CN104404171A - Liquid chip detection kit for H5N1 subtype influenza viruses - Google Patents
Liquid chip detection kit for H5N1 subtype influenza viruses Download PDFInfo
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Abstract
The invention relates to the field of biological medicines, in particular to a liquid chip detection kit for H5N1 subtype influenza viruses. The invention provides a primer pair and probe set for detecting H5 subtype influenza virus surface hemagglutinin antigen, a primer pair and probe set for detecting N1 subtype influenza virus neuraminidase antigen, a kit comprising the primer pairs and the probe sets, and a using method of the kit. The kit provided by the invention has favorable sensitivity, accuracy and specificity, is simple, convenient and fast to use, and can finish the detection of a to-be-detected sample within 4 hours. Experiments prove that the kit is free from a false positive when used for detecting a negative sample, achieves the detection rate 100% in detecting a sample only containing H5N1 subtype viruses, and can detect 100% of H5N1 viruses in a sample containing both H5N1 subtype viruses and H7N9 subtype viruses. Besides, the kit can effectively detect all to-be-detected samples containing more than five copies.
Description
Technical field
The present invention relates to biomedicine field, particularly relate to the liquid-phase chip detection kit of influenza virus H 5 N 1 hypotype.
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.
The influenza virus of all mankind can cause bird influenza, but not every avian influenza virus can cause Human Influenza, confirm that the avian influenza virus that can infect people has 8 kinds now, comprised H5N1, H5N2, H7N2, H7N3, H7N7, H9N2, H10N7, H7N9.Wherein H5 is highly pathogenic.H5N1 is the Notifiable disease of regulation report in the anti-therapy of Ministry of Health's New infectious disease, also known as human hepatic stellate cell.After H5N1 virus infected the mankind first from 1997, in the whole world, more than 60 country wreaks havoc, and mortality ratio is up to 60%.Therefore, infected by influenza H5N1 hypotype is made in early days, laboratory diagnosis is fast very urgent.
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 longer, and in general cell cultures needs 3d ~ 4d, and Serological testing needs two weeks.Therefore, these methods cannot realize the quick diagnosis of influenza virus.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 (Enzyme linked immunosorbentassays, ELISA).The sensitivity fluctuations of direct or indirect immunofluorescent test is 40% ~ 100%, and specificity 86% ~ 99%, but limits its application clinically owing to needing fluorescent microscope specific apparatus.To in the detection of viral nucleic acid and somatotype, there is a lot of method special fast.The particularly application of round pcr, plays increasing effect for influenza virus qualification and somatotype.These methods are as RT-polymerase chain reaction (reverse transcription polymerase chain reaction, RT-PCR), multiple reverse transcription polymerase chain reaction (multiplex reverse transcription-PCR, MRT-PCR), enzyme exempts from PCR (PCR-enzyme immunoassay, PCR-EIA), PCR in real time (real-time PCR, RT-PCR) etc. improve speed and the sensitivity of detection all greatly.
But, although increasing method is for detecting influenza virus, not yet there is a kind of method H5N1 hypotype that is quick, easy, infected by influenza accurately to distinguish.Therefore setting up the various type of a kind of infected by influenza, to carry out the method for Rapid identification just very important.
Liquid-phase chip (Multi-Analyte Suspension Array, MASA) realizes Multiple detection as a kind of novel low density biochip technology by suspension array.Molecular reaction occurs in through color-coded microsphere surface.Each target detect thing for test has thousands of nucleic acid (complementary strand) or albumen (as coated antibody) to covalently bind in color-coded microsphere surface.The detection thing that distinct colors coding stands is different.In suspension, microsphere is combined specifically with patient specimen, and adds fluorescent mark.Then, use multi-functional streaming dot matrix instrument to detect, first latex particle is restrainted laser by micro liquid transfer system defiled by two, and wherein redness judges the color of particle thus the specificity (qualitative) of decision analyte; Green measure Fluorescence labeling intensity on particulate thus quantitative to analyte.Finally, system software is understood gather data and is provided the result after processing by analysis.Because molecular hybridization or immune response carry out in aaerosol solution, detection speed is exceedingly fast, and in a micro-liquid reaction system, can detect nearly 100 indexs simultaneously, therefore has high-throughput, the feature that highly sensitive, speed is fast, easy and simple to handle.
Current liquid-phase chip technology has related in the research of the every field of life science, comprises clinical diagnosis, fundamental research, new drug development, judicial expertise, Food Hygiene Surveillance and biological weapon strick precaution etc.But the sensitivity of liquid-phase chip technology and accuracy height depend on the quality of primer and probe, primer and probe stable in properties must be ensured, do not produce hairpin structure or dimer, product length should more than 500bp, and will not ensure non-specific binding not to occur.Although these principles are uncomplicated for general design of primers, too complicated due to influenza virus sequences, there is no specificity at present and accuracy can reach the primer of the liquid-phase chip technology of clinical application requirement and the report of probe.Therefore, the primer of design high specific, accuracy and probe, detection liquid-phase chip technology being applied to H5N1 subtype influenza virus has very great meaning.
Summary of the invention
In view of this, technical problem to be solved by this invention is the liquid-phase chip detection kit providing influenza virus H 5 N 1 hypotype, and test kit provided by the invention is simple to operate, and specificity, accuracy are all higher.
The invention provides the primer pair detecting influenza surface haemagglutinin antigen H5 hypotype, the nucleotide sequence of upstream primer is as shown in SEQ ID NO:1; The nucleotide sequence of downstream primer is as shown in SEQ ID NO:2.
The invention provides the primer pair detecting influenza neuraminidase antigen N1 hypotype, the nucleotide sequence of upstream primer is as shown in SEQ ID NO:3; The nucleotide sequence of downstream primer is as shown in SEQ ID NO:4.
The invention provides the probe combinations detecting influenza surface haemagglutinin antigen H5 hypotype, comprise 2 probes of nucleotide sequence as shown in SEQ ID NO:5 ~ 6.
The invention provides the probe combinations detecting influenza neuraminidase antigen N1 hypotype, comprise 2 probes of nucleotide sequence as shown in SEQ ID NO:7 ~ 8.
Primer provided by the invention and probe design according to H5N1 virus, virus sequence is mainly from the nucleic acid database of NCBI and EBI, and with reference to WHO, the related data of U.S. CDC and Chinese CDC etc., confirm through experiment, primer provided by the invention, under the annealing temperature of 58 DEG C, does not produce non-specific band or primer dimer band through pcr amplification, proves that primer pair provided by the invention has good specificity.Probe provided by the invention does not produce non-specific hybridization under the hybridization temperature of 43 DEG C, proves that probe provided by the invention has good specificity.
Present invention also offers a kind of test kit detecting influenza virus H 5 N 1 hypotype, comprising: primer pair A, primer pair B, probe groups A and probe groups B;
In primer pair A, the nucleotide sequence of upstream primer is as shown in SEQ ID NO:1; The nucleotide sequence of downstream primer is as shown in SEQ ID NO:2;
In primer pair B, the nucleotide sequence of upstream primer is as shown in SEQ ID NO:3; The nucleotide sequence of downstream primer is as shown in SEQ ID NO:4;
Probe groups A comprises 2 probes of nucleotide sequence as shown in SEQ ID NO:5 ~ 6;
Probe groups B comprises 2 probes of nucleotide sequence as shown in SEQ ID NO:7 ~ 8.
In an embodiment, test kit provided by the invention detects for the Luminex of influenza virus H 5 N 1 hypotype.
In an embodiment of the present invention, test kit provided by the invention also comprises: pcr amplification reagent, probe binding reagents or microballoon hybridizing reagent.
In certain embodiments, pcr amplification reagent comprises: PCR premixed liquid, DNA Ploymerase and ddH
2o; Probe binding reagents comprises: latex beads; Microballoon hybridizing reagent comprises: 1.5 × TMAC hybridization buffer.
In certain embodiments, test kit provided by the invention also comprises: phycoerythrin.
Test kit provided by the invention has good sensitivity, accuracy and specificity.Confirm through test, with test kit provided by the invention, false positive is had no to the negative sample detection not containing virus and produce; Sample detection only containing H5N1 subtype virus is presented to the recall rate of 100%; Sample containing H5N1 hypotype and H7N9 subtype virus is detected, also can 100% the sample detected containing H5N1 virus hypotype.Visible, the detection of test kit infected by influenza H5N1 hypotype provided by the invention has good accuracy and specificity, and not by the impact that other subtype influenza virus exist.In addition, confirm through test, test kit provided by the invention can effectively detect containing 5 above testing samples of copy, illustrates that this test kit has higher sensitivity.
The using method of test kit provided by the invention, comprises the following steps:
Step 1: with testing sample cDNA for template, with the mixture of primer pair A and primer pair B for primer, obtains amplified production through PCR;
Step 2: probe groups A and probe groups B is combined with latex beads respectively, obtained bag is by microballoon;
Step 3: amplified production and bag are hybridized by microballoon, dyeing, detection fluorescent signal;
In primer pair A, the nucleotide sequence of upstream primer is as shown in SEQ ID NO:1; The nucleotide sequence of downstream primer is as shown in SEQ ID NO:2;
In primer pair B, the nucleotide sequence of upstream primer is as shown in SEQ ID NO:3; The nucleotide sequence of downstream primer is as shown in SEQ ID NO:4;
Probe groups A comprises 2 probes of nucleotide sequence as shown in SEQ ID NO:5 ~ 6;
Probe groups B comprises 2 probes of nucleotide sequence as shown in SEQ ID NO:7 ~ 8.
In an embodiment of the present invention, testing sample is viral cultures, animal tissues or pharynx nasalis secretory product.
In an embodiment of the present invention, the reaction system of PCR is:
Wherein, in the mix primer of primer pair A and primer pair B, in the mix primer of nucleotide sequence as shown in SEQ ID NO:1 ~ 4, the concentration of single primer is 2 μm of ol/L; In PCR reaction system, the concentration of the arbitrary primer of nucleotide sequence as shown in SEQ ID NO:1 ~ 4 is 0.2 μm of ol/L.
In an embodiment of the present invention, the annealing temperature of PCR is 58 DEG C.
In embodiments of the present invention, the reaction conditions of PCR is:
In an embodiment of the present invention, the system of hybridization is:
1.5 × TMAC hybridization buffer 21.9 μ L;
The microballoon 0.1 μ L of dressing probe;
Amplified production 3 μ L.
As preferably, in 1.5 × TMAC hybridization buffer, the concentration of each component is:
In an embodiment of the present invention, the condition of hybridization is:
95℃ 5min；
43℃ 60min。
In an embodiment of the present invention, the fluorescence dye of dyeing is phycoerythrin (SA-PE).
As preferably, SA-PE working fluid comprises:
SA-PE stoste 32 μ L;
1×TE(pH8.0) 4mL。
As preferably, the amount of SA-PE working fluid is 3 times of hybrid product volume.
As preferably, the temperature of dyeing is 43 DEG C, and the time is 15min.
In an embodiment of the present invention, detect fluorescent signal and adopt Luminex flow type analyzer.
In an embodiment of the present invention, the interpretation of result method detecting fluorescent signal is:
Signal value is less than 150, is judged to be feminine gender;
Signal value, between 150 ~ 200, is judged to be suspicious, continues experiment and confirms;
Signal value is greater than 200, is judged to be the positive;
In probe groups A, the detected result of arbitrary probe is judged to be the positive, then influenza surface haemagglutinin antigen H5 hypotype is positive;
In probe groups B, the detected result of arbitrary probe is judged to be the positive, then influenza neuraminidase antigen N1 hypotype is positive;
Same testing sample, influenza surface haemagglutinin antigen H5 hypotype and influenza neuraminidase antigen N1 hypotype are judged as the positive simultaneously, then containing influenza virus H 5 N 1 hypotype;
Same testing sample, only influenza surface haemagglutinin antigen H5 hypotype or only influenza neuraminidase antigen N1 hypotype be judged as the positive, then non-containing influenza virus H 5 N 1 hypotype.
Test kit provided by the invention, fast easy to use, in 4 hours, can complete detection to testing sample, accuracy in detection can reach 100%, does not produce false positive, and specificity is good, effectively can detect containing 5 above samples of copy, highly sensitive.
The invention provides the primer pair and probe groups that detect influenza surface haemagglutinin antigen H5 hypotype, for detecting primer pair and the probe groups of influenza neuraminidase antigen N1 hypotype, and comprising their test kit and the using method of test kit.Primer provided by the invention and probe design according to H5N1 virus, virus sequence is mainly from the nucleic acid database of NCBI and EBI, and with reference to WHO, the related data of U.S. CDC and Chinese CDC etc., confirm through experiment, primer provided by the invention, under the annealing temperature of 58 DEG C, does not produce non-specific band or primer dimer band through pcr amplification, proves that primer pair provided by the invention has good specificity.Probe provided by the invention does not produce non-specific hybridization under the hybridization temperature of 43 DEG C, proves that probe provided by the invention has good specificity.Test kit provided by the invention has good sensitivity, accuracy and specificity, and fast easy to use, can complete the detection to testing sample in 4 hours.Confirm through test, with test kit provided by the invention, false positive is had no to the negative sample detection not containing virus and produce; Sample detection only containing H5N1 subtype virus is presented to the recall rate of 100%; Sample simultaneously containing H5N1 hypotype and H7N9 subtype virus is detected, also can 100% detect the H5N1 virus wherein contained.Visible, the detection of test kit infected by influenza H5N1 hypotype provided by the invention has good accuracy and specificity, and not by the impact that other subtype influenza virus exist.In addition, confirm through test, test kit provided by the invention can effectively detect containing 5 above testing samples of copy, illustrates that this test kit has higher sensitivity.
Accompanying drawing explanation
Fig. 1 is the expanding effect of primer provided by the invention under different annealing temperature.
Embodiment
The invention provides the liquid-phase chip detection kit of influenza virus H 5 N 1 hypotype, those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in the present invention.Method of the present invention and application are described by preferred embodiment, related personnel obviously can not depart from content of the present invention, spirit and scope methods and applications as herein described are changed or suitably change with combination, realize and apply the technology of the present invention.
In the embodiment of the present invention, the synthesis of primer and probe is synthesized by prompt base (China) trade Co., Ltd in the English Weihe River, and way of purification is DHPL.
Other examination materials adopted in the embodiment of the present invention or instrument are all common commercially available product, all can buy in market.
Below in conjunction with specific embodiment, set forth the present invention further:
Embodiment 1: the invention provides the annealing temperature of primer, probe hybridization temperature, dyeing temperature screening
One, with influenza virus H 5 N 1 subtype cDNA for template, thermograde pcr amplification, annealing temperature arranges 50 DEG C ~ 66 DEG C, 4 DEG C of gradients.Amplified production is carried out agarose gel electrophoresis, and result as shown in Figure 1.From electrophoresis result, 54 DEG C can effectively be increased to 58 DEG C, consider sequence similarity between different virus hypotype, avoid non-specific amplification, so choose higher annealing temperature.
Two, being coated on microballoon by probe, is that increase under the 58 DEG C of conditions microballoon of the product that obtains and dressing probe combines, with 40 DEG C, 43 DEG C, 46 DEG C for hybridization temperature is hybridized by annealing temperature.Then dye.Result is as shown in table 1:
Table 1 probe hybridization temperature, dyeing temperature screen
From results of hybridization, 40 DEG C and 43 DEG C of results of hybridization all good, but in order to prevent non-specific hybridization.We can select any temperature high, namely 43 DEG C.From staining conditions, 43 DEG C of results of hatching are best.
Embodiment 2 the invention provides specificity, the repeatability detection of test kit
Use test kit provided by the invention, different subtype influenza sample is detected, to prove specificity of the present invention.And the sample to one of them H5N1 hypotype, detect in triplicate, qualification repeatability.
Concrete steps are as follows:
A, viral cDNA obtain: get Virus Sample, extract viral RNA according to RNeasy Mini Kit (Qiagen company, article No. 74104) specification sheets.According to the viral first chain cDNA of SuperScript III First-Strand SynthesisSystem (Invitrogen company, article No. 18080-051) specification sheets synthesis.
B, with the cDNA obtained in step a for template carries out PCR, in PCR pipe, add the following material of 20 μ L:
Primer is the mixture of 4 primers as shown in SEQ ID NO:1 ~ 4.
In mix primer, the concentration of single primer is the final concentration of a certain single primer in 2 μm of ol/L, PCR reaction solutions is 0.2 μm of ol/L.
C, PCR pipe is put into PCR instrument, condition is as follows:
The probe of nucleotide sequence shown in d, SEQ ID NO:5 ~ 6 is attached on the microballoon (small latex particle) of given color respectively, obtains bag by microballoon.
E, microballoon are hybridized:
The component of microballoon hybridization system and composition:
1.5×TMAC 21.9μL；
The each 0.1 μ L of microballoon of often kind of dressing probe;
PCR primer 3 μ L.
1. first added by microballoon mixed solution 22ul in the well on hybridization plate, the pcr amplification product 3 μ l then sampling this adds in well.
2. the shrouding paper of the corresponding size of clip, hybridizes plate and covers sealing by micropore.Please with finger pressure sealing for several times, guarantee to obturage, in case when high-temperature denatured and hybridization solution evaporation.
3. hybridization plate is placed in Metal constant temperature bath (can replace by PCR instrument), the lid of instrument is compressed the hybridization plate sealed, in case shrouding film opens after the heating, cause liquid evaporation.
4. crossover process runs following program:
95 DEG C of sex change in 5 minutes
43 DEG C of hybridization in 60 minutes
5. carefully torn by shrouding paper, every hole adds fluorescence dye phycoerythrin (SA-PE) 75 μ L.After adding, shrouding paper is glued again, continue to hatch 15 minutes at 43 DEG C;
Whole program is:
95 DEG C of sex change in 5 minutes
43 DEG C of hybridization in 60 minutes
43 DEG C of 15 minutes SA-PE are hatched
F, micropore hybridized plate fast transfer and read to preheated Luminex flow type analyzer.
G, result judge:
In an embodiment of the present invention, the interpretation of result method detecting fluorescent signal is:
Signal value is less than 150, is judged to be feminine gender;
Signal value, between 150 ~ 200, is judged to be suspicious, continues experiment and confirms;
Signal value is greater than 200, is judged to be the positive;
In probe groups A, the detected result of arbitrary probe is judged to be the positive, then influenza surface haemagglutinin antigen H5 hypotype is positive;
In probe groups B, the detected result of arbitrary probe is judged to be the positive, then influenza neuraminidase antigen N1 hypotype is positive;
Same testing sample, influenza surface haemagglutinin antigen H5 hypotype and influenza neuraminidase antigen N1 hypotype are judged as the positive simultaneously, then containing influenza virus H 5 N 1 hypotype.
Same testing sample, only influenza surface haemagglutinin antigen H5 hypotype or only influenza neuraminidase antigen N1 hypotype be judged as the positive simultaneously, then non-containing influenza virus H 5 N 1 hypotype.
Result is as shown in table 2.
Table 2 the invention provides specificity, the repeatability detection of test kit
Result shows, and can reach 100%, then show feminine gender to the influenza virus of other hypotypes to the recall rate of H5N1 hypotype.Illustrate that test kit provided by the invention has good specificity, detected result is not by the interference of other subtype influenza virus.Further, to simultaneously containing H5N1 and H7N9 pattern detection in, present method shows high accuracy.To the sample of one of them H5N1 hypotype, detect in triplicate, the signal value difference of acquisition is little, has good repeatability.
The susceptibility that embodiment 3 the invention provides test kit detects
First by real-time PCR method, the cDNA extracted from virus liquid is carried out quantitatively, then gradient dilution.Use test kit provided by the invention, carry out PCR respectively, molecular hybridization and upper machine testing, to identify the susceptibility that the invention provides test kit.The using method of test kit is with embodiment 2.Result is as shown in table 3:
The susceptibility of table 3 test kit provided by the invention detects
Result shows, and detect the cDNA after dilution with test kit provided by the invention, the sensitivity of H5N1 probe is very high, and single copy number can detect.Effectively can detect containing 5 above samples of copy.
The detection of embodiment 4 experiment sample
Utilize test kit provided by the invention to the nose swab of a large amount of infection ferret models that laboratory retains, the cDNA sample that throat swab and when dissected are respectively organized detects.The using method of test kit is with embodiment 2.Result is as shown in table 4:
Table 4 utilizes test kit test experience sample provided by the invention
In the 85 increment product detected, detect that, containing H5N1 subtype influenza virus in a sample, completely the same with expection, accuracy rate can reach 100%.In a copy of it sample, one of the probe signal value of H5 hypotype presents the positive, but the signal value of the probe of N1 hypotype is then negative simultaneously, finds in this sample containing H5N2 subtype influenza virus through order-checking.In addition, wherein one of the probe signal value of two increments N1 hypotype in this presents the positive, but the signal value of the probe of H5 hypotype is then negative simultaneously, finds in this sample containing H1N1 subtype influenza virus through order-checking.Illustrate that test kit provided by the invention has good accuracy and specificity, detected result is not by the interference of other subtype influenza virus.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. detect the primer pair of influenza surface haemagglutinin antigen H5 hypotype, it is characterized in that, the nucleotide sequence of upstream primer is as shown in SEQ ID NO:1; The nucleotide sequence of downstream primer is as shown in SEQ IDNO:2.
2. detect the primer pair of influenza neuraminidase antigen N1 hypotype, it is characterized in that, the nucleotide sequence of upstream primer is as shown in SEQ ID NO:3; The nucleotide sequence of downstream primer is as shown in SEQ IDNO:4.
3. detect the probe combinations of influenza surface haemagglutinin antigen H5 hypotype, it is characterized in that, comprise 2 probes of nucleotide sequence as shown in SEQ ID NO:5 ~ 6.
4. detect the probe combinations of influenza neuraminidase antigen N1 hypotype, it is characterized in that, comprise 2 probes of nucleotide sequence as shown in SEQ ID NO:7 ~ 8.
5. detect the test kit of influenza virus H 5 N 1 hypotype, it is characterized in that, comprising: primer pair A, primer pair B, probe groups A and probe groups B;
In described primer pair A, the nucleotide sequence of upstream primer is as shown in SEQ ID NO:1; The nucleotide sequence of downstream primer is as shown in SEQ ID NO:2;
In described primer pair B, the nucleotide sequence of upstream primer is as shown in SEQ ID NO:3; The nucleotide sequence of downstream primer is as shown in SEQ ID NO:4;
Described probe groups A comprises 2 probes of nucleotide sequence as shown in SEQ ID NO:5 ~ 6;
Described probe groups B comprises 2 probes of nucleotide sequence as shown in SEQ ID NO:7 ~ 8.
6. test kit according to claim 5, is characterized in that, also comprises: pcr amplification reagent, probe binding reagents or microballoon hybridizing reagent.
7. test kit according to claim 6, is characterized in that, described pcr amplification reagent comprises: PCR premixed liquid, DNA Ploymerase, ddH
2o; Described probe binding reagents comprises: latex beads; Described microballoon hybridizing reagent comprises: 1.5 × TMAC hybridization buffer.
8. the using method of the test kit as described in any one of claim 5 ~ 7, is characterized in that, comprises the following steps:
Step 1: with testing sample cDNA for template, with the mixture of described primer pair A and described primer pair B for primer, obtains amplified production through PCR;
Step 2: 4 probes in probe groups A and probe groups B are combined with latex beads respectively, obtained bag is by microballoon;
Step 3: described amplified production and described bag are hybridized by microballoon, dyeing, detection fluorescent signal;
In described primer pair A, the nucleotide sequence of upstream primer is as shown in SEQ ID NO:1; The nucleotide sequence of downstream primer is as shown in SEQ ID NO:2;
In described primer pair B, the nucleotide sequence of upstream primer is as shown in SEQ ID NO:3; The nucleotide sequence of downstream primer is as shown in SEQ ID NO:4;
Described probe groups A comprises 2 probes of nucleotide sequence as shown in SEQ ID NO:5 ~ 6;
Described probe groups B comprises 2 probes of nucleotide sequence as shown in SEQ ID NO:7 ~ 8.
9. the using method of test kit according to claim 8, is characterized in that, described testing sample is virus liquid, animal tissues or pharynx nasalis secretory product.
10. the using method of test kit according to claim 8, is characterized in that, the annealing temperature of described PCR 58 DEG C; The temperature of described dyeing is 43 DEG C, and the time is 15min.
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