CN104561377A - Real-time fluorescent multiplex PCR (polymerase chain reaction) based kit for rapidly detecting common respiratory pathogens - Google Patents
Real-time fluorescent multiplex PCR (polymerase chain reaction) based kit for rapidly detecting common respiratory pathogens Download PDFInfo
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Abstract
The invention belongs to the technical field of nucleic acid detection and particularly relates to a real-time fluorescent multiplex PCR (polymerase chain reaction) based kit for rapidly detecting common respiratory pathogens. By adopting the kit, the problem of mutual crosslinking of different primers/probes is solved, the sequences of the primers/probes in each group can not be crosslinked to other target and non-target nucleic acid sequences in homology, and a variety of common pathogens which can cause respiratory infection can be detected simultaneously through one-time reaction. The kit provided by the invention has the advantages of short detection time and high accuracy and functions as a powerful tool in clinical and detection laboratories.
Description
Technical field
The invention belongs to nucleic acid detection technique field, be specifically related to the real-time fluorescence multiple PCR fast detection kit of respiratory system common causative.
Background technology
Respiratory tract infection is common disease, the frequently-occurring disease of China, and the cause of disease of infection has bacterium, virus, mycoplasma etc., and the infected, from flu to pneumonia, can present different symptoms, cause serious complication even dead.
Respiratory tract infection is divided into upper respiratory tract infection and lower respiratory infection, and the former shows as acute rhinitis, pharyngitis and laryngitis, and the latter shows as acute tracheitis, bronchitis and pneumonia.The infection reason of upper respiratory tract infection can be bacterium or fungi.About 90% is caused by virus, after bacteriological infection is often secondary to virus infection.The cause of disease mainly bacterium and the fungi of lower respiratory infection.Because the type of virus is more, human body is more weak and of short duration to the immunizing power produced after various virus infection, there is no cross immunity, simultaneously in healthy population, has virus carrier, therefore people can have in 1 year and repeatedly falls ill.Economize Emergency department in hospital of TCM statistics according to certain, account for about 40% of emergency treatment outpatient service sum with the medical patient that generates heat, wherein Acute viral upper respiratory infection fever patient about just accounts for more than 80% wherein.Therefore, very necessary to the diagnosis fast and accurately of respiratory pathogen.In grownup and children, acute respiratory infection is the most general acute infectious disease, and Pathogen category is various, and this just brings huge challenge to diagnosis.
At present, respiratory pathogens body detecting method is virus culture the most accurately clinically, has the advantages that specificity is high, is clinical diagnosis " gold standard ".But culture method is consuming time, susceptibility is low, require high to experimental implementation.In addition also have microbial culture and susceptibility method, resistance situation can be monitored, but sample easily pollutes, large to Influence on test result; The mensuration of virus antigen can be used as infection evidence, detect fast, but susceptibility is low, and can not distinguish pollution and field planting bacterium; Detection of specific antibody, can do early diagnosis, specificity and susceptibility high, but be subject to the dynamic (dynamical) impact of antibody.It is high that fluorescence nucleic acid round pcr has susceptibility, detects the advantages such as quick, and expection will be widely used in clinical diagnosis.But traditional fluorescent real time PCR can only increase a kind of nucleic acid of pathogenic agent, and accurately will find out disease-producing pathogens need repeatedly attempt, the time and efforts of at substantial, and cannot effectively distinguish for the infection that multiple pathogens causes at every turn.Novel multiple real time fluorescence PCR effectively can make up these defects of substance PCR, is a kind of diagnostic method having very much application prospect.
Want successful implementation multiplex PCR, need to solve several technical problem:
because the best amplification condition of each amplicon is all different, need the condition coordinating each independent reaction to ensure that each amplicon can carry out Successful amplification under single amplification condition.
organizing primer/probe combinations because needs use more, produce mutually crosslinked possibility between different primers/probe very large, therefore needing when designing to consider to eliminate this possibility.
often organize primer/probe and can not have cross homology to other targets and non-targeted nucleotide sequence, to prevent false-positive result, affect the diagnosis to disease.
need certain method to get rid of the problem coming from nucleic acid extraction aspect, this just requires internal reference as a reference.
Summary of the invention
The object of the invention is to solve between different primers/probe and produce crosslinked problem mutually, the real-time fluorescence multiple PCR fast detection kit of respiratory system common causative is provided, can measures fast, accurately, effectively.
The present invention for the taked technical scheme that solves the problem is: the real-time fluorescence multiple PCR fast detection kit of respiratory system common causative, positive control is provided with in test kit, negative control and internal reference, also be provided with in this test kit for detecting influenza A virus, H1N1 subtype virus, influenza B virus, rhinovirus, HCoV-229E, HCoV-HKU1, coronavirus N L63, HCoV-OC43, parainfluenza virus 2, parainfluenza virus 3, parainfluenza virus 4, human bocavirus, human metapneumovirus, parainfluenza virus 1, mycoplasma pneumoniae, adenovirus, enterovirus, the multiple primer of one or more pathogenic agent and the mixture of probe in two Echo virus and respiratory syncytial virus, wherein, the primer sequence detected for influenza A virus is respectively SEQ ID NO: 1 and SEQ ID NO: 2, Taqman probe sequence is SEQ ID NO: 3, fluorescent mark is FAM, primer sequence for the detection of H1N1 subtype virus is respectively SEQ ID NO: 4 and SEQ ID NO: 5, Taqman probe sequence is SEQ ID NO: 6, and fluorescent mark is Cy5, primer sequence for influenza B Viral diagnosis is respectively SEQ ID NO: 7 and SEQ ID NO: 8, Taqman probe sequence is SEQ ID NO: 9, and fluorescent mark is ROX, primer sequence for rhinovirus detection is respectively SEQ ID NO: 10 and SEQ ID NO: 11, Taqman probe sequence is SEQ ID NO: 12, and fluorescent mark is VIC, primer sequence for HCoV-229E detection is respectively SEQ ID NO: 13 and SEQ ID NO: 14, Taqman probe sequence is SEQ ID NO: 15, and fluorescent mark is FAM, primer sequence for HCoV-HKU1 detection is respectively SEQ ID NO: 16 and SEQ ID NO: 17, Taqman probe sequence is SEQ ID NO: 18, and fluorescent mark is ROX, primer sequence for coronavirus N L63 detection is respectively SEQ ID NO: 19 and SEQ ID NO: 20, Taqman probe sequence is SEQ ID NO: 21, and fluorescent mark is VIC, primer sequence for HCoV-OC43 detection is respectively SEQ ID NO: 22 and SEQ ID NO: 23, Taqman probe sequence is SEQ ID NO: 24, and fluorescent mark is Cy5, primer sequence for parainfluenza virus 2 detection is respectively SEQ ID NO: 25 and SEQ ID NO: 26, Taqman probe sequence is SEQ ID NO: 27, and fluorescent mark is VIC, primer sequence for parainfluenza virus 3 detection is respectively SEQ ID NO: 28 and SEQ ID NO: 29, Taqman probe sequence is SEQ ID NO: 30, and fluorescent mark is FAM, primer sequence for parainfluenza virus 4 detection is respectively SEQ ID NO: 31 and SEQ ID NO: 32, Taqman probe sequence is SEQ ID NO: 33, and fluorescent mark is ROX, primer sequence for human bocavirus detection is respectively SEQ ID NO: 34 and SEQ ID NO: 35, Taqman probe sequence is SEQ ID NO: 36, and fluorescent mark is ROX, primer sequence for human metapneumovirus detection is respectively SEQ ID NO: 37 and SEQ ID NO: 38, Taqman probe sequence is SEQ ID NO: 39, and fluorescent mark is VIC, primer sequence for parainfluenza virus 1 detection is respectively SEQ ID NO: 40 and SEQ ID NO: 41, Taqman probe sequence is SEQ ID NO: 42, and fluorescent mark is FAM, primer sequence for mycoplasma pneumoniae detection is respectively SEQ ID NO: 43 and SEQ ID NO: 44, Taqman probe sequence is SEQ ID NO: 45, and fluorescent mark is Cy5, primer sequence for adenovirus detection is respectively SEQ ID NO: 46 and SEQ ID NO: 47, Taqman probe sequence is SEQ ID NO: 48, and fluorescent mark is Cy5, primer sequence for enterovirus detection is respectively SEQ ID NO: 49 and SEQ ID NO: 50, Taqman probe sequence is SEQ ID NO: 51, and fluorescent mark is ROX, the primer sequence detected for two Echo virus is respectively SEQ ID NO: 52 and SEQ ID NO: 53, Taqman probe sequence is SEQ ID NO: 54, and fluorescent mark is VIC, primer sequence for respiratory syncytial virus detection is respectively SEQ ID NO: 55 and SEQ ID NO: 56, Taqman probe sequence is SEQ ID NO: 57, and fluorescent mark is FAM, described internal reference is equine arteritis virus, and the primer sequence for equine arteritis virus detection is respectively SEQ ID NO: 58 and SEQ ID NO: 59, Taqman probe sequence is SEQ ID NO: 60, and fluorescent mark is Cy5.
beneficial effect
Detection kit involved in the present invention, avoid between different primers/probe and produce crosslinked problem mutually, often organize primer/probe and can not produce cross homology to other targets and non-targeted nucleotide sequence, primary first-order equation can be realized and detect the multiple common causative causing respiratory system infection simultaneously, detection required time is short, accuracy is high, for clinical and testing laboratory provide strong testing tool.
Embodiment
The real-time fluorescence multiple PCR fast detection kit of respiratory system common causative, positive control is provided with in test kit, negative control and internal reference, also be provided with in this test kit for detecting influenza A virus, H1N1 subtype virus, influenza B virus, rhinovirus, HCoV-229E, HCoV-HKU1, coronavirus N L63, HCoV-OC43, parainfluenza virus 2, parainfluenza virus 3, parainfluenza virus 4, human bocavirus, human metapneumovirus, parainfluenza virus 1, mycoplasma pneumoniae, adenovirus, enterovirus, the multiple primer of one or more pathogenic agent and the mixture of probe in two Echo virus and respiratory syncytial virus, wherein, the primer sequence detected for influenza A virus is respectively SEQ ID NO: 1 and SEQ ID NO: 2, Taqman probe sequence is SEQ ID NO: 3, fluorescent mark is FAM, primer sequence for the detection of H1N1 subtype virus is respectively SEQ ID NO: 4 and SEQ ID NO: 5, Taqman probe sequence is SEQ ID NO: 6, and fluorescent mark is Cy5, primer sequence for influenza B Viral diagnosis is respectively SEQ ID NO: 7 and SEQ ID NO: 8, Taqman probe sequence is SEQ ID NO: 9, and fluorescent mark is ROX, primer sequence for rhinovirus detection is respectively SEQ ID NO: 10 and SEQ ID NO: 11, Taqman probe sequence is SEQ ID NO: 12, and fluorescent mark is VIC, primer sequence for HCoV-229E detection is respectively SEQ ID NO: 13 and SEQ ID NO: 14, Taqman probe sequence is SEQ ID NO: 15, and fluorescent mark is FAM, primer sequence for HCoV-HKU1 detection is respectively SEQ ID NO: 16 and SEQ ID NO: 17, Taqman probe sequence is SEQ ID NO: 18, and fluorescent mark is ROX, primer sequence for coronavirus N L63 detection is respectively SEQ ID NO: 19 and SEQ ID NO: 20, Taqman probe sequence is SEQ ID NO: 21, and fluorescent mark is VIC, primer sequence for HCoV-OC43 detection is respectively SEQ ID NO: 22 and SEQ ID NO: 23, Taqman probe sequence is SEQ ID NO: 24, and fluorescent mark is Cy5, primer sequence for parainfluenza virus 2 detection is respectively SEQ ID NO: 25 and SEQ ID NO: 26, Taqman probe sequence is SEQ ID NO: 27, and fluorescent mark is VIC, primer sequence for parainfluenza virus 3 detection is respectively SEQ ID NO: 28 and SEQ ID NO: 29, Taqman probe sequence is SEQ ID NO: 30, and fluorescent mark is FAM, primer sequence for parainfluenza virus 4 detection is respectively SEQ ID NO: 31 and SEQ ID NO: 32, Taqman probe sequence is SEQ ID NO: 33, and fluorescent mark is ROX, primer sequence for human bocavirus detection is respectively SEQ ID NO: 34 and SEQ ID NO: 35, Taqman probe sequence is SEQ ID NO: 36, and fluorescent mark is ROX, primer sequence for human metapneumovirus detection is respectively SEQ ID NO: 37 and SEQ ID NO: 38, Taqman probe sequence is SEQ ID NO: 39, and fluorescent mark is VIC, primer sequence for parainfluenza virus 1 detection is respectively SEQ ID NO: 40 and SEQ ID NO: 41, Taqman probe sequence is SEQ ID NO: 42, and fluorescent mark is FAM, primer sequence for mycoplasma pneumoniae detection is respectively SEQ ID NO: 43 and SEQ ID NO: 44, Taqman probe sequence is SEQ ID NO: 45, and fluorescent mark is Cy5, primer sequence for adenovirus detection is respectively SEQ ID NO: 46 and SEQ ID NO: 47, Taqman probe sequence is SEQ ID NO: 48, and fluorescent mark is Cy5, primer sequence for enterovirus detection is respectively SEQ ID NO: 49 and SEQ ID NO: 50, Taqman probe sequence is SEQ ID NO: 51, and fluorescent mark is ROX, the primer sequence detected for two Echo virus is respectively SEQ ID NO: 52 and SEQ ID NO: 53, Taqman probe sequence is SEQ ID NO: 54, and fluorescent mark is VIC, primer sequence for respiratory syncytial virus detection is respectively SEQ ID NO: 55 and SEQ ID NO: 56, Taqman probe sequence is SEQ ID NO: 57, and fluorescent mark is FAM, described internal reference is equine arteritis virus, and the primer sequence for equine arteritis virus detection is respectively SEQ ID NO: 58 and SEQ ID NO: 59, Taqman probe sequence is SEQ ID NO: 60, and fluorescent mark is Cy5.
Embodiment one: detect influenza A virus: use influenza A virus positive control as sample.Embodiment two is shown in the acquisition of corresponding primer/probe combinations and positive control.First 2 μ l internal references (5 μMs) were added to sample and negative control (distilled water) before extraction.MagMAX Total Nucleic Acid Isolation Kit (Invitrogen) is used to extract nucleic acid from sample.Then the nucleic acid (10 μ l) adding each primer/probe combinations (1.5 μ l) respectively and extract from sample or negative control in TaqMan Fast Virus 1-Step Master Mix (Invitrogen, 13.5 μ l).Sample ultimate density is 5 μMs, and primer/concentration and probe concentration is 0.4 μM.Subsequently by each reaction tubes according to following program at suitable real-time fluorescence PCR instrument (as ABI 7500, Applied Biosystems) enterprising performing PCR reaction: 50 DEG C keep 15min (reverse transcription), and 95 DEG C keep 10min (warm start); 95 DEG C maintain 8s (sex change), and 60 DEG C maintain 34s (annealing/extend, gathers fluorescent signal), 40 circulations.Result can detect that influenza A virus exists.The detection of all the other each pathogenic agent is identical with embodiment 1.
Embodiment two: the detection kit of the various pathogenic agent of one-time detection respiratory system
1) primer/probe combinations is designed: according to the nucleotide sequence of each pathogenic agent, carry out the sequence alignment of of the same race to find conservative region, and use Beacon Designer software to carry out primer and the Taqman probe design of multiplex PCR to these conservative regions.Obtained each primer/probe combinations is carried out on NCBI website BLAST and analyze to guarantee cross reaction not to occur with other microorganisms that may exist in sample.Finally by its performance of experimental verification.The design of internal reference is adopted and is used the same method.
Each group of designed primer/probe is as follows:
Reaction one: containing influenza A and H1N1 subtype virus, influenza B virus, rhinovirus
Reaction two: containing HCoV-229E, NL63, OC43 and HKU1
Reaction three: containing parainfluenza virus 2,3,4 and equine arteritis virus (internal reference)
Reaction four: containing parainfluenza virus 1, human metapneumovirus, mycoplasma pneumoniae, human bocavirus
Reaction five: containing respiratory syncytial virus, adenovirus, enterovirus, two Echo virus
2) prepare each positive control: the nucleic acid being first separated various pathogenic agent, and prepare PCR reaction system, then various pathogen nucleic acid is added in reaction system respectively and carry out PCR reaction.Reaction product carries out reclaiming and using electroresis appraisal.Then use suitable TA Cloning Kit (as TOPO TA Cloning Kit for Subcloning, with TOP10 E. Coli, Invitrogen) and plasmid purification kit (as PureLink 96 Plasmid Purification System, Invitrogen) obtain the plasmid vector being connected with each target pathogen sequence, be used as the positive control of PCR reaction.
3) internal reference is prepared: use cell culture method to be separated EAV strain.
4) the main mixture of ready reaction: use TaqMan Fast Virus 1-Step Master Mix(Invitrogen), the Taq archaeal dna polymerase containing premix, ThermoScript II, the main mixture of the components such as dNTP, RNase inhibitor.
5) pcr amplification reaction is carried out: before extraction, first add 2 μ l internal references (5 μMs) to sample and negative control (distilled water).Suitable RNA/DNA extractive technique (as MagMAX Total Nucleic Acid Isolation Kit, Invitrogen) is used to extract nucleic acid from sample.Then the nucleic acid (10 μ l) adding each primer/probe combinations (1.5 μ l) respectively in the main mixture of reaction (13.5 μ l) and extract from sample or negative control.With identical ratio, married operation is carried out to positive control.Ultimate density positive control is 5 μMs, and primer/concentration and probe concentration is 0.4 μM.Subsequently by each reaction tubes according to following program at suitable real-time fluorescence PCR instrument (as ABI 7500, Applied Biosystems) enterprising performing PCR reaction: 50 DEG C keep 15min (reverse transcription), and 95 DEG C keep 10min (warm start); 95 DEG C maintain 8s (sex change), and 60 DEG C maintain 34s (annealing/extend, gathers fluorescent signal), 40 circulations.
Result judges: the signal value of all negative controls all should lower than threshold value.Index all should be had to rise for all positive control signals and Ct value must be less than 33.Index should be had to rise for internal reference signal and Ct value must be less than 33.Otherwise illustrate that amplification is unsuccessful.Fluorescent signal as corresponding in certain pathogenic agent produces index and rises, be then positive, otherwise is negative.
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The magnificent biotechnology company limited of <110>
The real-time fluorescence multiple PCR fast detection kit of <120> respiratory system common causative
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<210> 55
<211> 18
<212> DNA
<213> artificial sequence
<400> 55
cagcagggga tagatcag 18
<210> 56
<211> 18
<212> DNA
<213> artificial sequence
<400> 56
tggctacatc ctttggta 18
<210> 57
<211> 22
<212> DNA
<213> artificial sequence
<400> 57
cgcagtcatc agaagagcca ac 22
<210> 58
<211> 18
<212> DNA
<213> artificial sequence
<400> 58
cctcttttcg aaacggac 18
<210> 59
<211> 18
<212> DNA
<213> artificial sequence
<400> 59
cgatgatagc ctgagtgg 18
<210> 60
<211> 25
<212> DNA
<213> artificial sequence
<400> 60
ctacaagcta caatgaccta ctgcg 25
Claims (1)
1. the real-time fluorescence multiple PCR fast detection kit of respiratory system common causative, positive control is provided with in test kit, negative control and internal reference, it is characterized in that: be also provided with for detecting influenza A virus in this test kit, H1N1 subtype virus, influenza B virus, rhinovirus, HCoV-229E, HCoV-HKU1, coronavirus N L63, HCoV-OC43, parainfluenza virus 2, parainfluenza virus 3, parainfluenza virus 4, human bocavirus, human metapneumovirus, parainfluenza virus 1, mycoplasma pneumoniae, adenovirus, enterovirus, the multiple primer of one or more pathogenic agent and the mixture of probe in two Echo virus and respiratory syncytial virus, wherein, the primer sequence detected for influenza A virus is respectively SEQ ID NO: 1 and SEQ ID NO: 2, Taqman probe sequence is SEQ ID NO: 3, fluorescent mark is FAM, primer sequence for the detection of H1N1 subtype virus is respectively SEQ ID NO: 4 and SEQ ID NO: 5, Taqman probe sequence is SEQ ID NO: 6, and fluorescent mark is Cy5, primer sequence for influenza B Viral diagnosis is respectively SEQ ID NO: 7 and SEQ ID NO: 8, Taqman probe sequence is SEQ ID NO: 9, and fluorescent mark is ROX, primer sequence for rhinovirus detection is respectively SEQ ID NO: 10 and SEQ ID NO: 11, Taqman probe sequence is SEQ ID NO: 12, and fluorescent mark is VIC, primer sequence for HCoV-229E detection is respectively SEQ ID NO: 13 and SEQ ID NO: 14, Taqman probe sequence is SEQ ID NO: 15, and fluorescent mark is FAM, primer sequence for HCoV-HKU1 detection is respectively SEQ ID NO: 16 and SEQ ID NO: 17, Taqman probe sequence is SEQ ID NO: 18, and fluorescent mark is ROX, primer sequence for coronavirus N L63 detection is respectively SEQ ID NO: 19 and SEQ ID NO: 20, Taqman probe sequence is SEQ ID NO: 21, and fluorescent mark is VIC, primer sequence for HCoV-OC43 detection is respectively SEQ ID NO: 22 and SEQ ID NO: 23, Taqman probe sequence is SEQ ID NO: 24, and fluorescent mark is Cy5, primer sequence for parainfluenza virus 2 detection is respectively SEQ ID NO: 25 and SEQ ID NO: 26, Taqman probe sequence is SEQ ID NO: 27, and fluorescent mark is VIC, primer sequence for parainfluenza virus 3 detection is respectively SEQ ID NO: 28 and SEQ ID NO: 29, Taqman probe sequence is SEQ ID NO: 30, and fluorescent mark is FAM, primer sequence for parainfluenza virus 4 detection is respectively SEQ ID NO: 31 and SEQ ID NO: 32, Taqman probe sequence is SEQ ID NO: 33, and fluorescent mark is ROX, primer sequence for human bocavirus detection is respectively SEQ ID NO: 34 and SEQ ID NO: 35, Taqman probe sequence is SEQ ID NO: 36, and fluorescent mark is ROX, primer sequence for human metapneumovirus detection is respectively SEQ ID NO: 37 and SEQ ID NO: 38, Taqman probe sequence is SEQ ID NO: 39, and fluorescent mark is VIC, primer sequence for parainfluenza virus 1 detection is respectively SEQ ID NO: 40 and SEQ ID NO: 41, Taqman probe sequence is SEQ ID NO: 42, and fluorescent mark is FAM, primer sequence for mycoplasma pneumoniae detection is respectively SEQ ID NO: 43 and SEQ ID NO: 44, Taqman probe sequence is SEQ ID NO: 45, and fluorescent mark is Cy5, primer sequence for adenovirus detection is respectively SEQ ID NO: 46 and SEQ ID NO: 47, Taqman probe sequence is SEQ ID NO: 48, and fluorescent mark is Cy5, primer sequence for enterovirus detection is respectively SEQ ID NO: 49 and SEQ ID NO: 50, Taqman probe sequence is SEQ ID NO: 51, and fluorescent mark is ROX, the primer sequence detected for two Echo virus is respectively SEQ ID NO: 52 and SEQ ID NO: 53, Taqman probe sequence is SEQ ID NO: 54, and fluorescent mark is VIC, primer sequence for respiratory syncytial virus detection is respectively SEQ ID NO: 55 and SEQ ID NO: 56, Taqman probe sequence is SEQ ID NO: 57, and fluorescent mark is FAM, described internal reference is equine arteritis virus, and the primer sequence for equine arteritis virus detection is respectively SEQ ID NO: 58 and SEQ ID NO: 59, Taqman probe sequence is SEQ ID NO: 60, and fluorescent mark is Cy5.
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| CN105018488A (en) * | 2015-08-03 | 2015-11-04 | 博奥生物集团有限公司 | Kit used for detecting respiratory viruses and application thereof |
| CN105463129A (en) * | 2015-12-09 | 2016-04-06 | 深圳国际旅行卫生保健中心 | Double-fluorescent PCR detection primer, probe, reaction liquid and kit capable of detecting pathogens of respiratory tract |
| CN105648115A (en) * | 2016-02-26 | 2016-06-08 | 深圳市亿立方生物技术有限公司 | PCR (polymerase chain reaction) primer group, probe set and kit for detecting multiple respiratory pathogens |
| CN105713993A (en) * | 2016-02-26 | 2016-06-29 | 深圳市亿立方生物技术有限公司 | PCR primer set for detecting multiple respiratory viruses, probe set and kit |
| CN106868213A (en) * | 2017-03-21 | 2017-06-20 | 重庆迪安医学检验中心有限公司 | A kind of primer and its application for detecting parainfluenza virus and coronavirus simultaneously based on melting curve method single tube |
| CN107058622A (en) * | 2017-03-30 | 2017-08-18 | 德必碁生物科技（厦门）有限公司 | A kind of kit of multiple fluorescence PCR method joint-detection respiratory pathogen |
| CN107090519A (en) * | 2017-04-05 | 2017-08-25 | 苏州协云基因科技有限公司 | The multiple RT PCR Polymorphism chip inspecting reagent units of respiratory tract common causative |
| CN107254554A (en) * | 2017-07-05 | 2017-10-17 | 广州奥百阕谱生物科技有限公司 | Detect the suspension microballon array system of respiratory tract infection common causative |
| CN107365876A (en) * | 2017-08-07 | 2017-11-21 | 南京岚煜生物科技有限公司 | For detecting the kit and its application method of 10 respiratory tract infection pathogen |
| WO2017216204A1 (en) * | 2016-06-14 | 2017-12-21 | Roche Diagnostics Gmbh | Internal control probes for improving pcr assay performance |
| CN109609692A (en) * | 2018-12-25 | 2019-04-12 | 北京卓诚惠生生物科技股份有限公司 | For detecting the nucleic acid reagent, kit, system and method for respiratory tract infection pathogen |
| CN110408726A (en) * | 2019-07-23 | 2019-11-05 | 中国人民解放军军事科学院军事医学研究院 | The method for detecting 29 kinds of respiratory pathogens using Taqman low-density microfluidic chip technology |
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| CN116042921A (en) * | 2022-12-28 | 2023-05-02 | 圣湘生物科技股份有限公司 | Composition, kit and method for detecting viral encephalitis virus and application of composition |
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| CN107254554B (en) * | 2017-07-05 | 2020-11-03 | 广州奥百阕谱生物科技有限公司 | Suspended micro-bead array system for detecting common pathogens of respiratory tract infection |
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| CN110408726A (en) * | 2019-07-23 | 2019-11-05 | 中国人民解放军军事科学院军事医学研究院 | The method for detecting 29 kinds of respiratory pathogens using Taqman low-density microfluidic chip technology |
| CN110408726B (en) * | 2019-07-23 | 2022-10-04 | 中国人民解放军军事科学院军事医学研究院 | Method for detecting 29 respiratory pathogens by using Taqman low-density microfluidic chip technology |
| CN110468234A (en) * | 2019-08-09 | 2019-11-19 | 厦门安普利生物工程有限公司 | Multiple fluorescence quantitative PCR kit for 19 kinds of human respiratory viral diagnosis |
| CN111808989A (en) * | 2020-06-18 | 2020-10-23 | 重庆浦洛通基因医学研究院有限公司 | Coronavirus/influenza virus/rhinovirus nucleic acid combined detection kit and use method thereof |
| WO2024006845A1 (en) * | 2022-06-28 | 2024-01-04 | Life Technologies Corporation | Multiplex panel for upper respiratory pathogens |
| CN116042921A (en) * | 2022-12-28 | 2023-05-02 | 圣湘生物科技股份有限公司 | Composition, kit and method for detecting viral encephalitis virus and application of composition |
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