CN101948934B - Based on the kit for detecting seasonal influenza virus H 1 N 1 through real-time PCR of dye method - Google Patents
Based on the kit for detecting seasonal influenza virus H 1 N 1 through real-time PCR of dye method Download PDFInfo
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- CN101948934B CN101948934B CN201010278683.9A CN201010278683A CN101948934B CN 101948934 B CN101948934 B CN 101948934B CN 201010278683 A CN201010278683 A CN 201010278683A CN 101948934 B CN101948934 B CN 101948934B
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Abstract
The invention discloses a kind of real-time fluorescence quantitative PCR test kit detecting seasonal current Influenza Virus H1N1 based on dye method, comprise forward and reverse Auele Specific Primer and examination criteria product etc., also disclose the process of sample to be checked, Real-time? PCR reaction system and reaction conditions, the methods such as interpretation of result.Utilize this test kit can Quantitative detection seasonal current Influenza Virus H1N1, highly sensitive, relative inexpensiveness, can be used as the monitoring means of aided diagnosis method that basis and clinical labororatory infect seasonal current Influenza Virus H1N1 and clinical effectiveness, experimental implementation person is required relatively low, there is actual clinical value.
Description
Technical field
The present invention relates to a kind of real-time fluorescence quantitative PCR detection method and test kit of the Quantitative detection seasonal current Influenza Virus H1N1 nucleic acid based on dye method, belong to field of biomedicine technology.
Background technology
Influenza is the Acute respiratory infectious disease caused by influenza virus, and its epidemic characteristic propagates rapidly, and impact scope is wide.Up to now, influenza virus has caused four world's flu outbreaks, causes massive losses to the economy of human society and health.
Influenza virus belongs to orthomyxovirus section, and its genome is segmented strand RNA, can be divided into first, second, the third three kinds of types, all can infect people according to virus nucleocapsid albumen (NP) and stromatin (M) difference.First, Influenza B virus is popular virus main in crowd, and influenza A virus is divided into 16 HA hypotypes according to the difference of virus surface haemagglutinin antigen; 9 NA hypotypes are divided into according to the difference of neuraminidase antigen.H1N1 hypotype in influenza A virus once caused being very popular of world's influenza.After 1997, H1N1 hypotype and H3N2 hypotype replace popular in crowd.From 2002, the influenza of China is preponderated by H3N2 always, and after continue for nearly 3 years, after September in 2005, H1N1 subtype influenza strain becomes the advantage strain of the influenza pandemic of China again.
The laboratory diagnostic method of influenza virus comprises virocyte cultivation, serodiagnosis and Detection of antigen.Virus by infect responsive histocyte can separated qualification out, this can be the most direct as influenza infection, the most reliable foundation.In addition, acute phase serum has 4 times of increases to be also the standard of influenza infection compared with convalescent phase serum antibody.But both are consuming time, in general cell cultures needs 3 ~ 4d, and Serological testing needs two weeks.Its quick diagnosis clinically of this drawbacks limit.The direct-detection of antigen includes the detection to virus structural protein and nucleic acid.The detection of virus structural protein to add lustre to former or fluorescence dye based on the reaction of antigen-antibody, conjugate enzyme or chemistry, comprise immunofluorescence (1mmunofluorescence, IF), enzyme linked immunosorbent assay (Enzymelinkedimmunosorbentassays, ELISA), the sensitivity fluctuations of direct or indirect immunofluorescent test is 40% ~ 100%, specificity 86% ~ 99%, but limits its application clinically owing to needing fluorescent microscope specific apparatus.
The optimal detection means of current infected by influenza antigen nucleic acid are Fluorescent quantitative PCR (Real-timePCR), high sensitivity, high specific and high accuracy became one by it, overcome the shortcomings such as time-consuming, the easy pollution of normal PCR, small throughput, detection by quantitative accurately can be carried out to the influenza nucleic acids in sample.Learn from the Query Result of current document and patent, the method not yet having the real-time fluorescence quantitative PCR about seasonal influenza virus H1N1 to detect and the report of test kit.
Summary of the invention
The object of this invention is to provide a kind of simple to operate, application easily based on detection method and the test kit of the seasonal current Influenza Virus H1N1 real-time fluorescence quantitative PCR of dye method.
For reaching this object, the present invention takes following technical scheme:
One, for the Design and synthesis of the Auele Specific Primer of seasonal current Influenza Virus H1N1
The present invention selects the conservative gene NP gene of seasonal current Influenza Virus H1N1 as detection target, first from GenBank database, download the N gene complete sequence of the representative strains such as seasonal current Influenza Virus H1N1, H3N2, highly pathogenic bird flu virus H 5 N 1, novel A (H 1 N 1) virus, use ClustalW software to carry out sequence homology compare of analysis, filter out the specific conserved sequence of seasonal current Influenza Virus H1N1 as primer candidate region.
Application PrimerExpress and PrimerPremier5.0 software package matches further to candidate drugs and screens, and obtains optimum specific detection primer.Design of primers and select and follow following principle:
1. the primer selected is positioned at the conserved regions of seasonal current Influenza Virus H1N1NP gene, with other strain no cross reactions such as novel H1N1, H3N2, H5N1.
2. primer length is 18-25 base.
3. the GC% of primer requires at 40%-60%, theory T m > 50 DEG C.
4. avoid base pairing as far as possible between primer self.
5. the object fragment length of amplification is between 100-500bp.
Accordingly, the specific detection primer sequence that finally adopts of the present invention is as follows:
SF-F：5’-ctgagaagcagatactgggc-3’
SF-R：5’-ctgcattgtctccgaagaaat-3’
Two, the preparation of seasonal current Influenza Virus H1N1 examination criteria product
The mdck cell inoculation seasonal current Influenza Virus H1N1 representative strains that 1.24 porocyte culture plates are cultivated, after 48 hours, is got 100 μ l cells and supernatant, is extracted viral RNA according to RNeasyMiniKit (Qiagen company, article No. 74104) specification sheets.
2. according to the viral first chain cDNA of SuperScriptIIIFirst-StrandSynthesisSystem (Invitrogen company, article No. 18080-051) specification sheets synthesis.
3. design and synthesize PCR primer based on NP gene 5 ' and 3 ' terminal sequence, amplify NP gene by virus full length cDNA.
4. agarose gel electrophoresis detects the amplification situation of NP gene, and result display, except the specific band of 1497bp, also occurs at least 3 non-specific bands in addition.
5. the object band of couple 1497bp cuts glue recovery and purifying.
6. the NP full-length gene after pair recovery carries out nucleic acid concentration mensuration, and is converted into copy number.
Three, the foundation of real-time fluorescence quantitative PCR typical curve and sensitivity, specific detection
1. the standard substance calculating copy number are done 10 times of gradient dilutions, from 1 × 10
10copies/ μ l is diluted to 1 × 10
2copies/ μ l, totally 9 gradients.
2. configure Real-timePCRMix, add successively in order:
Mixed solution is divided equally to 48 hole 0.2mlPCR reaction tubes FastOptical48-wellRXNPlate (ABI companies, article No. 4375816) in, often manage and add different concns standard substance 2 μ l, stick light reaction epiphragma 48-WellOpticalAdhesiveFilm25PK (ABI company, article No. 4375928).
3. open ABISTEPONEPCR instrument and computer, run real-timePCR master routine.48 hole Sptting plates are placed in PCR instrument, start following PCR program:
Fluorescencemeasurementsweretakenaftereachcycle；
theTmvaluewasmeasuredthroughthemeltingcurvefrom60℃to95℃。
4. detection system sensitivity and specificity assessment
In the amplification curve, typical curve of standard substance, different gradient standard substance linearly relation (R
2) reaching 0.999, slope is between-3.0 to-3.5, and for-0.3433, amplification efficiency is 95.572%, is also positioned at standard amplification efficiency range.In melting curve, all there is point and narrow specificity melting peak at 83.2 DEG C in all standard substance.Fluorescent quantitative PCR result is carried out agarose gel electrophoresis, and specific band brightness reduces with template copy numbers gradient and equimultiple declines.To sum up assessment result, the sensitivity of this quantitative detection system reaches 10 as seen
2copies/ μ l, without non-specific amplification, quantitative result is true and reliable.
Advantage of the present invention is as follows:
1. the present invention is simple to operate, application is convenient;
2. the present invention selects the conservative gene N gene of seasonal current Influenza Virus H1N1 as detection target, obtains optimum specific detection primer through screening;
3. use the sensitivity of this detection method quick, clinical value is wide.
Accompanying drawing explanation
Fig. 1 agarose gel electrophoresis detects the amplification variation diagram of NP gene;
The amplification curve diagram of Fig. 2 standard substance;
Fig. 3 canonical plotting;
Fig. 4 standard substance melting curve figure;
Fig. 5 quantitative fluorescent PCR agarose gel electrophoresis result figure.
Embodiment 1: use the present invention to carry out real-time fluorescence quantitative PCR detection to seasonal current Influenza Virus H1N1 in unknown sample
1. use RNeasyMiniKit (Qiagen company, article No. 74104) to extract total serum IgE from sample to be checked.
2. use SuperScriptIIIFirst-StrandSynthesisSystem (Invitrogen company, article No. 18080-051) to synthesize the first chain cDNA.
3. standard substance are done 10 times of gradient dilutions, from 1 × 10
10copies/ μ l is diluted to 1 × 10
2copies/ μ l.
4. calculate the extra specimen amount of sample number N=sample to be checked+positive criteria product+2 negative controls+2.Configuration Real-timePCRMix, adds in order successively:
Mixed solution is divided equally to 48 hole 0.2mlPCR reaction tubes FastOptical48-wellRXNPlate (ABI companies, article No. 4375816) in, often manage and add different concns standard substance 2 μ l, stick light reaction epiphragma 48-WellOpticalAdhesiveFilm25PK (ABI company, article No. 4375928).
5. open ABISTEPONEPCR instrument and computer, run real-timePCR master routine.48 hole Sptting plates are placed in PCR instrument, start following PCR program:
Fluorescencemeasurementsweretakenaftereachcycle；
theTmvaluewasmeasuredthroughthemeltingcurvefrom60℃to95℃。
6. result judges: each experiment all needs again to carry out concentration gradient dilution, from 1 × 10 to standard substance
10copies/ μ l is diluted to 1 × 10
2copies/ μ l, negative control (H
2o) doing real-time fluorescence quantitative PCR after extracting RNA together with sample to be checked, obtaining virus load by reading instrument data.
Embodiment 2: application the present invention carries out real-time fluorescence quantitative PCR detection to the concha of seasonal current Influenza Virus H1N1 infection ferret model and lung tissue
After 16 ferret infection seasonal current Influenza Virus H1N1, scraping ferret concha secretory product, got lung tissue after the 5th day peaceful and comfortable ferret, extracted total serum IgE respectively 1-4 days every days, and reverse transcription is carry out real-time fluorescence quantitative PCR detection after cDNA, and result is as shown in the table.
Result display detects positive rate 100%, and virus load variation tendency and additive method detected result fit like a glove, and show that accuracy rate is also 100%.
The above; be only the present invention's preferably embodiment, but protection scope of the present invention is not limited thereto, any those skilled in the art of being familiar with are in the technical scope that this technology discloses; the change that can expect easily or replacement, all should be encompassed within protection scope of the present invention.
Claims (3)
1., based on a kit for detecting seasonal influenza virus H 1 N 1 through real-time PCR for dye method, it is characterized in that comprising a pair Auele Specific Primer and examination criteria product, primer sequence is:
SF-F：5’-ctgagaagcagatactgggc-3’
SF-R：5’-ctgcattgtctccgaagaaat-3’。
2., according to the kit for detecting seasonal influenza virus H 1 N 1 through real-time PCR based on dye method according to claim 1, it is characterized in that being prepared from through following steps:
(1) select the conservative gene NP gene of seasonal current Influenza Virus H1N1 as detection target, application ClustalW software carries out sequence homology compare of analysis, filter out the specific conserved sequence of seasonal current Influenza Virus H1N1 as primer candidate region, then apply PrimerExpress and PrimerPremier5.0 software package match further to candidate drugs and screen, obtain optimum specific detection primer, primer length is 18-25 base, the GC% of primer requires at 40%-60%, theoretical annealing temperature > 50 DEG C, the object fragment length of amplification is between 100-500bp,
(2) PCR primer is designed and synthesized based on NP gene 5 ' and 3 ' terminal sequence, NP gene is amplified by virus full length cDNA, agarose gel electrophoresis detects the amplification situation of NP gene and cuts glue recovery and purifying to object band, then nucleic acid concentration mensuration is carried out to the NP full-length gene after recovery, and be converted into copy number, as examination criteria product.
3. the kit for detecting seasonal influenza virus H 1 N 1 through real-time PCR based on dye method according to claim 2, it is characterized in that, the optimum condition of described step (2) is: primer length is 22 bases, the GC% of primer requires 50%, amplification object fragment length is 340bp, and annealing temperature is 55 DEG C.
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| CN101392302A (en) * | 2008-09-28 | 2009-03-25 | 中国疾病预防控制中心病毒病预防控制所 | Flu/human avian influenza virus detection gene chip and production method and use |
| CN101560574A (en) * | 2009-05-22 | 2009-10-21 | 中国疾病预防控制中心病毒病预防控制所 | Primers and probes for detecting influenza virus by rRT-PCR and method for detecting influenza virus |
| CN101649356A (en) * | 2009-07-24 | 2010-02-17 | 浙江省疾病预防控制中心 | Fluorescent quantitative detection kit of H1N1 influenza virus A and detection method thereof |
| CN101665842A (en) * | 2009-07-28 | 2010-03-10 | 中国检验检疫科学研究院 | Novel Kit for fluorescence quantitative PCR detection of influenza A H1N1 viruses and detecting method therefor |
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| CN101392302A (en) * | 2008-09-28 | 2009-03-25 | 中国疾病预防控制中心病毒病预防控制所 | Flu/human avian influenza virus detection gene chip and production method and use |
| CN101560574A (en) * | 2009-05-22 | 2009-10-21 | 中国疾病预防控制中心病毒病预防控制所 | Primers and probes for detecting influenza virus by rRT-PCR and method for detecting influenza virus |
| CN101649356A (en) * | 2009-07-24 | 2010-02-17 | 浙江省疾病预防控制中心 | Fluorescent quantitative detection kit of H1N1 influenza virus A and detection method thereof |
| CN101665842A (en) * | 2009-07-28 | 2010-03-10 | 中国检验检疫科学研究院 | Novel Kit for fluorescence quantitative PCR detection of influenza A H1N1 viruses and detecting method therefor |
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| 实时荧光定量 P C R检测 H1N1亚型猪流感病毒;段廷云等;《畜牧兽医学报》;20081231;第39卷(第6期);第7 5 2 — 7 5 6页 * |
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