CN104498629A - Duplex real-time fluorescence quantitative PCR (polymerase chain reaction) detection kit for H3N2 subtype avian influenza virus (AIV) - Google Patents

Duplex real-time fluorescence quantitative PCR (polymerase chain reaction) detection kit for H3N2 subtype avian influenza virus (AIV) Download PDF

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CN104498629A
CN104498629A CN201410735120.6A CN201410735120A CN104498629A CN 104498629 A CN104498629 A CN 104498629A CN 201410735120 A CN201410735120 A CN 201410735120A CN 104498629 A CN104498629 A CN 104498629A
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primer
probe
aiv
fluorescence quantitative
influenza virus
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谢芝勋
刘婷婷
谢志勤
邓显文
罗思思
刘加波
谢丽基
黄莉
黄娇玲
曾婷婷
王盛
张艳芳
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Guangxi Veterinary Research Institute
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Abstract

The invention discloses a duplex real-time fluorescence quantitative PCR (polymerase chain reaction) detection kit for an H3N2 subtype avian influenza virus (AIV). The duplex real-time fluorescence quantitative PCR detection kit for the H3N2 subtype AIV comprises a primer set and a probe set, wherein the primer set comprises a primer 1, a primer 2, a primer 3 and a primer 4 which respectively contain base sequences shown in SEQ.ID.No.1 and 2 and SEQ.ID.No.4 and 5, and the probe set comprises a probe A and a probe B which respectively contain base sequences shown in SEQ.ID.No.3 and 6. Experiments prove that the duplex real-time fluorescence quantitative PCR detection kit for the H3N2 subtype AIV has the advantages that detection time is short, detection sensitivity is high and specificity is strong; two pathogens, namely H3 subtype AIV and N2 subtype AIV, can be detected and identified at the same time, and corresponding pathogen contents can be determined, so that the duplex real-time fluorescence quantitative PCR detection kit for the H3N2 subtype AIV can be used for rapid diagnosis and monitoring of the H3N2 subtype AIV and has great significance in prevention and treatment of the H3N2 subtype AIV.

Description

H3N2 subtype avian influenza virus dual real-time fluorescence quantitative PCR detection kit
Technical field
The invention belongs to PCR and detect virus technology field, particularly relate to a kind of H3N2 subtype avian influenza virus dual real-time fluorescence quantitative PCR detection kit.
Background technology
Bird flu (Avian influenza, AI) be the class Important Infectious Diseases endangering poultry cultivation, its pathogenic agent AIV is the segmented RNA viruses of sub-thread minus strand, wherein hemagglutinin (Hemagglutinin, and neuraminidase (Neuraminidase HA), NA) be two important gene segments, the research of Susan etc. shows that there is certain balance between HA and NA gene has much relations to the pathogenic of virus.H3 hypotype AIV is low pathogenicity AIV, carries in recessiveness in bird, usually do not fall ill or Symptoms comparatively light, often not note by people, this resets for H3 hypotype AIV and other hypotype AIV producer and provides condition.Meanwhile, H3 hypotype AIV has the mammiferous abilities such as infected dogs, pig, horse, has potential significant damage to the public health security of human society.N2 hypotype AIV infects poultry (H9N2) and the common NA hypotype that can cause a disease, the AIV of different serotypes can be combined to form with most of HA hypotype AIV, and the AIV of traditional Serology test to polyinfection has limitation, therefore, a kind of energy is set up accurately, fast and the method that simultaneously can detect H3N2 hypotype AIV seems particularly important.
Real-time fluorescence quantitative PCR is combined with fluorescence detection method at regular-PCR method, adds and can indicate the probe of fluorophor with amplification template specific binding, accumulate Real-Time Monitoring PCR process by fluorescence in PCR reaction system.This method have easy and simple to handle, visual result, susceptibility are high, high specificity, can the feature such as detection by quantitative.Especially multiple real time fluorescence quantifying PCR has unique advantage in the differential diagnosis of clinical multiple cause of disease polyinfection.At present, report is had no for H3N2 hypotype AIV multiple fluorescence quantitative PCR method.
Summary of the invention
The technical problem to be solved in the present invention is to provide that a kind of susceptibility is good, specificity is high, accurately and reliably, H3N2 subtype avian influenza virus dual real-time fluorescence quantitative PCR detection kit quickly and easily, to realize detecting and quantitative H3N2 subtype avian influenza virus simultaneously, for the monitoring of H3N2 hypotype AIV provides technical support.
For solving the problems of the technologies described above, the present invention is by the following technical solutions: H3N2 subtype avian influenza virus dual real-time fluorescence quantitative PCR detection primer sets, comprise two pairs of Auele Specific Primers, be primer 1 and 2, primer 3 and 4 respectively, they have the base sequence of sequence table SEQ .ID.No.1 and 2, SEQ.ID.No.4 and 5 respectively.
H3N2 subtype avian influenza virus dual real-time fluorescence quantitative PCR detection kit, comprises primer sets and probe groups; Primer sets has primer 1 to 4, and they have the base sequence of sequence table SEQ .ID.No.1 and 2, SEQ.ID.No.4 and 5 respectively; Probe groups has probe A and probe B, and they have the base sequence of sequence table SEQ .ID.No.3 and 6 respectively.
5 ' the end of probe A is marked with reporter fluorescence dyestuff FAM, and 3 ' end is marked with quencher fluorescent dye ECLIPSE; 5 ' the end of probe B is marked with reporter fluorescence dyestuff ROX, and 3 ' end is marked with quencher fluorescent dye ECLIPSE.
The mol ratio of primer 1, primer 2, probe A, primer 3, primer 4 and probe B is 1:1:1:1:1:1.
This test kit contains following reagent:
A liquid: pcr amplification damping fluid, primer 1, primer 2, probe A, primer 3, primer 4, probe B;
B liquid: AIV-H3+AIV-N2 template, as positive control;
C liquid: ddH 2o, as negative control.
In A liquid, primer 1, primer 2, probe A final concentration are 0.4 μM, and primer 3, primer 4, probe B final concentration are 0.4 μM.
For lacking the technology effectively reliably of H3N2 subtype avian influenza virus being carried out to diagnosis and detection at present, contriver studies and has screened the two specific probe of cover and primers, and by carrying out the proportioning of different concns, obtain the concentration combination of best primer and probe, establish the detection method of the dual real-time fluorescence quantitative PCR of H3N2 subtype avian influenza virus accordingly, and prepare corresponding detection kit.Experiment proves, tool of the present invention has the following advantages:
1) detection time is short
Application the present invention can realize the object that a pipe two is examined, only need about 30 minutes, and reaction result can directly be observed by computer, and the RT-PCR method of routine needs 3.5 hours to carry out amplified reaction at least, then must spend 2 hours and carry out gel electrophoresis and carry out observations;
2) the high and high specificity of detection sensitivity
When H3 hypotype AIV and N2 hypotype AIV exists simultaneously, the present invention compares with the CT value that single virus detects its CT value detected, and result is substantially the same, does not affect the susceptibility detecting and detect to H3 hypotype AIV and N2 hypotype AIV.In addition, utilize the present invention also can carry out quantitatively to cause of disease content corresponding in sample, and detection sensitivity is very high, all 100 H3N2 hypotype AIV copied can be detected, higher than conventional PCR method susceptibility 100 times, thus can be used for quick diagnosis and the monitoring of H3N2 hypotype AIV, therefore the control of the present invention to H3N2 hypotype AIV is significant.
Accompanying drawing explanation
Fig. 1 is in susceptibility (FAM passage) result figure, the figure of fluorescence quantitative PCR detection H3 subtype avian influenza virus: 1 ~ 7 is respectively 1 × 10 8~ 1 × 10 2copy/μ l, 8 negative controls.
Fig. 2 is the typical curve of the susceptibility (FAM passage) of fluorescence quantitative PCR detection H3 subtype avian influenza virus.
Fig. 3 is in susceptibility (ROX passage) result figure, the figure of fluorescence quantitative PCR detection N2 subtype avian influenza virus: 1 ~ 7 is respectively 1 × 10 8~ 1 × 10 2copy/μ l, 8 negative controls.
Fig. 4 is the typical curve of the susceptibility (ROX passage) of fluorescence quantitative PCR detection N2 subtype avian influenza virus.
Fig. 5 is in specificity (FAM passage) result figure, the figure of fluorescence quantitative PCR detection H3 subtype avian influenza virus: 1-12 is respectively H3N2, H1N1, H3N6, H3N8, H4N2, H4N5, H5N1, H6N1, H6N6, H6N8, H7N2, H9N2 hypotype AIV; 13.NDV; 14.IBV; 15.ILTV; 16.MG; 17.ARV; 18. blanks.
Fig. 6 is in specificity (ROX passage) result figure, the figure of fluorescence quantitative PCR detection N2 subtype avian influenza virus: 1-12 is respectively H3N2, H1N1, H3N6, H3N8, H4N2, H4N5, H5N1, H6N1, H6N6, H6N8, H7N2, H9N2 hypotype AIV; 13.NDV; 14.IBV; 15.ILTV; 16.MG; 17.ARV; 18. blanks.
Fig. 7 is in crowd interior repeatability (FAM passage) result figure, figure of fluorescence quantitative PCR detection H3 subtype avian influenza virus: 1-5 is 1 × 10 8copy/μ L; 6. negative control.
Fig. 8 is in crowd interior repeatability (ROX passage) result figure, figure of fluorescence quantitative PCR detection N2 subtype avian influenza virus: 1-5 is 1 × 10 8copy/μ L; 6. negative control.
Embodiment
The experimental technique used in following examples if no special instructions, is ordinary method; Material used, reagent etc., if no special instructions, all can obtain from commercial channels.Concrete material therefor and reagent as follows:
Lightcycler2.0 quantitative real time PCR Instrument (Roche).Glue recovery test kit and plasmid extraction kit are purchased from Dongsheng bio tech ltd, Guangzhou; PMD-18T test kit, Reverse Transcription and fluorescent PCR Premix Ex Taq are purchased from the precious biotech firm in Dalian, and DNA/RNA extraction agent box is purchased from Beijing Quanshijin Biotechnology Co., Ltd.
H3N2, H6N8 subtype avian influenza virus is documented in " Visual detection of H3subtype avian influenzaviruses by reverse transcription loop-mediated isothermal amplification assay ", Virol J, 2011,8:337, the public can obtain from Veterinary Institute of Guangxi Zhuang Autonomous Region;
H1N1 subtype avian influenza virus is documented in " utilizing the somatotype of RT-LAMP Visual retrieval technology for detection H1 subtype avian influenza virus and N1, N2 hypotype ", virus journal, 2013,29 (2): 154-159, the public can obtain from Veterinary Institute of Guangxi Zhuang Autonomous Region;
H3N6 subtype avian influenza virus is documented in gene pool NCBI, and accession number is " KM186122-KM186129 ", and the public can obtain from Veterinary Institute of Guangxi Zhuang Autonomous Region;
H3N8 subtype avian influenza virus is documented in " Genetic Characterization of a NaturalReassortant H3N8Avian Influenza Virus Isolated from Domestic Geese in Guangxi; Southern China ", Genome Announc.2014,2 (4): e00747-14, the public can obtain from Veterinary Institute of Guangxi Zhuang Autonomous Region;
H4N2 subtype avian influenza virus is documented in gene pool NCBI, and accession number is " KJ881013-KJ881020 ", and the public can obtain from Veterinary Institute of Guangxi Zhuang Autonomous Region;
H4N5, H5N1, H7N2 subtype avian influenza virus is documented in " foundation of H7N9 hypotype AIV dual real-time fluorescence quantitative RT-PCR detecting method ", animal medicine is in progress, 2013,34 (12): 1-5, the public can obtain from Veterinary Institute of Guangxi Zhuang Autonomous Region;
H6N1 is documented in " Complete genome sequence analysis of an H6N1avian influenza virusisolated from Guangxi pockmark ducks ", J.Virol.2012,86:13868 – 13869, the public can obtain from Veterinary Institute of Guangxi Zhuang Autonomous Region;
H6N6 subtype avian influenza virus and mycoplasma gallisepticyum (MG) S6 strain are documented in " Epidemiological Surveillanceof Low Pathogenic Avian Influenza Virus (LPAIV) from Po μ Ltry in Guangxi Province; Southern China " PLOS ONE, 2013,8 (10): 1-6, the public can obtain from Veterinary Institute of Guangxi Zhuang Autonomous Region;
H9N2 avian influenza virus is documented in " Characterization of an avain influenza virus H9N2strainisolated from a wild bird in southern China.Genome Announc ", 2014,2:e00600-14, the public can obtain from Veterinary Institute of Guangxi Zhuang Autonomous Region;
Avian pneumo-encephalitis virus (NDV F48E9 strain) is documented in the foundation of detection method " the Avian pneumo-encephalitis virus highly-wetting liquid LAMP differentiate " Chinese veterinary science, 2011,41 (09): 917-922, the public can obtain from Veterinary Institute of Guangxi Zhuang Autonomous Region;
Avian infectious bronchitis virus (IBV H52 strain) is documented in " foundation of avian infectious bronchitis virus RT-LAMP visible detection method ", China's agronomy circular, 2012,28 (20): 83-87, the public can obtain from Veterinary Institute of Guangxi Zhuang Autonomous Region;
Avian infectious laryngotracheitis virus (ILTV Beijing Strain) is documented in " foundation of avian infectious laryngotracheitis virus LAMP detection method ". animal doctor's science and technology, 2012,44 (11): 52-55, the public can obtain from Veterinary Institute of Guangxi Zhuang Autonomous Region;
Avianreovirus (ARV S1733 strain) is documented in " preparation of Avianreovirus S1733 strain monoclonal antibody and the foundation of sandwich ELISA detection method ", herding and animal doctor, 2013,45 (5), 49-52, the public can obtain from Veterinary Institute of Guangxi Zhuang Autonomous Region.
The Design and synthesis of embodiment 1, primer and Taqman probe
According to the conserved sequence of H3 hypotype AIV and N2 hypotype AIV in GenBank, adopt Primer Express 3.0 software, devise many cover probes and primer, by analyzing the dimer between its primer, selected two pairs of Auele Specific Primers and two Taqmam probes (table 1).
Table 1 primer and TaqMan probe sequence (5 '-3 ')
The foundation that embodiment 2, fluorescence quantitative RT-RCR detect
One, the determination of fluorescence quantitative RT-PCR detecting method
1, the extracting of nucleic acid and reverse transcription
With reference to DNA/RNA extraction agent box specification sheets extracting ILTV, the DNA of MG, extract each hypotype AIV and NDV simultaneously, the RNA of IBV and ARV, and carry out reverse transcription with random primer, reverse transcription system is 50 μ L, comprise 5 μ L 10 × PCR damping fluids (precious biotechnology company limited in Dalian, catalog number: DRR0019A), 2 μ L 10mM dNTP (four kinds of bases), 1 μ L RNA inhibitor (the precious biotechnology company limited in Dalian, catalog number: DRR0019A), 1 μ L random primer (the precious biotechnology company limited in Dalian, catalog number: DRR0019A), 1 μ L AMV ThermoScript II (the precious biotechnology company limited in Dalian, catalog number: DRR0019A), total serum IgE template 40 μ L to be checked, wink is from rear water-bath 42 DEG C of 90min, the cDNA of preparation is put-30 DEG C save backup.
2, the preparation of standard substance
Respectively with the cDNA of H3N2 hypotype AIV obtained above for template carries out regular-PCR amplification, reaction system is that 50 μ L (comprise 25 μ L Ex Taq (TaKaRa RR902), 1 μ L influenza gene total length primer, 4 μ L cDNA, add ddH 2o supplies 50 μ L), reaction conditions is: 95 DEG C of denaturation 4min, 94 DEG C of 1min, 52 DEG C of 1min, 72 DEG C of 2min, totally 35 circulations, and 72 DEG C extend 10min, and 4 DEG C are terminated reaction.PCR primer is through 1.5% agarose gel electrophoresis, reclaim object segment rear clone to pMD-18T carrier, extract positive colony plasmid AIV-H3 and AIV-N2, deliver to precious raw biotechnology (Dalian) company limited to check order, and measure concentration and the purity of nucleic acid, calculate the copy number of each plasmid.
The establishment of primer and concentration and probe concentration in the reaction system of 3, fluorescence quantitative RT-RCR
Application AIV H3 and AIV N2 plasmid are as standard substance, adopt 20 μ L PCR reaction systems: 2 × premix Ex Taq enzyme 10 μ L (purchased from the precious biotech firm in Dalian), AIV-H3, AIV-N2 primer and probe final concentration adopt matrix method to adjust between 0.2 ~ 0.8 μm of ol/L, template 2 μ L, to supply 20 μ L without the ultrapure water of RNA enzyme.Filter out can detect H3, N2 hypotype AIV simultaneously according to result, and little primer and probe matched proportion density are affected on Ct value and amplification efficiency, set up bifluorescence quantitative RT-PCR method.
Result shows, different primers and probe final concentration affect larger on test-results, H3 hypotype AIV upstream and downstream primer and probe final concentration are respectively 0.4 μm of ol/L, N2 hypotype AIV upstream and downstream primer and probe final concentration are respectively 0.4 μm of ol/L, can obtain less Ct value to the detection of standard substance.
Therefore, the quantitative fluorescent PCR optimal reaction system optimized is 20 μ L, wherein 2 × premix Ex Taq enzyme 10 μ L, AIV-H3, AIV-N2 primer and probe are 0.4 μ L, template 2 μ L, to supply 20 μ L without the ultrapure water of RNA enzyme, Homogeneous phase mixing, put on Lightcycler quantitative real time PCR Instrument and carry out automatization amplified reaction.Temperature transition rate is 20 DEG C/s, at the end of the extension of each circulation, carry out fluorescent signal detection.Response procedures is 94 DEG C of 30s; 94 DEG C of 10s, 60 DEG C of 20s, 40 circulations; 40 DEG C are terminated reaction.
FAM, at 530nm exciting light fluoresces, is used for detecting H3 hypotype AIV; ROX, at 610nm exciting light fluoresces, is used for detecting N2 hypotype AIV.
Two, the sensitivity test of fluorescence quantitative RT-RCR
Respectively with the H3 hypotype AIV of 10 times of serial dilutions and N2 hypotype AIV plasmid, obtain copy number and be 1 × 10 8~ 1 × 10 2aIV-H3 and the AIV-N2 plasmid of copy/μ L, then the mixing of the plasmid of identical copies number is carried out bifluorescence quantitative pcr amplification as template, amplification system and condition are as above.
Result (FAM) under 530nm exciting light as illustrated in fig. 1 and 2; Result (ROX) under 610nm exciting light as shown in Figures 3 and 4.From the fluorescence curve figure, fluorescence curve is still had to detection 100 copy of H3 hypotype AIV and N2 hypotype AIV, show that this detection method is 100 copies to the sensitivity of H3N2 hypotype AIV, higher than conventional PCR method susceptibility 100 times, the result of duplicate detection is consistent.Increase linear as seen from typical curve, the concentration of Ct value and plasmid standard has good dependency, and the coefficient R 2 of AIV-H3 and AIV-N2 is respectively 0.9993,0.9995, and amplification efficiency is respectively 1.996,1.967.According to Ct value and the lg value of plasmid standard copy number, the typical curve regression equation of foundation is: H3 hypotype AIV is y=-3.23x+41.21; N2 hypotype AIV is y=-3.28x+41.31, and wherein X represents the lg value of template copy numbers, and y represents Ct value.Illustrate that set up method has good amplification efficiency.
Three, the specific test (Fig. 5 and Fig. 6) of fluorescence quantitative RT-RCR
Utilize the bifluorescence quantitative RT-PCR reaction system optimized, each hypotype AIV and above-mentioned common poultry diease pathogenic agent are increased, with ddH 2o be template as negative control, FAM and ROX two kinds of fluorescent signals are collected simultaneously to each detect aperture in instrument.
Result shows, H3N2 hypotype AIV all produces amplification curve at FAM and ROX fluorescence channel, amplification curve is there is in H3N6, H3N8 hypotype AIV at FAM fluorescence channel (under 530nm exciting light), be straight line at ROX fluorescence channel (under 610nm exciting light), amplification curve is there is in H4N2, H7N2, H9N2 hypotype AIV at ROX fluorescence channel, be straight line at FAM fluorescence channel, other hypotype AIV and common respiratory disease of birds virus detect at FAM and ROX fluorescence channel and are straight line, and this illustrates that the judgement of duplex fluorescent PCR reaction result is as follows:
Reaction result is straight line, be then negative; Reaction result is S type curve, be then positive;
If the reaction result of (FAM) is S type curve under 530nm exciting light, then contain H3 hypotype AIV in sample to be tested; Otherwise, then H3 hypotype AIV is not contained in sample; If the reaction result of (ROX) is S type curve under 610nm exciting light, then contain N2 hypotype AIV in sample to be tested; Otherwise, then N2 hypotype AIV is not contained in sample;
If under (ROX) and 530nm exciting light, the reaction result of (FAM) is S type curve under 610nm exciting light, then in sample containing H3N2 hypotype AIV; If under (ROX) and 530nm exciting light, the reaction result of (FAM) is not all S type curve under 610nm exciting light, then in sample all not containing H3 and N2 hypotype AIV.
Four, replica test
According to the dual real-time fluorescence quantitative PCR optimized, with 1 × 10 8aIV-H3 and the AIV-N2 plasmid mixture of copy/μ L, as template, is divided into 5 samples to detect simultaneously.Batch interior repeatability (Fig. 7-8, table 2) of real-time fluorescence quantitative PCR is verified by the standard deviation (SD) and the variation coefficient (CV) calculating Ct value.After the 4th day, 7 days, duplicate detection is stored in the template of-20 DEG C, the stability of validation template and real-time fluorescence quantitative PCR batch between repeatability (table 3).
In batch, repeated detected result is shown in Fig. 7, under 530nm exciting light (FAM), detect H3 hypotype AIV passage and detect N2 hypotype AIV passage under 610nm exciting light (ROX), table 2 is the Ct value of the multiple duplicate detection of same concentration, between batch, repeatability is in table 3, for the Ct value of same template different detection time, can find out, batch in repeatability and batch between the repeated variation coefficient be all less than 3%.Illustrate that this method has good accuracy and repeatability.
Replica test in table 2 group
Replica test between table 3 group
The assembling of embodiment 3, detection kit
According to the result of study of embodiment 1 and 2, assembling detection kit is with easy to use.
A liquid: pcr amplification damping fluid, primer 1, primer 2, probe A, primer 3, primer 4, probe B; In A liquid, primer 1, primer 2, probe A final concentration are 0.4 μM, and primer 3, primer 4, probe B final concentration are 0.4 μM.
B liquid: AIV-H3+AIV-N2 template, as positive control;
C liquid: ddH 2o, as negative control.
The detection of embodiment 4, clinical pathological material of disease
Sample to be tested is 96 parts of clinical samples that Guangxi province is collected, and extracts the RNA of sample respectively and reverse transcription is cDNA.Use the test kit of embodiment 3, carry out fluorescence quantitative RT-RCR according to the reaction system of the fluorescence quantitative RT-RCR of above-mentioned optimization and response procedures.
Detected result is evaluated as follows with reference to above-mentioned judging criterion:
Detect H3N2 hypotype AIV 4 parts (positive rate is 4.17%), detect single H3 hypotype AIV 1 part (positive rate is 1.04%), detect single N2 hypotype AIV 17 parts (positive rate is 17.70%) other sample standard deviations and do not detect H3 hypotype AIV and N2 hypotype AIV.Detected result is consistent with Virus Isolation result, and the foundation of this bifluorescence quantitative RT-PCR method is described, has directive significance to the prevention and control of Guangxi province H3N2 hypotype AIV.

Claims (6)

1.H3N2 subtype avian influenza virus dual real-time fluorescence quantitative PCR detection primer sets, it is characterized in that comprising two pairs of Auele Specific Primers, be primer 1 and 2, primer 3 and 4 respectively, they have the base sequence of sequence table SEQ .ID.No.1 and 2, SEQ.ID.No.4 and 5 respectively.
2. a H3N2 subtype avian influenza virus dual real-time fluorescence quantitative PCR detection kit, is characterized in that comprising primer sets and probe groups; Primer sets has primer 1 to 4, and they have the base sequence of sequence table SEQ .ID.No.1 and 2, SEQ.ID.No.4 and 5 respectively; Probe groups has probe A and probe B, and they have the base sequence of sequence table SEQ .ID.No.3 and 6 respectively.
3. H3N2 subtype avian influenza virus dual real-time fluorescence quantitative PCR detection kit according to claim 1, is characterized in that: the 5 ' end of described probe A is marked with reporter fluorescence dyestuff FAM, and 3 ' end is marked with quencher fluorescent dye ECLIPSE; 5 ' the end of described probe B is marked with reporter fluorescence dyestuff ROX, and 3 ' end is marked with quencher fluorescent dye ECLIPSE.
4. H3N2 subtype avian influenza virus dual real-time fluorescence quantitative RT-PCR detecting kit according to claim 2, is characterized in that: the mol ratio of described primer 1, primer 2, probe A, primer 3, primer 4 and probe B is 1:1:1:1:1:1.
5. H3N2 subtype avian influenza virus dual real-time fluorescence quantitative PCR detection kit according to claim 3, is characterized in that this test kit contains following reagent:
A liquid: pcr amplification damping fluid, primer 1, primer 2, probe A, primer 3, primer 4, probe B;
B liquid: AIV-H3+AIV-N2 template, as positive control;
C liquid: ddH 2o, as negative control.
6. H3N2 subtype avian influenza virus dual real-time fluorescence quantitative PCR detection kit according to claim 5, it is characterized in that: in described A liquid, primer 1, primer 2, probe A final concentration are 0.4 μM, primer 3, primer 4, probe B final concentration are 0.4 μM.
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CN108085421A (en) * 2018-01-24 2018-05-29 北京毅新博创生物科技有限公司 Method of mass spectrography detection influenza A virus multiple PCR products and products thereof
CN108359744A (en) * 2018-01-24 2018-08-03 北京毅新博创生物科技有限公司 Detect the mass spectrometry method and products thereof of H3N2 segment multiple PCR products
CN108359744B (en) * 2018-01-24 2021-08-06 北京毅新博创生物科技有限公司 Mass spectrometry method for detecting H3N2 fragment multiplex PCR product and product thereof
CN110643725A (en) * 2019-10-16 2020-01-03 河南牧业经济学院 PCR (polymerase chain reaction) special primer and molecular beacon TaqMan probe combination for simultaneously detecting two kinds of anaplasma and detection method

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