CN102337351B - Typing detection kit for influenza virus - Google Patents
Typing detection kit for influenza virus Download PDFInfo
- Publication number
- CN102337351B CN102337351B CN201010229496.1A CN201010229496A CN102337351B CN 102337351 B CN102337351 B CN 102337351B CN 201010229496 A CN201010229496 A CN 201010229496A CN 102337351 B CN102337351 B CN 102337351B
- Authority
- CN
- China
- Prior art keywords
- influenza
- virus
- probe
- specific
- influenza virus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Abstract
The invention relates to a typing detection kit for an influenza virus, especially to a kit for detecting influenza virus A, influenza virus B and influenza virus A (H1N1) with a multiple real-time fluorescent polymerase chain reaction technology. The kit mainly comprises an RT-PCR (real-time polymerase chain reaction) reaction solution, a mixed solution of primer and probe, RT-PCR reaction enzymes, DEPC H2O, and packing boxes for separation and centralized packaging of the reagent bottles or tubes. Able to distinguish influenza virus A, influenza virus B and influenza virus A (H1N1) accurately, the kit of the invention can be widely applied in multiple fields like differential diagnosis and epidemic prevention and control of influenza caused by the above influenza viruses.
Description
Technical field
The present invention relates to the test kit that a kind of influenza virus somatotype detects, particularly relate to a kind of test kit that utilizes multiple real time fluorescence polymerase chain reaction technology to detect influenza A virus, Influenza B virus and H1N1virus.This test kit can accurately be distinguished A type, Influenza B virus and H1N1virus, can be widely used in by a plurality of fields such as the differential diagnosis of the caused influenza of above-mentioned influenza virus, epidemic prevention and controls.
Background technology
Seasonal influenza is a kind of common Acute respiratory infectious disease being caused by influenza virus.The northern area of China, how occurred frequently in the winter time seasonal influenza is; And in southern area, the four seasons have case to occur, there will be epidemic peak in summer and winter.The general susceptible of mankind's infected by influenza.Influenza virus mainly coughs by the infected or the propagation of sneezing, and also has people because touch the article (as tableware, teacup or toy etc.) that polluted influenza virus, then the mouth of contact oneself or nose and infect virus again.Be about 1～3 day the latent period of seasonal influenza, and Acute onset is usually expressed as high heat, headache, myalgia, the toxicity symptom such as weak, can be with symptoms such as pharyngalgia, runny nose, dry cough, gastrointestinal upsets.Main complication is bacterial pneumonia, ear infection, sinus infection, dehydration, and old man, children, with serious complication easily occurs after some basic disease or the person's of having a delicate constitution influenza virus infection are even dead.Although can obtain certain immunizing power after being ill, because influenza virus sub-strain inside often can occur to make a variation by a small margin, i.e. " antigenic drift ", people is to the virus after making a variation still susceptible.Flu outbreak refers to the Influenza Outbreak of global range.It can be brand-new causing pandemic influenza virus, can be also that old influenza virus reappears.In human body, almost do not resist the immunizing power of this virus, there is no again effective vaccine.Influenza virus can be in crowd fast propagation, can there is serious disease in the infected, even dead.Disease can involve whole country and even the whole world at short notice.
Influenza virus is in viral taxonomy Shang Shu orthomyxoviridae family (Orthomyxoviridae).Its genome is segmented (first, B-mode strain are containing 8 sections, and the third type only contains 7 sections, the sections of a few coding neuraminic acid zymoprotein), the RNA of sub-thread minus strand.First, B-mode strain genome encode respectively 10 and 11 kind of albumen.Because genome is segmented, therefore easily produce not gene resortment between homophyletic of homotype.Influenza virus, especially the constant origination point sudden change of influenza virus A hominis HA gene, cause the HA protein molecular upper amino acid sequence of its coding to be replaced, cause constant the drifting about of its antigenicity, each antigenic drift often brings influenza pandemic in various degree.Influenza A virus can be divided into many hypotypes again according to its surperficial hemagglutinin (H) and neuraminidase (N) protein structure and genetic characteristics thereof, so far the blood clotting that influenza A virus has been found have 15 hypotypes (H1-H15), and neuraminidase has 9 hypotypes (N1-N9).H1N1virus is that influenza A virus belongs to (Influenza virus A), and typical virion is spherical, and diameter is 80nm-120nm, has cyst membrane.The projection glycoprotein that has many radial arrangement on cyst membrane is respectively red corpuscle hemagglutinin (HA), neuraminidase (NA) and matrix protein 2.In virion, be nucleocapsid, symmetrical in the shape of a spiral, diameter is 10nm, is sub-thread minus-stranded rna virus, and genome is about 13.6kb, 8 independent segments that differ in size, consists of.Virus is responsive to common disinfectantses such as ethanol, Iodophor, the tincture of iodine; To thermo-responsive, 56 ℃ of conditions lower 30 minutes can deactivation.On March 18th, 2009, Mexico finds H 1 N 1 influenza A virus infection case successively, and there are several deaths, and Influenza A H1N1 epidemic situation occurs in a big way in the whole world subsequently, the World Health Organization was also once being carried flu outbreak warning level to 6 grades.China has included Influenza A H1N1 in the Category B notifiable disease of the law on the prevention and control of infectious diseases > > of < < People's Republic of China (PRC) regulation, and takes the preventive and control measure of category A infectious disease.After 2010, Influenza epidemic situation steps into the mitigation stage.
The method of general evaluation influenza virus mainly contains following several method: (1) Virus Isolation.Adopt chicken embryo and/or mdck cell to carry out influenza virus separation.Sample is made suspension inoculation in 10～11 age in days chick embryo allantoic cavities, collects allantoic fluid after 48h, can with mdck cell, cultivate to gather in the crops virus-culturing fluid or carry out titre evaluation again.The method is long and higher to operator's requirement detection time; (2) serological method, is also the conventional means that virus is identified, by serological identification, judges viral kind and hypotype.But be often subject to the aspects such as nonspecific agglutination factor, antibodies specific in experimentation, disturb, the aspects such as the stdn of antigen, monoclonal antibody technology of preparing are proposed to stricter requirement.(3) molecular biology for detection.Protocols in Molecular Biology has been widely used in the Rapid&Early diagnosis of influenza.Reverse transcription polymerase chain reaction is that RT-PCR has high degree of specificity and susceptibility, to develop at present the most ripe molecular diagnostic techniques, can detect Influenza Virus RNA from gene level, greatly shortened the detection time of cause of disease, for the diagnosis of influenza virus provides more responsive, method faster.Present most of laboratory all adopts polymerase chain reaction to carry out viral evaluation.Influenza virus is single stranded RNA, and therefore suitable employing reverse transcriptase polymerase chain reaction carries out the amplification of viral nucleic acid.
The advantages such as real-time fluorescence PCR technology is a kind of direct-detection nucleic acid the Protocols in Molecular Biology that carries out nucleic acid quantification, has highly sensitively, and specificity is good, simple, cheap.It adds fluorophor a kind of in PCR reaction system, utilizes fluorescent signal accumulation Real-Time Monitoring PCR reaction process, by result data analysis, starting template is carried out quantitatively.The present invention adopts the multiple real time fluorescence PCR method in real-time fluorescence PCR technology, three kinds of influenza viruses (influenza A virus, Influenza B virus, H1N1virus) nucleic acid is increased, according to the amplification situation of the three fluorescence of mark, differentiate three kinds of influenza viruses.Test kit of the present invention can apply to the Influenza Surveillance being caused by influenza A virus, Influenza B virus and H1N1virus widely.
Summary of the invention
The object of the present invention is to provide a kind of test kit that utilizes multiple real time fluorescence polymerase chain reaction technology to carry out the detection of influenza virus somatotype, utilize this test kit can accurately distinguish influenza A virus, Influenza B virus and H1N1virus.
On the basis that the nucleotide sequence of all known influenza A viruss, Influenza B virus and H1N1virus strain is compared on to GENBANK, find respectively the specificity conserved regions of three kinds of nucleic acid sequences, and for conserved regions design target nucleotide primer and probe.These primer probes are containing hot resistant DNA polymerase, the RT-PCR reaction enzymes of reversed transcriptive enzyme, high quality deoxyribonucleoside triphosphate (dNTPs) system and contain Mg
2+in RT-PCR reaction solution Deng composition, by fluorescent PCR instrument, realize the cyclic amplification of external nucleic acid.
Test kit involved in the present invention mainly comprises: 1) RT-PCR reaction solution, primer probe mixed solution, RT-PCR reaction enzymes system, DEPC H
2o, and 2) separate and concentrate the packing box of these reagent bottles of packing or pipe.
A preferred embodiment of the present invention is that primer probe mixed solution is just specific by a pair of influenza A virus, reverse primer, , the specific probe of influenza A virus, a pair of Influenza B virus is just specific, reverse primer, , the specific probe of Influenza B virus, a pair of influenza A H 1 N 1 virus specific just, reverse primer, article one, the probe of influenza A H 1 N 1 virus specific forms, the sequence that it is characterized in that the specific forward of influenza A virus and reverse primer is respectively 5 '-TAAAGATGAGTCTTCTAACCGAG-3 ' (SEQ ID NO:1) and 5 '-CAAGATCTGTGTTCTTTCCTGC-3 ' (SEQID NO:2), the sequence of the specific probe of influenza A virus is 5 '-CGAAACGTACGTTCTTTCTATCATCCC-3 ' (SEQ ID NO:3), and the two ends of probe are combined with respectively fluorescence generation group FAM and fluorescent quenching group B HQ1, the sequence of the specific forward of Influenza B virus and reverse primer is respectively 5 '-GTTGGTAAACGGAACATTCCTCAAAC-3 ' (SEQ ID NO:4) and 5 '-GCAACAAGCCTTCCACTCTGGTC-3 ' (SEQ ID NO:5), the sequence of the specific probe of Influenza B virus is 5 '-CCCAATGGATACAAGTCCTTATCAACTC-3 ' (SEQID NO:6), and the two ends of probe are combined with respectively fluorescence generation group HEX and fluorescent quenching group Eclipse, the forward of influenza A H 1 N 1 virus specific and the sequence of reverse primer are respectively 5 '-GGCCATTGCCGGTTTCA-3 ' (SEQ ID NO:7) and 5 '-TTGTTAGTAATCTCGTCAATGGCATT-3 ' (SEQ ID NO:8), the sequence of the probe of influenza A H 1 N 1 virus specific is 5 '-ATATGCAGCCGACCTGAAGAGCACACA-3 ' (SEQ ID NO:9), and the two ends of probe are combined with respectively fluorescence generation group Cy5 and fluorescent quenching group Eclipse.
Another preferred embodiment of the present invention is that in primer probe mixed solution, primer concentration is 0.2mmol/L, and concentration and probe concentration is 0.1mmol/L.
Another preferred embodiment of the present invention is that RT-PCR reaction solution is by Tris-HCl (50mmol/L, pH8.0), MgCl
2(8mmol/L), KCl (250mmol/L) forms.
Another preferred embodiment of the present invention is that RT-PCR reaction enzymes system is comprised of warm start Taq enzyme, reversed transcriptive enzyme, dNTPs.Reversed transcriptive enzyme can be selected the conventional reversed transcriptive enzymes such as mMLV.Warm start Taq enzyme, reversed transcriptive enzyme, dNTPs all can adopt commercially available prod, and as the product of Qiagen company, wherein in every person-portion RT-PCR reaction enzymes system, the consumption of warm start Taq enzyme is 5U, and reversed transcriptive enzyme consumption is 10U, and dNTPs consumption is 10mmol.
The condition that another preferred embodiment of the present invention is pcr amplification is: 50 ℃ 15 minutes, 95 ℃ 15 minutes; 94 ℃ 15 seconds, 58 ℃ 45 seconds, 40 circulations (58 ℃ time collect fluorescent signal).
A preferred embodiment of the present invention is that test kit provides extra quality control product, is respectively negative quality control product and positive quality control product.Negative quality control product is physiological saline, positive quality control product is the virus-culturing fluid of deactivation, by influenza A virus and Influenza B virus two-strain, formed, virus strain is purchased from ATCC, wherein influenza A virus can be selected one of following numbering: VR-1284, VR-1285, VR-1287, VR-1289, VR-1469, VR-1520, VR-1608, VR-1609, VR-1641, VR-1642, VR-1679, VR-1680, VR-1682, VR-1683, VR-1737, VR-219, VR-333, VR-544, VR-546, VR-547, VR-776, VR-777, VR-810, VR-822, VR-825, VR-897, VR-95, VR-96, VR-97, VR-98, VR-99.Influenza B virus can be selected one of following numbering: VR-101, VR-102, VR-103, VR-1535, VR-1735, VR-295, VR-296, VR-523, VR-786, VR-787, VR-788, VR-789, VR-790.Two-strain concentration is 5.0 * 10
3～5.0 * 10
6between pFU/mL, preferably 5.0 * 10
3pFU/mL, and mix and be prepared into positive quality control product with volume ratio 1:1.In test kit, carry out Samples detection to be checked and can carry out the detection of two special quality control product simultaneously, only when positive quality control product detects, FAM, HEX and CY5 passage fluorescent signal are all positive, when three passage fluorescent signals of negative quality control product detection are all negative, the detected result of sample to be checked is just effective.
The test kit of the present invention sample to be checked that increases is completed automatically by commercially available quantitative real time PCR Instrument, simple to operate, consuming time few, and has reduced to greatest extent the generation of polluting.Detected result can be used for a plurality of area researches such as influenza virus somatotype, auxiliary clinical diagnosis and routine monitoring.
The present invention compared with prior art, advantage is: 1. infected by influenza nucleic acid amplification level detects, the state that can reflect A type in patient body, B-mode and H 1 N 1 influenza A virus infection, can be used for the Supervise prevention and cure of influenza, contributes to the early diagnosis and therapy of disease; 2. multiple real time fluorescence round pcr, for multiple viral nucleic acid specific sequence design primer, probe, has higher flux, and easy and simple to handle, the advantage that relatively reduces costs, is more suitable for Most patients and detects; 3. whether be the type of influenza infection and infection to a sample one-time detection if can distinguish, and greatly improved detection efficiency.
Accompanying drawing explanation
Fig. 1 shows the reaction conditions of pcr amplification.
The amplification curve of the negative quality control product of Fig. 2 visualizingre agent box.In figure, there is no S type amplification curve, illustrate that FAM, HEX and CY5 passage fluorescent signal are all negative.
The amplification curve of Fig. 3 visualizingre agent box positive quality control product.In figure, the amplification curve of FAM passage is S type, illustrates that the fluorescent signal of this passage is positive.
The amplification curve of Fig. 4 visualizingre agent box positive quality control product.In figure, the amplification curve of HEX passage is S type, illustrates that the fluorescent signal of this passage is positive.
The amplification curve of Fig. 5 visualizingre agent box positive quality control product.In figure, the amplification curve of CY5 passage is S type, illustrates that the fluorescent signal of this passage is positive.
Fig. 6 shows the amplification curve of 3 routine negative sample.In figure, there is no S type amplification curve, illustrate and detect in sample without three kinds of influenza nucleic acids.
Fig. 7 shows the amplification curve of 1 routine positive throat swab sample.In figure, the amplification curve of FAM passage is S type, illustrates that this sample has the amplification of influenza A virus nucleic acid, represents to have influenza A virus in sample.
Fig. 8 shows the amplification curve of 1 routine positive throat swab sample.In figure, the amplification curve of HEX passage is S type, illustrates that this sample has the amplification of Influenza B virus nucleic acid, represents to have Influenza B virus in sample.
Fig. 9 shows the amplification curve of 1 routine positive throat swab sample.In figure, the amplification curve of FAM passage and Cy5 passage is S type, illustrates that this sample has the amplification of H1N1virus nucleic acid, represents to have H1N1virus in sample.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.
1, preparation comprises the test kit of following moiety: primer probe mixed solution (25 μ l/ pipe) 1 pipe, RT-PCR reaction solution (250 μ l/ pipe), RT-PCR reaction enzymes system (75 μ l/ pipe) 1 pipe, positive quality control product (200 μ l/ pipe) 1 pipe, negative quality control product (200 μ l/ pipe) 1 pipe, DEPC H
2o (2000 μ l/ pipe) 1 pipe.
2, the collection of sample, storage and transport
2.1 sample types: nose swab, throat swab, nasopharynx are drawn the respiratory tract samples such as thing.
2.2 collections of specimens, preserve and transport: with disinfecting silk or cotton swab, get nasal cavity or throat secretory product, and drop into and be equipped with in the small test tube of sterile saline or Eagle liquid or 0.5% lactoalbumin hydrolysate Hanks liquid 2mL immediately, post label, put in curling stone, take to laboratory or low temperature (20 ℃～-70 ℃) frozen.The long-distance employing dry ice that transports of sample.
3, nucleic acid extraction:
Can adopt commercially available RNA to extract test kit, suggestion is used QIAamp Viral RNA Mini Kit to extract test kit, and operates according to the specification sheets of test kit, finally collects 50 μ l RNA solution, directly detects or be stored in-20 ℃.
4, real-time fluorescence quantitative PCR amplification and detection
4.1 reagent are prepared: primer probe mixed solution, RT-PCR reaction solution, the RT-PCR reaction enzymes of getting in proportion respective amount are that (primer probe mixed solution 1 μ l/ person-portion+RT-PCR reaction solution 10 μ l/ person-portion+RT-PCR enzymes are 3 μ l/ person-portion+DEPC H
2o16 μ l/ person-portion), after fully mixing, by 30 μ l/ pipes, be distributed into PCR reaction tubes, standby.
4.2 application of samples: in PCR reaction tubes, add respectively positive and negative quality control product, sample rna solution 20 μ l after extraction, cover tightly pipe lid, put into instrument sample cell.
4.3 editors: (ABI Prism7500 quantitative real time PCR Instrument)
Open Setup window, by correspondence, positive and negative quality control product and sample to be measured are sequentially set, and sample title is set in Name hurdle.Choose all sample wells that arrange, double-click, select Add Detector, selecting Reporter is that FAM and Quencher are BHQ1, then to select Reporter be that HEX and Quencher are Eclipse; Then selecting Reporter is that Cy5 and Quencher close window after Eclipse.In Passive Reference, select (none).Open instrument window cycling condition be set: 50 ℃ 15 minutes, 95 ℃ 15 minutes; 94 ℃ 15 seconds, 58 ℃ 45 seconds, 40 circulations (seeing accompanying drawing 1).Preservation file after all settings complete, operation.
4.4 interpretations of result:
Reaction finishes rear preservation and detects data file.Under Results, open Amp plot window.The object sample position of selection analysis.Change Baseline numerical value into start:3, stop:10, and open manual and set Threshold:1.5 ± 100000.Double-click numerical value on Rn coordinate and open Graph settings window, change Log in Post Run Settings into Linear, after OK, open Analysis preferences window, under Analysis menu, select Analyze automatic analysis result.
Choose 3 examples and be accredited as influenza virus feminine gender through virus culture method, 3 examples are accredited as the sample of the influenza virus positive through virus culture method, the nucleic acid extraction of sample, and pcr amplification and interpretation of result step are carried out with reference to embodiment 1, carry out the moon, the detection of positive quality control product simultaneously.
Detected result: the amplification curve of negative quality control product not S-type (seeing accompanying drawing 2), the amplification curve of positive quality control product is obvious S type curve (seeing accompanying drawing 3,4,5), positive and negative quality control product all meets the Quality Control requirement of test kit, and therefore the detected result of sample to be checked is effective.The amplification (seeing accompanying drawing 6,7,8,9) of corresponding A type, B-mode and H1N1virus nucleic acid by known test kit of detected result of sample to be checked, all detected.
In this test, detected result and the virus culture qualification result of 6 routine samples fits like a glove, and illustrate that it is feasible utilizing the detection of this test kit and differentiation A type, B-mode and H1N1virus.This test kit is easy and simple to handle, and detection time is short, can realize high throughput testing, and it is cheap in addition, is expected to be applied to the monitoring of clinical detection and the seasonal influenza of influenza.
Claims (5)
1. a typing detection kit for influenza virus, comprising: 1) RT-PCR reaction solution, primer probe mixed solution, RT-PCR reaction enzymes system, DEPC H
2o, with 2) separate and concentrate the packing box of packing these reagent bottles or pipe, wherein primer probe mixed solution is by the specific forward and reverse primer of a pair of influenza A virus,, the specific probe of influenza A virus, the specific forward and reverse primer of a pair of Influenza B virus, a specific probe of Influenza B virus, the forward and reverse primer of a pair of influenza A H 1 N 1 virus specific, article one, the probe of influenza A H 1 N 1 virus specific forms, and it is characterized in that:
The sequence of the specific forward of influenza A virus and reverse primer is respectively 5 '-TAAAGATGAGTCTTCTAACCGAG-3 ' and 5 '-CAAGATCTGTGTTCTTTCCTGC-3 ', the sequence of the specific probe of influenza A virus is 5 '-CGAAACGTACGTTCTTTCTATCATCCC-3 ', and the two ends of probe are combined with respectively fluorescence generation group FAM and fluorescent quenching group B HQ1;
The sequence of the specific forward of Influenza B virus and reverse primer is respectively 5 '-GTTGGTAAACGGAACATTCCTCAAAC-3 ', with 5 '-GCAACAAGCCTTCCACTCTGGTC-3 ', the sequence of the specific probe of Influenza B virus is 5 '-CCCAATGGATACAAGTCCTTATCAACTC-3 ', and the two ends of probe are combined with respectively fluorescence generation group HEX and fluorescent quenching group Eclipse;
The forward of influenza A H 1 N 1 virus specific and the sequence of reverse primer are respectively 5 '-GGCCATTGCCGGTTTCA-3 ' and 5 '-TTGTTAGTAATCTCGTCAATGGCATT-3 ', the sequence of the probe of influenza A H 1 N 1 virus specific is 5 '-ATATGCAGCCGACCTGAAGAGCACACA-3 ', and the two ends of probe are combined with respectively fluorescence generation group Cy5 and fluorescent quenching group Eclipse.
2. test kit according to claim 1, is further characterized in that in primer probe mixed solution, primer concentration is 0.2mmol/L, and concentration and probe concentration is 0.1mmol/L.
3. test kit according to claim 1, is further characterized in that 50mmol/LTris-HCl, 8mmol/L MgCl that RT-PCR reaction solution is 8.0 by pH value
2, 250mmol/L KCl forms.
4. test kit according to claim 1, wherein RT-PCR reaction enzymes system is comprised of warm start Taq enzyme, reversed transcriptive enzyme, dNTPs, the consumption that is characterised in that warm start Taq enzyme in every person-portion RT-PCR reaction enzymes system is 5U, and reversed transcriptive enzyme consumption is 10U, and dNTPs is 10mmol.
5. test kit according to claim 1, is further characterized in that the optimum reaction condition of pcr amplification is: 50 ℃ of 15min, 1 circulation; 95 ℃ of 15min, 1 circulation; 94 ℃ of 15s, 58 ℃ of 45s, 40 circulations.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010229496.1A CN102337351B (en) | 2010-07-16 | 2010-07-16 | Typing detection kit for influenza virus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010229496.1A CN102337351B (en) | 2010-07-16 | 2010-07-16 | Typing detection kit for influenza virus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102337351A CN102337351A (en) | 2012-02-01 |
| CN102337351B true CN102337351B (en) | 2014-04-02 |
Family
ID=45513300
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201010229496.1A Active CN102337351B (en) | 2010-07-16 | 2010-07-16 | Typing detection kit for influenza virus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102337351B (en) |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104032034A (en) * | 2014-03-07 | 2014-09-10 | 王全意 | Method for simultaneously detecting influenza A virus, influenza B virus and influenza C virus and kit |
| CN104388587A (en) * | 2014-11-03 | 2015-03-04 | 深圳市生科源技术有限公司 | One-step detection kit for influenza B virus and influenza B virus detection method |
| CN107287346B (en) * | 2016-03-30 | 2020-11-06 | 广东省疾病预防控制中心 | Multiplex fluorescence quantitative PCR detection kit for avian influenza virus N subtype genotyping and application thereof |
| CN110408697A (en) * | 2016-07-08 | 2019-11-05 | 中山大学达安基因股份有限公司 | NPM1 gene mutation typing detection method and kit |
| CN106854682A (en) * | 2016-12-27 | 2017-06-16 | 上海派森诺生物科技股份有限公司 | A kind of method that parting is carried out using gene infected by influenza |
| CN107034310A (en) * | 2017-01-24 | 2017-08-11 | 南方医科大学 | It is a kind of while detecting the primer sets of a variety of influenza viruses, probe groups and its kit |
| CN107893130A (en) * | 2017-12-26 | 2018-04-10 | 湖南圣湘生物科技有限公司 | The application method of the primer and probe of influenza virus parting detection, kit and kit |
| CN113249519A (en) * | 2021-04-21 | 2021-08-13 | 深圳市儿童医院 | Adenovirus detection typing kit and method for peripheral blood sample |
| CN113278735A (en) * | 2021-05-26 | 2021-08-20 | 首都医科大学附属北京朝阳医院 | Human respiratory virus nucleic acid multiplex detection primer, probe and kit |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1865937A (en) * | 2005-05-20 | 2006-11-22 | 程小雯 | Real-time fluorescent quantitative detection method for simultaneous detection of A-type and B-type influenza virus and kit therefor |
| CN1904067A (en) * | 2006-05-10 | 2007-01-31 | 浙江省疾病预防控制中心 | B type grippal virus fluorescent augmentation detection kit and detection method |
| CN101363063A (en) * | 2007-08-10 | 2009-02-11 | 程小雯 | Primer, probe, kit and method for detecting A, B and H5 subtype influenza virus by triple fluorescent quantitative RT-PCR |
-
2010
- 2010-07-16 CN CN201010229496.1A patent/CN102337351B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1865937A (en) * | 2005-05-20 | 2006-11-22 | 程小雯 | Real-time fluorescent quantitative detection method for simultaneous detection of A-type and B-type influenza virus and kit therefor |
| CN1904067A (en) * | 2006-05-10 | 2007-01-31 | 浙江省疾病预防控制中心 | B type grippal virus fluorescent augmentation detection kit and detection method |
| CN101363063A (en) * | 2007-08-10 | 2009-02-11 | 程小雯 | Primer, probe, kit and method for detecting A, B and H5 subtype influenza virus by triple fluorescent quantitative RT-PCR |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102337351A (en) | 2012-02-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102337351B (en) | Typing detection kit for influenza virus | |
| CN104513865B (en) | Reverse transcription PCR detects test kit and the detection method thereof of Chikungunya virus | |
| CN103060451B (en) | A kind of mycoplasma pneumoniae MP detection kit | |
| CN101942525B (en) | One-tube method with multiplex detection for human Influenza A and B and new Influenza A H1N1 virus and kit | |
| CN104630388A (en) | Dengue virus rapid classification identification detection kit | |
| CN101550452B (en) | Human bocavirus real-time fluorescence PCR detection reagent kit | |
| CN105441589A (en) | Human parainfluenza virus I, II, III, and IV-type quadruple-PCR detection kit, and detection method thereof | |
| CN102206713B (en) | Triple fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) detection kit and use thereof | |
| CN102251061A (en) | Nucleic acid dual fluorescence PCR (Polymerase Chain Reaction) detection kit for influenza A/B virus | |
| CN102286639A (en) | Type A H1N1/influenza A virus nucleic acid dual fluorescent polymerase chain reaction (PCR) detection kit | |
| CN104498622A (en) | Primers, probes, and method used for influenza virus typing | |
| CN103352088A (en) | Primer pair, probe, reagent kit and method for detecting avian influenza virus H7 subtype | |
| CN102002539B (en) | Porcine parvovirus assay kit and application thereof | |
| CN103993102A (en) | Multiple fluorescent PCR method and kit for simultaneous detection of human adenovirus, human mycoplasma pneumonia and bocavirus | |
| CN102337352B (en) | Kit for detecting multiple influenza viruses by polymerase chain reaction (PCR) microarray | |
| CN101392299A (en) | Equine influenza detection kit and detection method | |
| CN107236827B (en) | Kit and method for detecting transmissible gastroenteritis virus of swine | |
| CN105463131A (en) | Human bocavirus LAMP (loop-mediated isothermal amplification) detection kit | |
| CN102816865B (en) | H1N1, PRRSV and CSFV quadruple detection kit | |
| CN102181576B (en) | Primer, probe and method for detecting respiratory infectious disease pathogen by using liquid chip | |
| CN101230405B (en) | Type H5 and H9 avian influenza virus and newcastle disease virus multiple detection kit and method therefor | |
| WO2016078215A1 (en) | Primers, probes and kit for detecting and typing five ebola virus subtypes by one-step method reverse transcription pcr | |
| CN102605105B (en) | Diagnostic kit used for identifying reverse transcription-composite nested polymerase chain reaction of swine fever vaccine strains and pandemic strains and detection method | |
| CN102776294B (en) | Quick detecting kit for C-type foot and mouth disease virus | |
| CN101886136B (en) | Kit for detecting H1N1 influenza A virus by real-time fluorescence RT-PCR |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20160831 Address after: 510665 Guangzhou high tech Industrial Development Zone, Guangdong, Xiang Shan Road, No. 19 Patentee after: Daan Gene Co., Ltd., Zhongshan Univ. Patentee after: Chengdu high-new Da An medical test company limited Address before: 510665 Guangzhou high tech Industrial Development Zone, Guangdong, Xiang Shan Road, No. 19 Patentee before: Daan Gene Co., Ltd., Zhongshan Univ. |
|
| CP01 | Change in the name or title of a patent holder | ||
| CP01 | Change in the name or title of a patent holder |
Address after: 510665 No. 19 incense Hill Road, hi tech Industrial Development Zone, Guangdong, Guangzhou Patentee after: Guangzhou Da'an gene Co.,Ltd. Patentee after: CHENGDU GAOXIN DAAN MEDICAL SCIENCE INSPECTION Co.,Ltd. Address before: 510665 No. 19 incense Hill Road, hi tech Industrial Development Zone, Guangdong, Guangzhou Patentee before: DA AN GENE CO., LTD. OF SUN YAT-SEN University Patentee before: CHENGDU GAOXIN DAAN MEDICAL SCIENCE INSPECTION Co.,Ltd. |