CN103497947B - The reagent of a kind of external novel quick loop-mediated isothermal amplification method, vitro detection H1N1virus and method - Google Patents
The reagent of a kind of external novel quick loop-mediated isothermal amplification method, vitro detection H1N1virus and method Download PDFInfo
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- CN103497947B CN103497947B CN201310419597.9A CN201310419597A CN103497947B CN 103497947 B CN103497947 B CN 103497947B CN 201310419597 A CN201310419597 A CN 201310419597A CN 103497947 B CN103497947 B CN 103497947B
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Abstract
The invention provides a kind of external novel quick loop-mediated isothermal amplification method, the reagent of vitro detection H1N1virus and method, it comprises outer primer F3, outer primer B3, inner primer FIP, inner primer BIP, ring primer Loop-f, ring primer Loop-r, cover primers F NP, cover primer BNP.External novel quick loop-mediated isothermal amplification method provided by the present invention makes the contact site of primer and template increase thus substantially increase amplification efficiency, the proliferation time reacted than common LAMP shortens about 50%, shorten about 30% than the LAMP reaction times adding ring primer, the quick diagnosis of disease is had important practical significance.Detection H1N1virus reagent provided by the present invention, relation between its primer adopts the scheme in external novel quick loop-mediated isothermal amplification method provided by the present invention, only need can complete for about 40-45 minute to the detection of sample, have easy and simple to handle, efficient, quick, special advantage.
Description
Technical field
The present invention relates to a kind of external novel quick amplification method of ring mediated isothermal amplification method (LAMP), the reagent of vitro detection H1N1virus and method.
Background technology
Ring mediated isothermal amplification (Loop-mediated isothermal amplification, LAMP) depend on 4 and can identify the primer of 6 specific regions on target sequence and a kind of archaeal dna polymerase with strand displacement characteristic, do not need the DNA of thermally denature as template, just can carry out efficient, quick, high amplified target sequence specifically at a constant temperature.Therefore the method has highly application value, and is not only suitable for scientific effort, can also carry out instrument that is conventional and Emergent detection as the grass-roots unit related personnel that appointed condition is relatively poor.
LAMP reaction method adds ring primer exactly the most fast at present, and the object of this research is the reaction times how shortening isothermal duplication, reaches than adding ring primer amplification efficiency faster.LAMP reaction is basis for estimation with the amount of product, in common LAMP, with template contacts be pair of primers, four sites, a kind of amplification program, this program determines the inherent amplification efficiency of this reaction, and the condition optimizing of such as primer concentration in addition etc. and the amount etc. of template amount and enzyme determine external amplification efficiency, and the object of this research improves the inherent amplification efficiency of reaction.Add in LAMP reaction system ring primer add two with the nucleic acid-templated site contacted, so improve amplification efficiency, but do not change the kind of product.If the binding site of primer and template is become 8 from 4, so, the product of reaction will become 4 kinds from a kind, significantly can increase amplification efficiency in theory.
The H1N1virus of outburst in 2009 is popular causes global tens of millions of people to be very popular, and death toll reaches thousands.This virus has the gene fragment that this virus stain includes porcine influenza, bird flu and human influenza three kinds of influenza viruses, infects this virus and occurs heating, cough, laryngalgia, physical distress, has a headache, feels cold and the symptom such as fatigue.This virus is still popular at present, and within 2013, the domestic death that still has is reported.This research, for H1N1virus, studies Novel isothermal amplification detection method and reagent.
Summary of the invention
First object of the present invention is to provide a kind of external novel quick loop-mediated isothermal amplification method, makes a ring mediated isothermal react the time of having increased shorter than the time adding ring primer used.The present invention adopts following design for this reason:
In goal gene, the primer of conventional ring mediated isothermal amplification method (LAMP) is designed at two ends, be for a pair wherein outer primer F3, B3 and another is to being inner primer FIP, BIP, and reserve enough bases of design cover inner primer FNP, BNP and ring primer Loop-f, ring primer Loop-r; Add ring primer Loop-f, ring primer Loop-r, during LAMP can be made to react, primer increases by 2 with the nucleic acid-templated position contacted, and improves amplification efficiency;
A pair cover inner primer FNP, BNP is designed between inner primer FIP and BIP of outside, now isothermal amplification not only can increase according to nucleic acid-templated with cover inner primer FNP and BNP, also can combine with the product of the conventional LAMP primer amplification in outside and increase simultaneously, make to turn increase 4 with the nucleic acid-templated position contacted;
Cover inner primer FNP not only can combine with the BNP of cover inner primer and carries out amplified reaction, also can combine with the conventional inner primer BIP in outside simultaneously and increase, increase the chance with nucleic acid-templated combination and amplification, otherwise, cover inner primer BNP also can combine with the FNP of cover inner primer and carries out amplified reaction, also can combine with the conventional inner primer FIP in outside simultaneously and carry out amplified reaction;
Utilize above-mentioned primer pair goal gene to carry out ring mediated isothermal amplification, conventional LAMP reaction and the amplified production of LAMP adding ring primer only have the cyclic sequence of a kind of product, add the sequence that ring primer does not change product; After adding cover inner primer, product is exactly the cyclic sequence of 4 kinds of amplified productions, i.e. the amplified production of inner primer FIP, BIP, the amplified production of cover inner primer FNP, BNP, the amplified production of inner primer FIP and cover inner primer BNP, the amplified production of cover inner primer FNP and inner primer BIP.
Second object of the present invention is to provide a kind of detection H1N1virus reagent being suitable for above-mentioned external novel quick ring mediated isothermal amplification method, may be used for detecting A (H 1 N 1) virus rapidly and accurately.For this reason, the present invention is by the following technical solutions:
It comprises outer primer F3, outer primer B3, inner primer FIP, inner primer BIP, ring primer Loop-f, ring primer Loop-r, cover primers F NP, cover primer BNP; Primer gene order is as follows:
Outer primer F3:AAGACAAGTTCATGGCCCAATC;
Outer primer B3:CGGCTGCATATCCTGACCC;
Inner primer FIP:
CCGGCTTGAACTTCTTGCTGTAGGGGCATTCACCATCCATCT；
Inner primer BIP:
GTGCTATAAACACCAGCCTCCCAGGCCAGTCTCAATTTTGTGCTT；
Ring primer Loop-f:GCATTCTGATAGAGACTTTGTTGGT;
Ring primer Loop-r:TTCAGAATATACATCCGATCACAAT;
Cover primers F NP:
TGGACACTAGTAGAGCCGGGAGACAGACCCAAAGTGAGGGATCAAGAA；
Cover primer BNP:
TCATTTCAGATACACCAGTTCACGATTGCCTTGGGTGTCTGACAAGTTGTATTG。
The present invention's the 3rd object is to provide the method using the external new and effective isothermal duplication of mentioned reagent to detect H1N1virus, can detect H1N1virus rapidly and accurately.For this reason, the present invention is by the following technical solutions:
Primer mixture 1 μ L, 10 × buffer2.5 μ L, template ribonucleic acid 1 μ L, 8U BstDNA polysaccharase 1 μ L, 10U AMV reversed transcriptive enzyme 1 μ L, concentration is the trimethyl-glycine 2 μ L of 8mol/L, and concentration is the dNTP2 μ L of 10mmol/L, and concentration is the MgSO of 100mmol/L
41.2 μ L, Eva_green1 μ L, adds DEPC-H
2o supplies 25 μ L and mixes;
Each primer concentration in primer mixture is as follows respectively: F3, B3 each 1 μm of ol/L, FIP, BIP each 15 μm of ol/L, Loop-f, Loop-r each 5 μm of ol/L, FNP, each 15 μm of ol/L of BNP;
The step of described method is: negative control, positive control and sample to be tested are hatched 63 DEG C by above-mentioned reaction system respectively and within 40-45 minute, reacts; Wherein negative control adopts distilled water; Result judges can in the following ways:
Quantitative real time PCR Instrument observes real-time fluorescence curves, occurs that S shape amplification curve person contains H1N1virus, result is positive, does not occur S shape amplification curve person, then for not having H1N1virus, result is negative; Or:
Get amplified production and observe electrophoresis result, Positive control wells produces stair-stepping band, and negative control hole does not then have band to produce, and detects as produced stepped band then containing H1N1virus in sample aperture, otherwise does not have H1N1virus; Or:
After reaction terminates, the centrifugal 6000rpm of reaction tubes, 3 minutes, observations, positive control pipe visible white precipitates, and negative control pipe does not produce precipitation, as produced white precipitate then containing H1N1virus in sample tube, otherwise there is no H1N1virus; Or:
Under ultraviolet lamp, observe the change of fluorescence intensity, positive control produces intense green fluorescence, and negative control fluorescence intensity does not change, and then has H1N1virus, otherwise do not have H1N1virus in sample tube as produced intense green fluorescence.
External novel quick loop-mediated isothermal amplification method provided by the present invention is improvement innovation to the carrying out of original LAMP technology, the contact site of primer and template is made to increase thus substantially increase amplification efficiency, the proliferation time reacted than common LAMP shortens about 50%, shorten about 30% than the LAMP reaction times adding ring primer, the quick diagnosis of disease is had important practical significance.
Detection H1N1virus reagent provided by the present invention, relation between its primer adopts the scheme in external novel quick loop-mediated isothermal amplification method provided by the present invention, the novel constant-temperature amplification influenza A H1N1 virus detection method utilizing such scheme to set up has easy and simple to handle, efficient, quick, special advantage; The foundation of the method, only needs can complete for about 40-45 minute to the detection of sample.
Accompanying drawing explanation
Fig. 1 a is the specific test product electrophoresis result of H1N1virus isothermal duplication, and M is marker, and 1 is positive control, 2-3 is H1N1virus, and 4-5 is H3N2 influenza virus, and 6-7 is Influenza B virus, 8 is varicella virus, and 9 is yellow fever virus vaccine, and 10 is negative control.
Fig. 1 b is the specific test precipitin reaction result of H1N1virus isothermal duplication, and M is marker, and 1 is positive control, 2-3 is H1N1virus, and 4-5 is H3N2 influenza virus, and 6-7 is Influenza B virus, 8 is varicella virus, and 9 is yellow fever virus vaccine, and 10 is negative control.
Fig. 1 c is the specific test fluoroscopic examination result of H1N1virus isothermal duplication, and M is marker, and 1 is positive control, 2-3 is H1N1virus, and 4-5 is H3N2 influenza virus, and 6-7 is Influenza B virus, 8 is varicella virus, and 9 is yellow fever virus vaccine, and 10 is negative control.
Fig. 2 is the sensitivity test real-time fluorescence result of H1N1virus isothermal duplication, and M is marker, and 2 is 1 × 10
6copy/μ L, 3 is 1 × 10
5copy/μ L, 4 is 1 × 10
4copy/μ L, 5 is 1 × 10
3copy/μ L, 6 is 1 × 10
2copy/μ L, 7 is 1 × 10
1copy/μ L.
Fig. 3 is the sensitivity test product electrophoresis result of H1N1virus isothermal duplication, and M is marker, and 2 is 1 × 10
6copy/μ L, 3 is 1 × 10
5copy/μ L, 4 is 1 × 10
4copy/μ L, 5 is 1 × 10
3copy/μ L, 6 is 1 × 10
2copy/μ L, 7 is 1 × 10
1copy/μ L.
Fig. 4 is H1N1virus different modes isothermal duplication comparative result, and 1 is novel quick LAMP amplified reaction, and 2 for adding the LAMP amplified reaction of ring primer, and 3 is common LAMP amplified reaction.
Embodiment
The detection of embodiment 1 H1N1virus
1. design of primers
According to H1N1virus HA gene, designing a series of primer, is outer primer F3, outer primer B3, inner primer FIP, inner primer BIP, ring primer Loop-f, ring primer Loop-r respectively, cover primers F NP, cover primer BNP.Primer gene order is as follows:
Outer primer F3:AAGACAAGTTCATGGCCCAATC;
Outer primer B3:CGGCTGCATATCCTGACCC;
Inner primer FIP:
CCGGCTTGAACTTCTTGCTGTAGGGGCATTCACCATCCATCT；
Inner primer BIP:
GTGCTATAAACACCAGCCTCCCAGGCCAGTCTCAATTTTGTGCTT；
Ring primer Loop-f:GCATTCTGATAGAGACTTTGTTGGT;
Ring primer Loop-r:TTCAGAATATACATCCGATCACAAT;
Cover primers F NP:
TGGACACTAGTAGAGCCGGGAGACAGACCCAAAGTGAGGGATCAAGAA；
Cover primer BNP:
TCATTTCAGATACACCAGTTCACGATTGCCTTGGGTGTCTGACAAGTTGTATTG。
In above-mentioned primer, outer primer F3, B3 and inner primer FIP, BIP are the primers of conventional ring mediated isothermal amplification method (LAMP);
Add ring primer Loop-f, ring primer Loop-r, during LAMP can be made to react, primer increases by 2 with the nucleic acid-templated position contacted; Isothermal amplification not only can increase according to nucleic acid-templated with cover inner primer FNP and BNP, also can combine with the product of the conventional LAMP primer amplification in outside and increase simultaneously, make to turn increase 4 with the nucleic acid-templated position contacted;
Cover inner primer FNP not only can combine with the BNP of cover inner primer and carries out amplified reaction, also can combine with the conventional inner primer BIP in outside simultaneously and increase, increase the chance with nucleic acid-templated combination and amplification, otherwise, cover inner primer BNP also can combine with the FNP of cover inner primer and carries out amplified reaction, also can combine with the conventional inner primer FIP in outside simultaneously and carry out amplified reaction;
Utilize above-mentioned primer pair goal gene to carry out ring mediated isothermal amplification, conventional LAMP reaction and the amplified production of LAMP adding ring primer only have the cyclic sequence of a kind of product, add the sequence that ring primer does not change product; After adding cover inner primer, product is exactly the cyclic sequence of 4 kinds of amplified productions, i.e. the amplified production of inner primer FIP, BIP, the amplified production of cover inner primer FNP, BNP, the amplified production of inner primer FIP and cover inner primer BNP, the amplified production of cover inner primer FNP and inner primer BIP.
2. the extraction of viral RNA
The Nasopharyngeal swabs stroke-physiological saline solution of the A (H 1 N 1) virus positive is mixed wash-out, draws 100 μ l and add in 1mL trizol solution, extract viral RNA by test kit specification sheets, be dissolved in 100 μ l without in RNase water ,-70 DEG C of preservations.
3. the reaction system that increases of novel quick ring mediated isothermal (LAMP) and reaction conditions
Reaction system is as follows: 10 × buffer2.5 μ L, the each primer of primer mixture 1 μ L(concentration in primer mixture is as follows respectively: each 1 μm of ol/L of F3, B3, FIP, BIP each 15 μm of ol/L, Loop-f, Loop-r each 5 μm of ol/L, FNP, each 15 μm of ol/L of BNP), template ribonucleic acid 1 μ L, 8U BstDNA polysaccharase 1 μ L, 10U AMV reversed transcriptive enzyme 1 μ L, trimethyl-glycine (8mol/L) 2 μ L, dNTP (10mmol/L) 2 μ L, MgSO
4(100mmol/L) 1.2 μ L, Eva_green1 μ L, adds DEPC-H
2o supplies 25 μ L, and mixing, 63 DEG C of isothermal reactions 45 minutes.
4. specific test
Influenza B virus, H3N2 influenza virus, varicella virus, yellow fever virus vaccine nucleic acid extraction test kit are extracted nucleic acid according to specification sheets, detects with H1N1virus simultaneously.Amplification reaction system is 10 × buffer2.5 μ L, the each primer of primer mixture 1 μ L(concentration in primer mixture is as follows respectively: each 1 μm of ol/L of F3, B3, FIP, BIP each 15 μm of ol/L, Loop-f, Loop-r each 5 μm of ol/L, FNP, each 15 μm of ol/L of BNP), template ribonucleic acid 1 μ L, 8U BstDNA polysaccharase 1 μ L, 10U AMV reversed transcriptive enzyme 1 μ L, trimethyl-glycine (8mol/L) 2 μ L, dNTP (10mmol/L) 2 μ L, MgSO
4(100mmol/L) 1.2 μ L, Eva_green1 μ L, adds DEPC-H
2o supplies 25 μ L, and mixing, 63 DEG C of isothermal reactions after 45 minutes.Reaction electrophoresis result is shown in Fig. 1 a, and precipitin reaction the results are shown in Figure 1b, observes fluorescence results and see Fig. 1 c under ultraviolet lamp.
Reaction result illustrates except H1N1virus nucleic acid occurs reacting except the positive findings of the closeer stepped band of band arrangement than common LAMP, the detected result of negative control, H3N2 influenza virus and Influenza B virus nucleic acid, varicella virus, yellow fever virus does not all amplify band, is shown as feminine gender; Visible on quantitative real time PCR Instrument, only have H1N1virus to occur amplification curve, other viruses are and occur amplification curve, show the method and have good specificity.
5. sensitivity test
With the random priming in Reverse Transcription box, the RNA reverse transcription of having extracted is become cDNA, take cDNA as template, increase by PCR primer, PCR primer purifying is reclaimed test kit and carries out purifying recovery, OD value is measured to the PCR primer after purifying, carry out copy number calculating according to product length and OD value, product is finally diluted to 1 × 10
8copy/ul.CDNA is carried out 10 times of dilutions of going forward one by one, get 1 μ l and add in reaction system as template, under 63 DEG C of constant temperatures, reaction 60min.Quantitative real time PCR Instrument is observed real-time fluorescence response diagram, the results are shown in Figure 3; And get 2 μ l reaction product and carry out 1% agarose gel electrophoresis, seeing Fig. 2, all there is scalariform band in 1-6 swimming lane, indicates the reaction of existing positive amplification.Can judge that the limit of detection of present method is about 1 × 10
2copy/μ L.
6. common LAMP reaction, add ring primer LAMP reaction and compare with the proliferation time that novel quick LAMP reacts
By common LAMP, add ring primer LAMP and novel rapid isothermal amplified reaction and adopt same sample, same reaction conditions to detect on quantitative real time PCR Instrument simultaneously, reaction conditions is 63 DEG C, 55s, fluorescent collecting 5s, 100 circulations.Common LAMP reaction system is: 10 × buffer2.5 μ L, the each primer of primer mixture 1 μ L(concentration in primer mixture is as follows respectively: each 1 μm of ol/L of F3, B3, the each 15 μm of ol/L of FIP, BIP), template ribonucleic acid 1 μ L, 8U BstDNA polysaccharase 1 μ L, 10U AMV reversed transcriptive enzyme 1 μ L, trimethyl-glycine (8mol/L) 2 μ L, dNTP (10mmol/L) 2 μ L, MgSO
4(100mmol/L) 1.2 μ L, Eva_green1 μ L, adds DEPC-H
2o supplies 25 μ L.The reaction system adding ring primer is: 10 × buffer2.5 μ L, the each primer of primer mixture 1 μ L(concentration in primer mixture is as follows respectively: each 1 μm of ol/L of F3, B3, the each 15 μm of ol/L of FIP, BIP, the each 5 μm of ol/L of Loop-f, Loop-r), template ribonucleic acid 1 μ L, 8U BstDNA polysaccharase 1 μ L, 10U AMV reversed transcriptive enzyme 1 μ L, trimethyl-glycine (8mol/L) 2 μ L, dNTP (10mmol/L) 2 μ L, MgSO
4(100mmol/L) 1.2 μ L, Eva_green1 μ L, adds DEPC-H
2o supplies 25 μ L.The reaction system of novel rapid isothermal amplification: 10 × buffer2.5 μ L, the each primer of primer mixture 1 μ L(concentration in primer mixture is as follows respectively: each 1 μm of ol/L of F3, B3, FIP, BIP each 15 μm of ol/L, Loop-f, Loop-r each 5 μm of ol/L, FNP, each 15 μm of ol/L of BNP), template ribonucleic acid 1 μ L, 8U BstDNA polysaccharase 1 μ L, 10U AMV reversed transcriptive enzyme 1 μ L, trimethyl-glycine (8mol/L) 2 μ L, dNTP (10mmol/L) 2 μ L, MgSO
4(100mmol/L) 1.2 μ L, Eva_green1 μ L, adds DEPC-H
2o supplies 25 μ L.The results are shown in Figure 4.Approximately control at about 40-45 minute by the reaction times of the visible FAST-LAMP of real-time fluorescence response diagram, the LAMP reaction times adding ring primer is about 65 minutes, and the common LAMP reaction times was at about 90 minutes.
Embodiment 2, the detection of entry and exit heating personnel H1N1virus
Adopt fever patient Nasopharyngeal swabs 30 person-portion of airport immigration, RNA extraction agent box is utilized to extract RNA according to specification sheets, reaction system is as follows: 10 × buffer2.5 μ L, in primer mixture 1 μ L(primer mixture, each primer concentration is as follows respectively: F3, the each 1 μm of ol/L of B3, FIP, the each 15 μm of ol/L of BIP, Loop-f, the each 5 μm of ol/L of Loop-r, FNP, the each 15 μm of ol/L of BNP), template ribonucleic acid 1 μ L, 8U BstDNA polysaccharase 1 μ L, 10U AMV reversed transcriptive enzyme 1 μ L, trimethyl-glycine (8mol/L) 2 μ L, dNTP (10mmol/L) 2 μ L, MgSO
4(100mmol/L) 1.2 μ L, Eva_green1 μ L, adds DEPC-H
2o supplies 25 μ L, and mixing, with sterilizing distilled water for negative control.On quantitative real time PCR Instrument, response procedures is 63 DEG C, 55 seconds, 63 DEG C, 5 seconds, reads fluorescent value, 60 circulations.Have 4 examples to occur amplification curve in 30 routine fever patients, Novel isothermal amplification A (H 1 N 1) virus detected result is positive, and other do not occur amplification curve, and result is negative.Result conforms to H1N1 quantitative fluorescent PCR reagent detected result.
<110> Zhejiang International Travel Healthcare Center
The reagent of the external novel quick loop-mediated isothermal amplification method of <120> mono-kind, vitro detection H1N1virus
And method
<130>
<160> 8
<170> PatentIn version 3.3
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aagacaagtt catggcccaa tc 22
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cggctgcata tcctgaccc 19
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ccggcttgaa cttcttgctg taggggcatt caccatccat ct 42
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gtgctataaa caccagcctc ccaggccagt ctcaattttg tgctt 45
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gcattctgat agagactttg ttggt 25
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ttcagaatat acatccgatc acaat 25
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tcatttcaga tacaccagtt cacgattgcc ttgggtgtct gacaagttgt attg 54
Claims (2)
1. the detection H1N1virus reagent of external novel quick ring mediated isothermal amplification method, it is characterized in that: it comprises outer primer F3, outer primer B3, inner primer FIP, inner primer BIP, ring primer Loop-f, ring primer Loop-r, cover primers F NP, cover primer BNP, primer gene order is as follows:
Outer primer F3:AAGACAAGTTCATGGCCCAATC;
Outer primer B3:CGGCTGCATATCCTGACCC;
Inner primer FIP:
CCGGCTTGAACTTCTTGCTGTAGGGGCATTCACCATCCATCT；
Inner primer BIP:
GTGCTATAAACACCAGCCTCCCAGGCCAGTCTCAATTTTGTGCTT；
Ring primer Loop-f:GCATTCTGATAGAGACTTTGTTGGT;
Ring primer Loop-r:TTCAGAATATACATCCGATCACAAT;
Cover primers F NP:
TGGACACTAGTAGAGCCGGGAGACAGACCCAAAGTGAGGGATCAAGAA；
Cover primer BNP:
TCATTTCAGATACACCAGTTCACGATTGCCTTGGGTGTCTGACAAGTTGTATTG。
2. use the reaction system of the external novel rapid isothermal augmentation detection H1N1virus of reagent described in claim 1, it is characterized in that:
Reaction system is as follows: primer mixture 1 μ L, 10 × buffer2.5 μ L, template ribonucleic acid 1 μ L, 8U BstDNA polysaccharase 1 μ L, 10U AMV reversed transcriptive enzyme 1 μ L, concentration is the trimethyl-glycine 2 μ L of 8mol/L, concentration is the dNTP 2 μ L of 10mmol/L, and concentration is the MgSO of 100mmol/L
41.2 μ L, Eva_green1 μ L, adds DEPC-H
2o supplies 25 μ L and mixes;
Each primer concentration in primer mixture is as follows respectively: F3, B3 each 1 μm of ol/L, FIP, BIP each 15 μm of ol/L, Loop-f, Loop-r each 5 μm of ol/L, FNP, each 15 μm of ol/L of BNP.
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| CN103497947B (en) * | 2013-09-13 | 2015-08-12 | 浙江国际旅行卫生保健中心 | The reagent of a kind of external novel quick loop-mediated isothermal amplification method, vitro detection H1N1virus and method |
| KR101652806B1 (en) | 2015-08-21 | 2016-09-01 | 주식회사 엠모니터 | Primers used for LAMP reaction for the detection of influenza viruses and its use |
| CN105483211B (en) * | 2015-10-26 | 2018-11-06 | 浙江国际旅行卫生保健中心 | A kind of reagent of the detection legionella pneumophilia of external shell type ring mediated isothermal amplification method and the method for detecting legionella pneumophilia |
| CN108384837A (en) * | 2018-03-28 | 2018-08-10 | 厦门蓝特生物科技有限公司 | A kind of LAMP quantitative approach based on fluorescent quantitation system |
| CN110106289A (en) * | 2019-05-28 | 2019-08-09 | 上海市嘉定区中心医院 | Childrens respiratory tract secretion Type A Influenza H1N1 viral diagnosis primer sets |
| CN113249516B (en) * | 2020-02-10 | 2023-04-25 | 华南理工大学 | Primer group of 2019-nCoV and 2019-nCoV nucleic acid detection kit |
| CN111944925A (en) * | 2020-08-12 | 2020-11-17 | 深圳市儒翰基因科技有限公司 | Primer combination and kit for detecting H1N1 influenza A virus |
| CN113136454B (en) * | 2021-04-15 | 2022-07-12 | 厦门健康工程与创新研究院 | Multiple LAMP primer combination for detecting multiple influenza viruses and application thereof |
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