WO2005038427A2 - Identification rapide des virus responsables des infections des voies respiratoires superieures, y compris des coronavirus responsables du syndrome respiratoire aigu severe - Google Patents

Identification rapide des virus responsables des infections des voies respiratoires superieures, y compris des coronavirus responsables du syndrome respiratoire aigu severe Download PDF

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WO2005038427A2
WO2005038427A2 PCT/US2004/017767 US2004017767W WO2005038427A2 WO 2005038427 A2 WO2005038427 A2 WO 2005038427A2 US 2004017767 W US2004017767 W US 2004017767W WO 2005038427 A2 WO2005038427 A2 WO 2005038427A2
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sequence
sars
pathogen
pcr
amplification
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PCT/US2004/017767
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WO2005038427A3 (fr
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Joseph Wing On Tam
Hok Sin Woo
Wing Sze Lo
Sin Fun Sia
Tsz Kam Fang
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Medical Gene Center Ltd.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the present invention relates to method of making definitive identification of upper respiratory viruses and Severe Acute Respiratory Syndrome (SARS) causing viruses such as coronus and related viruses in human and animals by DNA analysis and the device thereof.
  • SARS Severe Acute Respiratory Syndrome
  • SARS Severe Acute Respiratory Syndrome
  • the present invention is based on a low-density array membrane flow-through system which provides a better detection format .
  • the present invention not only provides a fast, simple format for identifying the coronus virus with adequate controls, but also provides simultaneous identification of the known genotypes of the causative pathogens for SARS. Moreover, if needed, additional pathogens, whether SARS related or unrelated, can alsobedetectedinthe same reaction and/or in the same membrane or matrixes simultaneously. Once the results are known, appropriate and potentially life-saving treatment regimen specific to the identified pathoge (s) can be prescribed for the patient.
  • this invention provides a novel method of analysis for SARS which can also be used for the analyses of other gene(s) or pathogens .
  • the method is based on our patented principle of Direct Flow-through DNA Hybridization (Tarn JWO, US Pat. No. 5,471,547 (1998) & 6,020, 187 (2000) ) .
  • Direct Flow-through DNA Hybridization is the fastest annealing process that uses a very inexpensive device for accurate mutation detection, genotyping and fingerprinting analysis.
  • the present invention presents the scheme and data obtained from analyzing the SARS pathogen and SARS causative pathogens by RT-PCR and Allele (or gene or sequence) Specific Oligonucleotides (ASO) probes (oligo-probes) using the Flow-through format .
  • ASO Oligonucleotides
  • ASO-RDB Allelic-Specific-Oligonucleotide Reversed-Dot- Blotting
  • Data obtained refer to the specific segments of coronus virus at multiple regions that are able to provide accurate determination of the genotypes and/or multiple species of pathogens, such as the upper respiratory track infectious viruses, using the fast hybridization procedures, and the ASO oligo-probes in a low-density array format of the present invention.
  • PCR amplification using multiple pairs of PCR primers was used.
  • Nucleic acids amplification can be performed using other amplification methods, such as strand displacement amplification (SDA) , transcription mediated amplification (TMA) , loop-mediated amplification (LAMP) and other amplification methods already established or new amplification technology to be invented in the future.
  • SDA strand displacement amplification
  • TMA transcription mediated amplification
  • LAMP loop-mediated amplification
  • the primers and oligo-probes shown in the sequence listing described herein and attached have been tested and confirmed to be useful for the upper respiratory viruses including SARS causing viruses.
  • primers and oligo-probes are used as examples and, therefore, are not the only primers and oligo-probes which can be used with this invention; other primers and oligo-probes can be created by the methods set out in this invention as soon as the sequences of the viruses are known.
  • PCR was used as the amplification method, any other method that can produce specific target sequences in enough quantity and/or without amplification if naturally occurring in high enough quantity for testing can also be used for the ASO-RDB flow through Hybridization determination.
  • the detection can be by labeling of the target DNA or conjugates through any known or those developed in the future appropriate for present method of detection.
  • Fig. 1 is the diagram of the method for obtaining the ASO probes and PCR primers data base of the invention.
  • Fig. 2 is one of the examples the hybridization device already in production and registered elsewhere.
  • Fig. 3 is the agarose gel image showing the detection limit of SARS coronavirus down to 2 copies.
  • Fig. 4 shows the result of SARS coronus virus detection by flow-through hybridization. Positive signals are represented by blue dots on the pre-probed membrane.
  • Fig. 5 is the arrangement of the ASO probes of the membrane array.
  • Fig. 6 is the hybridization result of the SARS coronus virus variant (genotyping identification) detection.
  • Table below shows three known strains or variants CA, CB, Tor (as the wide type) , and the mutated sequence probes that were used to test the correspondent mutants. The probe arrangement is given along with resulted image.
  • Fig. 7 is the arrangement of the viral detection array for the upper respiratory track infection.
  • Respiratory syncytial virus (RSV) 2. Enterovirus (Entero) 3. Adenovirus (Adeno) 4. Parainfluenza virus type 1 (PIV1) 5. Parainfluenza virus type 3 (PIV 3) 6. Parainfluenza virus type 3B (PIV 3B) 7. Influenza A (Inf A) 8. Influenza B (Inf B) 9. SARS coronavirus (SARS) 10. Metapneumovirus (Meta) 11. Mycoplasma pneumoniae (M. Pneu) 12. Chlamydia pneumoniae (CP)
  • Fig. 8 shows the hybridization results of viral detection array after multiplex PCR: A) SARS coronavirus; B) Influenza A virus; C) Parainfluenza type 1 virus; D) Respiratory syncytical virus.
  • the test specificity was evaluated with theuseofnucleicacid samples and probes of 10 other respiratory tract pathogens (respiratory syncytial virus (RSV) , parainfluenza virus type 1 & 3 (PIV 1 & 3) , Mycoplasma pneumoniae, Chlamydia pneumoniae, enterovirus, influenza A & B, adenovirus, metapneumovirus). No cross-reaction was detected.
  • This figure shows examples of the specific HybriMax-based hybridization of SARS coronavirus and other pathogens PCR products to SARS coronavirus probe and other specific probes respectively.
  • Adenovirus (Adeno) 4. Parainfluenza virus type 1 (PIV1)
  • Chlamydia pneumoniae (CP)
  • Analytical sensitivity was determined by RT-PCR of serial dilution of in vitro transcribed cloned RT-PCRproducts .
  • the detection limits were as follows :
  • This invention provides a method for the detection of a pathogen through analysis of nucleic acid sequences, comprising the steps of: (a) selecting at least one appropriate target sequence from the pathogen; (b) screening said sequence to obtain at least one appropriate primer for amplification; (c) selecting an appropriate probe from the sequence; (d) immobilizing the sequence-specific-oligonucleotide site of each sequence onto a solid matrix suitable for capturing the target sequence; (e) amplifying the sequence (s) wherein the steps (d) and (e) are performed separately;
  • steps (f) and (e) are performed sequentially or concurrently.
  • the sequence is a gene, pathogen or allelic sequence.
  • the solidmatri is a low-density array for detecting multiple SARS variant.
  • the solid matrix is a viral detection array for detecting upper respiratory tract infection.
  • the array is on a single membrane .
  • the pathogen includes but is not limited to the upper respiratory viruses.
  • the pathogen is a coronavirus.
  • the pathogen is an avian influenza virus.
  • the primer and the sequence-specific-oligonucleotide site are obtained by screening data from the GenBank or by sequencing the target sequence or in combination thereof.
  • target sequence comprises a gene, nucleic acid or DNA sequence.
  • the probe is a single nucleotide polymorphism probe.
  • the solid matrix is a membrane or nylon.
  • a target sequence having a concentration less than 0.5 f moles is amplified by the above describe method.
  • the amplification step can be performed by PCR amplification, isothermal amplification, nested RT-PCR amplification, two-step nested RT-PCR amplification.
  • the upper respiratory viruses include but is not limited to a group selected from respiratory syncytial virus (RSV) , parainfluenza virus type 1 (PIV1) , parainfluenza virus type
  • PAV3 parainfluenza virus type 3B
  • MP Mycoplasma pneumoniae
  • CP Chlamydia pneumoniae
  • EN Enterovirus
  • EN Influenza A
  • Inf B Influenza B
  • Adenovirus Adeno
  • Mea Metapneumovirus
  • SARS SARS coronavirus
  • the invention provides a method for the detection of more than one pathogen through analysis of nucleic acid sequences, comprising the steps as described above. However, more than one pair of primers can also be used.
  • the detection procedure can be completed within two hours . In another embodiment, the detection procedure is finished within minutes . In a further embodiment, the detection procedure is finished within 20 minutes using enzyme link color development. Preferably, the detection procedure can be completed within between 2 to 3 minutes with using use colloidal gold or florescence dye.
  • This invention further provides a method for the detection of pathogens including but not limited to SARS causative pathogens.
  • This invention further provides novel polymerase chain reaction (PCR) primers and allele-, gene- or sequence-specific oligonucleotides
  • ASO oligo-probes
  • This invention further provides a method for identifying novel probes and primers which can be used to detecting pathogen (s) .
  • This invention provides a kit for detection of pathogen comprising a compartment containing the primer (s) or probe (s) or a combination thereof, which are disclosed herein or developed by the methods as describe above.
  • This invention provides a machine capable of detecting pathogens through analysis of nucleic acid sequences. In an embodiment, the machine can be programmed to automatically carryout the methods as described above.
  • This invention provides a method for amplifying the target sequence of a pathogen for hybridization comprising the steps of: selecting an appropriate primer pair; performing reverse transcription on the primer pair; and performing the first and second round of PCR in a single tube .
  • the second round of PCR is performed with a thermal cycling program having the following thermal cycling profile: (1) 95°C, 5 min; (2) 95°C, 10 sec; (3) 56°C, lOsec; (4) 72°C, 30sec; (5) 39 cycle repeats on steps 2 to 4, followed by 72°C, 10 min final extension time.
  • the second round PCR was done using lul of the 1:10 dilution of the first round product in 50ul reaction volume .
  • the reverse transcription is performed with positive control RNA of about 2xl0 "5 copies using the Thermoscript RT-PCR system and Platinum Taq DNA polymerase.
  • thermal cycling profile (1) 95°C - 3min; (2) 95°C , lOsec; (3) 60°C, lOsec (drop by l°C/cycle) ; (4) 72°C, 30 sec; (5) 9 cycle repeats on steps 2 to 4; (6) 95°C, lOsec; (7) 56°C, lOsec; (8) 72°C, 30sec; (9) 9 cycle repeats on steps 6 to 8; and (10) Store at 4°C.
  • the following is one of the general procedures for the present invention:
  • (a) Select gene segments and determine the PCR primers and the ASO sites: 1. Select the appropriate target sequences to be analyzed by either screening data from the GenBank and/or by sequencing the target genes or target DNA segments to get the SNP (or ASO) profile related to the pathogen (s) . 2. From these data, determine the ASO sites to be used for genotyping based on the sequence data to evaluate if the sites are indeed unique for the genotype or variant within target species to be tested. 3. Determine the number of ASO capture probes.
  • the following protocol is an example: i) Denature and drop the target DNA solution onto the membrane; ii) Wash and develop the color for visual inspection or spectrometric measurements (The target DNA can be labeled with fluorescence dye in which case direct spectrometric determination other than color development by enzyme linked immuno-specific assay is needed. Other developing methods like colloidal Gold or magnetic bead or quantum-dot or any other conjungate labeling systems already used or developed in the future can also be used) . iii) Results are compared with known sequence data for accuracy evaluation, iv) Modify the probes and testing conditions for accuracy. The RDB-ASO data are verified by DNA sequencing.
  • the ASO or SNP probe sequences and specific primers sequences to be used can be designed appropriately for any given organism with which the nucleic sequence data is known or can be determined accurately to perform the test in the present invention.
  • a concentration of the target nucleic acid should be above 0.5 f moles for avidin-AP conjugate enzyme link color development (other labeling development system may be more sensitive) .
  • amplification must be performed before the hybridization process.
  • the PCR amplifications were used for examples given here.
  • Other amplification methods such as those mentioned elsewhere in this text can also generate similar concentration and therefore should be feasible .
  • isothermal amplification methods are preferable because the relatively expensive PCR equipment (Thermal Cycler) will not be needed, and the amplification process is much faster.
  • a two-step nested RT-PCR protocol was used for initial development of the SARS diagnosis kit.
  • the reversed transcription was done with the first primer pair: 5' -ATGAATTACCAAGTCAATGGTTAC-3' and 5' -CATAACCAGTCGGTACAGCTAC-3' with positive control RNA of about 2xl0 "5 copies using the Thermoscript RT-PCR system and Platinum Taq DNA polymerase, followed by the first round of PCR.
  • the 2 nd round PCR was done using lul of the 1:10 dilution of the first round product in 50ul reaction volume.
  • the thermal cycling program used for both round was the same : (1)95°C, 5 min; (2) 95°C, 10 sec;(3)56°C, lOsec; (4) 72°C, 30sec (5)39 cycle repeats on steps 2 to 4, followed by 72°C, 10 min final extension time.
  • the process was a two-stage single program nested PCRamplification in which the outside primer pair was designed to have a higher annealing temperature for the initial amplification of 10 to 20 cycles followed by a lower annealing temperature amplification cycles . As shown in Figure 3, this process has produced a much more specific (less non-specific band(s) was obtained other than the target sequence) result even at the limit of its detection.
  • ASO or SNP oligo-probes and PCRprimers, which are listed in the Appendix, were designed.
  • ASO or SNPs probe (s) and amplification primers of any organisms with adequate sequence data can be designed to perform such genetic analysis, and can be detected by the flow-through hybridization method. The results of hybridization were obtained within minutes.
  • SNP oligo-probes were used as the low-density array for detecting multiple SARS variants on a single membrane for the SARS causing coronus viral nucleic acid diaqnosis.
  • Figure 5 showed the array of ASO probes dotted onto the membrane for hybridization detection with target samples.
  • the protocol of the fast flow-through hybridization was developed successfully. The typical result is shown in Figure 6.
  • 10 random clinical samples were tested and the results were confirmed by DNA sequencing.
  • Table IV indicated 100% agreement.
  • Table III Nested PCR Amplification Cycling Program Specifically for SARS Corona Virus
  • test specificity was evaluated with the use of nucleic acid samples and probes of 10 other respiratory tract pathogens (respiratory syncytial virus (RSV) , parainfluenza virus type 1, 3 & 3b (PIV 1, 3 & 3b) , Mycoplasma pneumoniae, Chlamydia pneumoniae, enterovirus, influenza A & B, adenovirus, metapneumovirus).
  • RSV respiratory tract pathogens
  • PAV 1, 3 & 3b parainfluenza virus type 1, 3 & 3b
  • Mycoplasma pneumoniae Chlamydia pneumoniae
  • enterovirus influenza A & B
  • adenovirus adenovirus
  • metapneumovirus metapneumovirus
  • Membrane positions of probes are as follows: 1 Respiratory syncytial virus (RSV); 2 Enterovirus (EN); 3 Adenovirus (Adeno) ; 4 Parainfluenza virus type 1 (PIV1); 5 Parainfluenza virus type 3 (PIV3); 6 Parainfluenza virus type 3b (PIV3b) ;7 InfluenzaA (Inf A) ; 8 Influenza B (Inf B) ; 9 SARS coronavirus (SARS);10 Metapneumovirus (Meta); 11 Mycoplasma pneumoniae (MP) ; 12 Chlamydia pneumoniae (CP) .
  • Total volume 25 ⁇ l *6-plex primer mix contains primers for SARS coronavirus (SARS1 & SARS2), influenza A & B viruses, parainfluenza virus type 1 & 3, respiratory syncytial virus.
  • SARS coronavirus SARS1 & SARS2
  • influenza A & B viruses influenza A & B viruses
  • parainfluenza virus type 1 & 3 respiratory syncytial virus.
  • the ability to simultaneously and rapidly detect the other viruses causing the SARS like syndrome is of utmost important because this will be the most effective way for triage patients for effective treatment and prevent unnecessary spread of the serious communicable infectious SARS disease.
  • the present invention can also be extended to include simultaneously identify variants of these viruses in one single membrane test strip to enable physicians to prescribe the most appropriate therapy to save lives .
  • the rapid and simplicity of test which can be carried out onsite is unique to the present invention.
  • NPA nasopharyngeal aspirates
  • Probe 1. Amino modified oligonucleotide capture probes - BNIcapture (stock at (given in appendix) , -20°C, 1 nmol/ ⁇ l, dilute to 10 ⁇ M with 0.5 M sodium bicarbonate)
  • Biodyne C membrane (Pa ll) (used for probe immobiliza tion to capt ure the target labeled DNA samples used a s the ma trix for the low-densi ty array)
  • Oligonucleotide application buffer 0.5 M sodium bicarbonate, pH 8.4
  • StabilGuard ® - (SurModics SGOl-1000) (stored at 4°C)
  • DNA HybriMax DNA Flow-Through Hybridization Device
  • Memebrane Array preparation Covalent attachment of amino modified probes to Biodyne C membrane filter. 1. Briefly rinse the membrane with 0.1 M HCl, blot, then soak in freshly prepared 20% EDAC (lg EDAC in 5 ml Milli-Q dH 2 0) for 15 min .
  • SARS and other upper respiratory track infectious viral PCR products in the examples shown in this invention were labeled with biotin-labeled primers.
  • the PCR amplification was done using either the MJ p200 or the ABI 9700 thermalcycler (in principle, any other thermal cyclers can be used for the amplification with appropriate optimization of the cycling protocols) .
  • Single or nested PCR have been tried successfully with the one-step or the two-step processes.
  • DrostenC. etal. identificationofa novel coronavirus in patients with severe acute respiratory ayndrome . N. Engl . J. med., 348: 1967-1976.
  • Coronavirus (SARS) Variants used in the invention Known genoptypes : Urbani ; HKU-39849; CUHK-sulO; SIN2500; SIN2677; SIN2679; SIN2748; SIN2774; T 1; ZJ01; TOR2 ; CUHK- 1 ; BJ01
  • SARS Coronavirus
  • Variants used in the invention Known genoptypes : Urbani ; HKU-39849; CUHK-sulO; SIN2500; SIN2677; SIN2679; SIN2748; SIN2774; T 1; ZJ01; TOR2 ; CUHK- 1 ; BJ01
  • Tor2WTCAP - ACCTTGCTCTTTTGG 7.
  • RSV - CCTGCATTAACACTAAATTC
  • PIV3 AAAATTCCAAAAGAGACCGGC 10.
  • PIV3B - ACAGAACACCARAACAACAA
  • Influenza A GTCCTCATCGGAGGACTTGAATGGAATGAT 15. Influenza B - GTCAAGAGCACCGATTATCAC
  • Influenza B forward - ATGGCCATCGGATCCTCAAC

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Abstract

La présente invention concerne un procédé qui permet de détecter un pathogène par l'analyse de séquences nucléotidiques, selon lequel : on choisit au moins une séquence cible appropriée du pathogène ; on crible ladite séquence afin d'obtenir au moins une amorce appropriée à l'amplification ; on choisit une sonde appropriée dans la séquence ; on immobilise le site de la sonde oligonucléotidique spécifique d'allèle de chaque séquence sur une matrice solide permettant la capture de la séquence cible ; on amplifie la ou les séquences, les étapes (d) ou (e) pouvant être effectuées simultanément ou séquentiellement ; on effectue des analyses par buvardage sur chaque séquence ; et on compare les résultats de l'analyse par buvardage avec des témoins positifs ou négatifs. L'invention porte en outre sur un procédé qui permet d'amplifier la séquence cible d'un pathogène afin de l'hybrider, selon lequel on choisit une paire d'amorces appropriées ; on effectue une transcription inverse de la paire d'amorces ; et on effectue le premier et le second cycle de la PCR dans un seul tube.
PCT/US2004/017767 2003-06-05 2004-06-04 Identification rapide des virus responsables des infections des voies respiratoires superieures, y compris des coronavirus responsables du syndrome respiratoire aigu severe WO2005038427A2 (fr)

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10294534B2 (en) 2011-12-09 2019-05-21 The Secretary Of State For Health Respiratory infection assay
US11697843B2 (en) 2012-07-09 2023-07-11 Tecan Genomics, Inc. Methods for creating directional bisulfite-converted nucleic acid libraries for next generation sequencing
US10619206B2 (en) 2013-03-15 2020-04-14 Tecan Genomics Sequential sequencing
US10760123B2 (en) 2013-03-15 2020-09-01 Nugen Technologies, Inc. Sequential sequencing
US11098357B2 (en) 2013-11-13 2021-08-24 Tecan Genomics, Inc. Compositions and methods for identification of a duplicate sequencing read
US10570448B2 (en) 2013-11-13 2020-02-25 Tecan Genomics Compositions and methods for identification of a duplicate sequencing read
US11725241B2 (en) 2013-11-13 2023-08-15 Tecan Genomics, Inc. Compositions and methods for identification of a duplicate sequencing read
WO2016022833A1 (fr) * 2014-08-06 2016-02-11 Nugen Technologies, Inc. Mesures numériques à partir de séquençage ciblé
US10102337B2 (en) 2014-08-06 2018-10-16 Nugen Technologies, Inc. Digital measurements from targeted sequencing
US11099202B2 (en) 2017-10-20 2021-08-24 Tecan Genomics, Inc. Reagent delivery system
US12059674B2 (en) 2020-02-03 2024-08-13 Tecan Genomics, Inc. Reagent storage system
CN111378018A (zh) * 2020-03-28 2020-07-07 江苏省疾病预防控制中心(江苏省公共卫生研究院) 一种检测新型冠状病毒抗体的试纸条及其制备方法和应用
CN111378789A (zh) * 2020-06-01 2020-07-07 广州凯普医药科技有限公司 一种呼吸道感染病原体核酸联合检测试剂盒
CN112280897A (zh) * 2020-06-01 2021-01-29 上海市浦东新区周浦医院 一种呼吸道感染病原体核酸联合检测试剂盒

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