L volumetric reaction liquid, in ABI7500 type quantitative real time PCR Instrument, is undertaken by following reaction parameter: 50 ℃ of reverse transcription 30min, 95 ℃ of denaturation 10min; 95 ℃ of sex change 15s, 55 ℃ of annealing 45s, 45 circulations of increasing.
3, determine that test kit forms
Test kit forms (25 μ l reaction * 50 times): according to above-mentioned test-results, the reaction conditions that, simultaneous test preferred according to reaction conditions and proof test are determined is formulated as follows the test kit component:
| Solution A (2 * RT-PCR mixed solution contains primer and probe) | 1mL |
| Solution B (Taq archaeal dna polymerase, AMV ThermoScript II) | 30μL |
| Solution C (DEPC-H 2O) | 1mL |
| Positive control | 1mL |
| Negative control | 1mL |
Mentioned solution A comprises and takes from AgPath-ID
TM2 * RT-PCR mixed solution and Auele Specific Primer and fluorescent probe in the One-Step RT-PCR Kit test kit, as preferably, the corresponding 1 μ l Auele Specific Primer (F) of 2 * RT-PCR mixed solution of 12.5 μ l, 1 μ l Auele Specific Primer (R) and 0.5 μ l fluorescent probe in the solution A, namely wherein volume proportion is 12.5: 1: 1: 0.5.
Solution B is taken from AgPath-ID
TMOne-Step RT-PCR Kit test kit comprises Taq archaeal dna polymerase and AMV ThermoScript II.
Above-mentioned AgPath-ID
TMOne-Step RT-PCR Kit test kit is available from ABI company.
Solution C is DEPC-H
2O.
Positive control is influenza split vaccine diluent, available from the Beijing Kexing Biotech Products Co., Ltd.
Negative control is the SPF chick embryo allantoic liquid, available from Beijing Cimmeria Wei Tong company.
Three, detection method
1, the extraction of sample RNA
The extraction of RNA can be by a kind of realization in following two kinds of methods:
(1) with reference to " practical molecular biology method handbook, phenol/chloroform nucleic acid extraction process extracts RNA.
(2) also can buy the commercial kit that extracts for all kinds of sample RNA from biological reagent company, extract test kit, the RNA of animal tissues extraction test kit, tissue juice RNA extraction test kit and cell culture fluid RNA such as virus genome RNA and extract test kit etc.Wherein, positive control in the one-step method real-time fluorescent RT-PCR test kit of the present invention need use viral RNA extraction test kit to carry out the extraction of positive control RNA, every part of positive control is got 100 μ L, is dissolved in 30 μ L DEPC water or the TE damping fluid stand-by behind the extraction RNA.
2, the preparation of reaction component
The reaction system cumulative volume is 25 μ l, adds following component in the reaction tubes:
3, fluorescence RT-PCR response procedures
By following parameter the fluorescence RT-PCR response procedures is set:
4, interpretation of result and judgement
(1) interpretation of result condition is set
Fluorescein kind according to amplification curve directly reads detected result.Baseline and threshold setting principle are adjusted according to the noise of instrument situation, are as the criterion with the vertex of threshold line just above normal negative control amplification curve.
(2) quality control standard
Negative control: without the CT value, and without amplification curve, be a sea line substantially.
Positive control: the CT value is less than 35.0, and typical amplification curve occurs, and the amplification curve of the corresponding derived component of 2 positive control pipes overlaps substantially, particularly near Threshold (fluorescence threshold value).Otherwise it is invalid that this time experiment is considered as.
(3) result describes and judges
Negative criterion: the CT value is greater than 38.0 or without amplification curve, and H1N1virus nucleic acid is negative in the expression sample.
Positive judgement standards: the amplification curve CT value that represents FAM is less than or equal to 35.0, and typical amplification curve occurs, and H1N1virus nucleic acid is positive in the expression sample.
Effective principle: according to the detection screening to a large amount of samples, the sample that the CT value of discovery amplification curve is between 35.0～38.0 is considered as suspicious specimen, and suggestion is reformed.Reform the CT value as a result greater than 38.0 or negative without the amplification curve person, otherwise positive.
The Performance Detection experiment of embodiment 2 single stage method fluorescence RT-PCR stdn test kits
One, specificity comparative experiments
(all RNA sample standard deviations extract from inactivation antigen to the RNA sample of H3N2, H5N1, H9N2, H1N1, Avian pneumo-encephalitis virus, available from the Harbin veterinary institute) detect, carry out the specificity comparative experiments of H1N1virus one-step method real-time fluorescent RT-PCR method.
Two, susceptibility test experience
PH1N1-2009 Influenza Virus RNA (being influenza split vaccine diluent) is carried out extracting RNA behind 10 times of serial dilutions, detect with pH1N1-2009 influenza virus one-step method real-time fluorescent RT-PCR method, determine sensitivity.
Three, stability and repeated test experience
When the circulation ratio of assessing pH1N1-2009 influenza virus one-step method real-time fluorescent RT-PCR method, stability, with primer, probe positive control (being influenza split vaccine diluent) RNA is detected, the above-mentioned product that respectively detects is carried out detected through gel electrophoresis, determine further whether the clip size of positive amplified production is consistent with expection.Under same reaction conditions, repeatedly detect, 6 parallel reactor pipes are set, each test repeats 12 times.
The control experiment of embodiment 3 single stage method fluorescence RT-PCR stdn test kits and ordinary method test sample
Experiment arranges: adopt respectively pH1N1-2009 influenza virus one-step method real-time fluorescent RT-PCR method and ordinary method that 200 parts of samples to be checked are detected, the detected result of two kinds of different methods of contrast.
Detection method: single stage method fluorescence RT-PCR stdn test kit, conventional PCR;
Experimental procedure: detecting the pH1N1-2009 Influenza Virus RNA that adopts in the test with pipe equivalent at each (is influenza split vaccine diluent, together lower) sample, every kind of method of every part of pH1N1-2009 Influenza Virus RNA sample detects and must repeat 2 times, 2 parallel tests all are set at every turn, only have when 2 revision tests of every kind of method and all parallel test results are consistent, just determine the detected result of this part pH1N1-2009 Influenza Virus RNA sample, otherwise need re-start detection.Whether at last, it is consistent to the detected result of every a pH1N1-2009 Influenza Virus RNA sample to compare respectively two kinds of methods, calculates the detected result coincidence rate of two kinds of methods.
Interpretation of result: the result shows, is detected the CT value of sample in one step of pH1N1-2009 influenza virus, the real-time fluorescence RT-PCR method detected that all is shown as the positive by two kinds of methods and all is less than or equal to 35.0; The CT value of negative control sample is tested high specificity all greater than 38.0 or without the CT value.Through the statistical study to a large amount of positives and negative sample detected result, determine that CT value 38.0 can be used as positive and negative threshold value (cut-off).The CT value is greater than 38.0 or can be judged to feminine gender without the CT value, and the CT value is less than or equal to 35.0 can be judged to the positive, is suspicious between 35.0-38.0, must again test.Therefore, pH1N1-2009 influenza virus one-step method real-time fluorescent RT-PCR method detects the high specificity of pH1N1-2009 influenza virus, and susceptibility is high, good reproducibility.
Claims (7)
1. the single stage method fluorescent RT-PCR detection reagent box for detection of the pH1N1-2009 influenza virus comprises solution A, and this solution A comprises a pair of Auele Specific Primer and a fluorescent probe, and the sequence of described Auele Specific Primer and fluorescent probe is:
Auele Specific Primer: F:5 '-CAACACCAACTT TGC TGC-3 '
R：5’-GGAACCGATTCT TCACTG TTGTC-3’
Fluorescent probe: probe:5 '-F-CAGTCAGTGGTTCCGTGGAAATTAGC-Q-3 ', wherein F is the fluorescence report group, Q is quenching group.
2. single stage method fluorescent RT-PCR detection reagent box according to claim 1 is characterized in that: the fluorescence report group F of this fluorescent probe 5 ' end mark is 6-Fluoresceincarboxylic acid FAM, and the quenching group Q of 3 ' end mark is tertiarybutylhydroquinone BHQ.
3. single stage method fluorescent RT-PCR detection reagent box according to claim 2, it is characterized in that: described solution A also comprises AgPath-ID
TM2 * RT-PCR mixed solution in the One-Step RT-PCR Kit test kit.
4. single stage method fluorescent RT-PCR detection reagent box according to claim 3, it is characterized in that: also comprise solution B, this solution B comprises Taq archaeal dna polymerase and AMV ThermoScript II.
5. single stage method fluorescent RT-PCR detection reagent box according to claim 4, it is characterized in that: also comprise solution C, this solution C is DEPC-H
2O.
6. single stage method fluorescent RT-PCR detection reagent box according to claim 5, it is characterized in that: also comprise positive control, this positive control is influenza split vaccine diluent.
7. single stage method fluorescent RT-PCR detection reagent box according to claim 6, it is characterized in that: also comprise negative control, this negative control is the SPF chick embryo allantoic liquid.
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| CN103911460B (en) * | 2013-11-28 | 2016-03-30 | 中国科学院广州生物医药与健康研究院 | H1 type influenza virus isothermal amplification detection kit |
| CN104450956A (en) * | 2014-11-12 | 2015-03-25 | 中国农业科学院哈尔滨兽医研究所 | Real-time fluorescence RT-PCR detection kit for H1N1 type A swine influenza virus and application of detection kit |
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| CN101886136A (en) * | 2009-05-12 | 2010-11-17 | 中山大学达安基因股份有限公司 | Kit for detecting H1N1 influenza A virus by real-time fluorescence RT-PCR |
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| 莫秋华等.一步法多重RT-PCR快速筛查A型、B型和新型甲型H1N1流感病毒.《南方医科大学学报》.2009,第29卷(第8期),1545-1547. * |
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