WO2021174984A1 - 一种新型冠状病毒rt-pcr检测方法及试剂盒 - Google Patents

一种新型冠状病毒rt-pcr检测方法及试剂盒 Download PDF

Info

Publication number
WO2021174984A1
WO2021174984A1 PCT/CN2020/141570 CN2020141570W WO2021174984A1 WO 2021174984 A1 WO2021174984 A1 WO 2021174984A1 CN 2020141570 W CN2020141570 W CN 2020141570W WO 2021174984 A1 WO2021174984 A1 WO 2021174984A1
Authority
WO
WIPO (PCT)
Prior art keywords
probe
kit
seq
primer
primer pair
Prior art date
Application number
PCT/CN2020/141570
Other languages
English (en)
French (fr)
Inventor
蒋析文
廖芷卉
范建
Original Assignee
广州达安基因股份有限公司
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by 广州达安基因股份有限公司 filed Critical 广州达安基因股份有限公司
Priority to US17/905,662 priority Critical patent/US20230114907A1/en
Priority to EP20922897.2A priority patent/EP4116440A4/en
Publication of WO2021174984A1 publication Critical patent/WO2021174984A1/zh

Links

Images

Classifications

    • 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
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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
    • 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
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention belongs to the field of biotechnology and molecular diagnosis. Specifically, the present invention relates to a real-time fluorescent RT-PCR detection method and kit for a novel coronavirus 2019-nCoV.
  • Real-time fluorescent RT-PCR is one of the commonly used methods for coronavirus nucleic acid detection, which can be used for laboratory examinations, epidemiological studies, and exclusion diagnosis.
  • the method has high sensitivity and good specificity, and can accurately and quickly provide the detection results of the target virus.
  • the routine detection method for novel coronavirus infection is through real-time fluorescent RT-PCR identification, mainly for the novel coronavirus genome open reading frame 1a/b (open reading frame 1ab, ORF1ab) and nucleocapsid protein (N) .
  • N nucleocapsid protein
  • the present invention develops a novel coronavirus diagnostic system for the envelope protein (E) of the novel coronavirus genome.
  • E envelope protein
  • a primer pair set for detecting nucleic acid of the novel coronavirus 2019-nCoV is provided, and the primer pair set includes:
  • a first primer pair set, the first primer pair set includes:
  • the primer pair set further includes:
  • An internal standard primer pair set includes:
  • the second aspect of the present invention provides a probe set for multiplex detection of nucleic acid of the novel coronavirus 2019-nCoV, the probe set including the first probe whose nucleotide sequence is shown in SEQ ID NO.3.
  • the probe set further includes an internal control probe whose nucleotide sequence is shown in SEQ ID NO.6.
  • the 5'end of each probe contains a fluorescent reporter group; and/or the 3'end of each probe contains a fluorescence quenching group.
  • the fluorescent reporter groups labeled with the probes are different from each other.
  • the third aspect of the present invention provides a kit for multiple detection of nucleic acid of the novel coronavirus 2019-nCoV, the kit comprising the primer pair set described in the first aspect of the present invention.
  • the kit further includes the probe set according to the second aspect of the present invention.
  • the kit includes a first container, and the first container contains a primer-probe mixture, and the primer-probe mixture contains the polynucleoside shown in SEQ ID NO. 1-6. Acid sequence.
  • the kit further includes a second container, and the second container contains an RT-PCR reaction enzyme system, and the RT-PCR reaction enzyme system includes a hot-start enzyme and a reverse transcriptase C- MMLV; preferably also includes dNTPs.
  • the kit further includes a third container, and the third container contains an RT-PCR reaction solution.
  • the kit further includes a fourth container, and the fourth container contains DEPC H 2 O.
  • the fourth aspect of the present invention provides a method for multiple detection of nucleic acid of the novel coronavirus 2019-nCoV, the method comprising the steps:
  • the RT-PCR reaction system includes: the nucleic acid sample provided in step (1), the primer pair set described in the first aspect of the present invention, and the probe set described in the second aspect of the present invention.
  • the nucleic acid sample may be from a throat swab sample, an alveolar lavage fluid sample, a blood sample, a sputum sample, a stool sample, or an environmental sample.
  • the method is a detection method for non-diagnostic purposes.
  • the RT-PCR reaction system further includes a positive quality control product and/or a negative quality control product.
  • the RT-PCR reaction system further includes an RT-PCR enzyme system.
  • the fifth aspect of the present invention provides the use of the primer pair set according to the first aspect of the present invention and/or the probe set according to the second aspect of the present invention for preparing a PCR detection kit, the PCR detection The kit is used to detect the nucleic acid of the new coronavirus 2019-nCoV.
  • Figure 1 The amplification curve of the optimized detection system
  • Figure 6 The detection result of the control primer pair E-NCOV-F2 and E-NCOV-R2;
  • Figure 7 The detection results of the control primer pair E-NCOV-F3 and E-NCOV-R3.
  • the inventors obtained a kit and method for multiple detection of the nucleic acid of the novel coronavirus 2019-nCoV.
  • the targeted detection of the nucleic acid target of the membrane protein of the novel coronavirus 2019-nCoV it can be highly efficient and highly specific.
  • the detection of patients infected with the new coronavirus 2019-nCoV can be carried out in an efficient and low-cost manner, and to prevent false negatives that may occur due to virus variability, and to confirm missed detections that may be caused by mutations, which significantly improves the accuracy of virus identification .
  • Multiplex PCR also known as multiple primer PCR or multiple PCR
  • multiple PCR is a PCR reaction in which two or more pairs of primers are added in the same PCR reaction system to amplify multiple nucleic acid fragments at the same time.
  • the reaction principle, reaction reagents and operation The process is the same as general PCR.
  • the reaction system is unbalanced.
  • the unbalance of the reaction system leads to rapid amplification of some dominant primers and their templates in the previous rounds of reactions, and a large amount of amplification products are obtained, and these amplification products are also DNA polymerases.
  • a good inhibitor Therefore, with the emergence of a large number of amplified products, the polymerization ability of the polymerase is more and more strongly inhibited. Therefore, the primers and their templates that are inferior in the early stage are more difficult to react at this time, and finally the amount of amplified products is very large. It is too small to detect.
  • the optimal annealing temperature is inconsistent. Put multiple pairs of primers into one system for amplification. Since the annealing temperature for the PCR reaction is the same, the optimal annealing temperature of each pair of primers is required to be close.
  • Primer dimers including the dimers between primers and the hairpin structure formed by the primers themselves, and a third-party DNA-mediated polymer. These dimers and non-specific primers will interfere with the primers and Competition for target binding sites affects amplification efficiency.
  • the present invention relates to the field of biological detection technology and medical diagnosis, in particular to a real-time fluorescent RT-PCR detection method for a novel coronavirus (2019-nCoV).
  • This kit mainly includes RT-PCR reaction solution, primer probe mixture, RT-PCR reaction enzyme system, DEPC H 2 O, and a packaging box that separates and centrally packs these reagent bottles or tubes.
  • the new coronavirus nucleic acid test is the gold standard for diagnosis.
  • the present invention develops a detection kit for detecting the E gene of a new type of coronavirus.
  • This kit also includes an endogenous internal standard detection system for monitoring specimen collection, nucleic acid extraction process and PCR amplification process, which can reduce The appearance of false negative results.
  • This kit is a dual fluorescence detection kit with the characteristics of high sensitivity, strong specificity, and good repeatability.
  • the present invention provides a kit for specifically detecting the E gene of 2019 novel coronavirus in a sample, wherein the primer sequence for detecting the E gene is:
  • SEQ ID NO. 1 TTTTCTTGCTTTCGTGGTA and SEQ ID NO. 2: GGTTTTACAAGACTCACGTT,
  • the sequence of the corresponding detection probe is SEQ ID NO. 3: ATTGCAGCAGTACGCACACA.
  • the kit further includes internal standard quality control and amplification primers and detection probes; the internal standard quality control amplification primer sequences are:
  • SEQ ID NO. 4 CAAGTAAGTTTCTCCGAATCCC and SEQ ID NO. 5: GCTGAAGTCCCATGACCGT,
  • sequence of the corresponding detection probe is SEQ ID NO. 6: CAACTGGAGGTAGAGACGGACTGCG.
  • the kit includes a positive quality control product (a pseudovirus containing E gene amplification target sequence) and a negative control product (hela cell nucleic acid).
  • the kit includes an RT-PCR reaction solution, a primer probe mixture, an RT-PCR reaction enzyme system, and DEPC H 2 O.
  • the RT-PCR reaction solution includes: Tris-HCl with pH 8.8, Mg 2+ , and Tween-20.
  • the primer-probe mixture includes: the primer-probe sequence shown in SEQ ID NO.: 1-6.
  • the RT-PCR reaction enzyme system includes: hot-start Taq enzyme, reverse transcriptase and dNTPs.
  • the present invention also provides a new type of coronavirus (2019-nCoV) detection method, including the following steps: extract the sample to be tested (the extraction reagent uses the nucleic acid extraction or purification reagent produced by Sun Yat-sen University Daan Gene Co., Ltd. (Yue Sui Prepare No. 20170583) to obtain a nucleic acid sample; mix with RT-PCR reaction solution, primer probe mixture, RT-PCR reaction enzyme system and DEPC H 2 O, perform amplification reaction in a real-time fluorescent PCR machine, and PCR amplification program as follows;
  • test results can be used for auxiliary diagnosis of new coronavirus infection and observation of drug efficacy, providing a reliable basis for research.
  • GISAID BetaCov/Wuhan/WH01/2019
  • oligonucleotide sequence information of its E gene please refer to the literature (Roujian Lu, Xiang Zhao, Juan Li, et .al, Genomic characterisation and epidemiology of 2019novel coronavirus: implications for virus origins and receptor binding. Lancet. 2020 Jan 30).
  • composition of this kit is shown in Table 1 and Table 2. It can detect the E gene of the new coronavirus 2019-nCoV and confirm the missed detection that may be caused by mutation, which can significantly improve the accuracy of virus identification.
  • Primer probe name Primer/probe sequence SEQ ID NO. E-NCOV-F TTTTCTTGCTTTCGTGGTA 1 E-NCOV-R GGTTTTACAAGACTCACGTT 2 E-NCOV-P ATTGCAGCAGTACGCACACA 3 RP30-F CAAGTAAGTTTCTCCGAATCCC 4 RP30-R GCTGAAGTCCCATGACCGT 5 RP30-P CAACTGGAGGTAGAGACGGACTGCG 6
  • the fluorescent group is selected from the following group: FAM, VIC, HEX, NED, ROX, TET, JOE, TAMRA, CY3, CY5.
  • the quenching gene is selected from the following group: MGB, BHQ-1, BHQ-2, BHQ-3.
  • the specific primers and probes are screened through a large number of experiments, and they are combined, optimized, and verified. Finally, the combination will not interfere with each other, the amplification efficiency is high, and the specificity is good for double detection. Optimal lead-exploration combination.
  • the method of using the kit of the present invention includes the following steps:
  • the first stage 50°C2-15min, 95°C10-15min, 1 cycle;
  • the second stage 94°C10-15s, 55-60°C45s, 45 cycles.
  • the negative or positive nucleic acid of the corresponding pathogen is judged according to the different fluorescence channel curves and Ct value to give the detection result.
  • the present invention can detect patients infected with the new coronavirus 2019-nCoV with high efficiency, high specificity and low cost, and prevent possible mutations caused by the virus. False negatives due to sex can confirm missed detections that may be caused by mutations, which significantly improves the accuracy of virus identification.
  • the detection kit of the present invention also includes the membrane protein nucleic acid target detection system of the new coronavirus 2019-nCoV and the endogenous internal standard detection system. It is a dual fluorescence detection kit that can perform sample collection, nucleic acid extraction processes and The PCR amplification process is monitored to reduce the occurrence of false negative results.
  • the present invention obtains a primer-probe combination with high sensitivity, strong specificity, good reproducibility, and suitable for multiple detection from a large number of primer-probe combinations.
  • the invention is suitable for the detection of the nucleic acid of the novel coronavirus 2019-nCoV, provides a reliable basis for virus identification and prevention and control, and is worthy of popularization and application.
  • the method of the present invention is also suitable for non-diagnostic purposes. For example, in the process of epidemic prevention and control, the detection method of the present invention is used to detect viral nucleic acids in the environment, and the viral nucleic acid information can be used for public health management.
  • the conserved regions of its genome are analyzed. In these conserved regions, dozens of sets of specific primers and probe sequences for the detection of the 2019 new coronavirus E gene have been designed.
  • ribonuclease P RPP30
  • primers and probes try to avoid the formation of hairpin structures, primer internal dimers, inter-primer dimers, and mismatches.
  • NCBI Blast online database https://blast.ncbi.nlm.nih.gov/Blast.cgi
  • NCBI Blast online database https://blast.ncbi.nlm.nih.gov/Blast.cgi
  • the 5'-end fluorescent group of E-NCOV-P is FAM, and the 3'-end quenching group is BHQ1; the 5'-end fluorescent group of RP30-P is VIC, and the 3'-end quenching group is BHQ1.
  • the principle of using Taqman probe to detect new coronavirus RNA is: first use the added primer sequences E-NCOV-F and E-NCOV-R as reverse transcription primers to reverse transcribe the extracted viral RNA into anchor sequences cDNA, and then use E-NCOV-F and E-NCOV-R as the forward and reverse primers for real-time PCR detection for amplification.
  • This method is a one-step amplification method, that is, the two steps of reverse transcription of viral RNA into cDNA and DNA amplification are combined without changing the tube for operation.
  • the detection results are shown in Figure 1. From left to right, the results of the pseudovirus gradient detection when the primer concentration is 15pmol, 10pmol, and 8pmol, and the probe concentration is 8pmol, 5pmol, and 3pmol are shown from left to right.
  • the final reaction system (25 ⁇ L) is as follows:
  • the RT-PCR reaction enzyme system is composed of hot-start Taq enzyme, reverse transcriptase, and dNTPs.
  • the reverse transcriptase can be selected from commonly used reverse transcriptases such as mMLV.
  • Hot-start Taq enzymes, reverse transcriptases, and dNTPs can all be commercially available products, such as products from Qiagen, in which the amount of hot-start Taq enzyme in the RT-PCR reaction enzyme system per person is 8U, and the amount of reverse transcriptase is 3U.
  • the amount of dNTPs is 10 mmol.
  • the final reaction program on the ABI7500 fluorescent PCR machine is determined as follows: 50°C for 15 minutes, 1 cycle; 95°C for 15 minutes, 1 cycle; 94°C for 15 seconds ⁇ 55°C for 45 seconds (collect Fluorescence), 45 cycles.
  • the target fragment was ligated into the constructed pET28a-MS2 vector and transformed into the expression host bacteria BL21 competent cells, picked a single clone and sequenced for verification and then induced expression.
  • the fragmented expression product was digested with RNaseA and DNaseI. Virus-like particles containing the fragment of interest.
  • the concentrations are 1.00E+06, 1.00E+05, 1.00E+04, 1.00E+03, and 1.00E+02copies/ml.
  • Influenza A/B, Coronavirus 229E, Coronavirus NL63, Coronavirus OC43, Coronavirus HKU1, Respiratory Syncytial Virus, Adenovirus, Mycoplasma Pneumoniae, and Chlamydia Pneumoniae were tested as specific samples (Figure 3).
  • test results show that the test specific reference products (influenza A/B, coronavirus 229E, coronavirus NL63, coronavirus OC43, coronavirus HKU1, respiratory syncytial virus, adenovirus, Mycoplasma pneumoniae, Chlamydia pneumoniae), test The results are all negative, indicating that the kit of the present invention has good specificity.
  • the negative and positive of the corresponding pathogen nucleic acid can be judged through different fluorescence channel curves and Ct values.
  • Sequencing verification results show that the detection accuracy of the detection system of the present invention reaches 100%, which further proves the clinical detection accuracy of the detection system of the present invention.
  • the inventors screened dozens of PCR primers and probes against the target nucleic acid sequence of the new coronavirus 2019-nCoV. After a large number of tests, they finally obtained sensitivity and specificity that can meet the needs of clinical testing and can perform multiplexing. The combination of primers and probes for detection.
  • Control upstream primer E-NCOV-F2 CTTTCGTGGTATTCTTGCTA (SEQ ID NO.7)
  • Control downstream primer E-NCOV-R2 CTCACGTTAACAATATTGCAG (SEQ ID NO.8)
  • Control upstream primer E-NCOV-F3 AGTTACACTAGCCATCCTT (SEQ ID NO.9)
  • Control downstream primer E-NCOV-R3 TTTAACACGAGAGTAAACGTA (SEQ ID NO.10)
  • E-NCOV-F3 and E-NCOV-R3 show that the primer pair has better specificity and sensitivity to the E gene target nucleic acid in a single detection system, but the E gene target low concentration nucleic acid amplification in a multiple detection system The increase was significantly inhibited, and the results of single and multiple systems are shown in Figure 7. It shows that the control primer pair E-NCOV-F3 and E-NCOV-R3 cannot be used in multiple detection systems.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Virology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

一种新型冠状病毒2019-nCoV实时荧光RT-PCR检测方法及试剂盒，具体的地，涉及检测新型冠状病毒2019-nCoV的E基因核酸的试剂盒及方法，具有极高的灵敏度和特异性，并且能够显著提高病毒鉴定的准确性。

Description

一种新型冠状病毒RT-PCR检测方法及试剂盒 技术领域
本发明属于生物技术和分子诊断领域，具体地说，本发明涉及一种新型冠状病毒2019-nCoV实时荧光RT-PCR检测方法及试剂盒。
背景技术
2019年12月以来，陆续发现了多例不明原因肺炎病例，现已证实为一种新型冠状病毒感染引起的急性呼吸道传染病。基于目前的流行病学调查，潜伏期一般为3-7天，最长不超过14天。此次疫情临床表现以发热、乏力、干咳为主要表现。少数患者伴有鼻塞、流涕、腹泻等症状。部分患者仅表现为低热、轻微乏力等，无肺炎表现，多在1周后恢复。
鉴于该病潜伏期患者也可进行人传人现象，目前迫切需要一种灵敏和精确的方法，用于快速检测2019新型冠状病毒感染所引起的肺炎的。而《新型冠状病毒感染的肺炎诊疗方案》明确规定疑似病例，需要具备以下病原学证据之一者才能确诊：1.呼吸道标本或血液标本实时荧光RT-PCR检测新型冠状病毒核酸阳性；2.呼吸道标本或血液标本病毒基因测序，与已知的新型冠状病毒高度同源。
实时荧光RT-PCR是冠状病毒核酸检测常用方法之一，可用于实验室检查、流行病学研究、排除诊断等。该方法灵敏度高、特异性好，可以准确快速提供目标病毒的检测结果。目前，对新型冠状病毒感染的常规检测方法是通过实时荧光RT-PCR鉴定，主要针对新型冠状病毒基因组开放读码框1a/b(openreading frame 1ab，ORF1ab)和核壳蛋白(nucleocapsid protein，N)。但是，鉴于RNA病毒变异速度较快，容易对突变病毒造成漏检，不利于病毒的及时发现与防控。
因此，有必要开发针对性的检测体系，预防可能因突变而引起的漏检。
发明内容
本发明针对新型冠状病毒基因组膜蛋白(envelope protein，E)开发了新型冠状病毒诊断体系。以便能够高效率、高特异性、低成本的对新型冠状病毒2019-nCoV感染患者进行检测，并预防可能出现的变异性。
在本发明的第一方面，提供了一种用于检测新型冠状病毒2019-nCoV核酸的引物对集，所述引物对集包括：
第一引物对组，所述第一引物对组包括：
如SEQ ID NO.1所示的正向引物；和，如SEQ ID NO.2所示的反向引物。
在另一优选例中，所述引物对集还包括：
内标引物对组，所述内标引物对组包括：
如SEQ ID NO.4所示的正向引物；和，如SEQ ID NO.5所示的反向引物。
本发明的第二方面，提供了一种多重检测新型冠状病毒2019-nCoV核酸的探针组，所述探针组包括核苷酸序列如SEQ ID NO.3所示的第一探针。
在另一优选例中，所述探针组还包括核苷酸序列如SEQ ID NO.6所示的内控探针。
在另一优选例中，各探针的5’端包含有荧光报告基团；和/或，各探针的3’端包含有荧光淬灭基团。
在另一优选例中，各探针标记的荧光报告基团互不相同。
本发明的第三方面，提供了一种用于多重检测新型冠状病毒2019-nCoV核酸的试剂盒，所述试剂盒包括本发明第一方面所述的引物对集。
在另一优选例中，所述试剂盒还包括本发明第二方面所述的探针组。
在另一优选例中，所述试剂盒包括第一容器，所述第一容器内包含引物探针混合液，所述引物探针混合液中包含SEQ ID NO.1-6所示的多核苷酸序列。
在另一优选例中，所述试剂盒还包括第二容器，所述第二容器内包含RT-PCR反应酶系，所述RT-PCR反应酶系包括热启动酶、和逆转录酶C-MMLV；优选地还包括dNTPs。
在另一优选例中，所述试剂盒还包括第三容器，所述第三容器内包含RT-PCR反应液。
在另一优选例中，所述试剂盒还包括第四容器，所述第四容器内包含DEPC H 2O。
本发明的第四方面，提供了一种多重检测新型冠状病毒2019-nCoV核酸的方法，所述方法包括步骤：
(1)提供待检测对象的核酸样本；
(2)制备RT-PCR反应体系并进行RT-PCR检测：
其中，所述RT-PCR反应体系包括：步骤(1)提供的所述核酸样本、本发明第一方面所述的引物对集、和本发明第二方面所述的探针组。
在另一优选例中，所述核酸样本可来自咽拭子样本、肺泡灌洗液样本、血液样本、痰液样本、粪便样本或环境样本。
在另一优选例中，所述方法为非诊断目的的检测方法。
在另一优选例中，所述RT-PCR反应体系还包括阳性质控品，和/或阴性质控品。
在另一优选例中，所述RT-PCR反应体系还包括RT-PCR酶系。
本发明的第五方面，提供了本发明第一方面所述的引物对集、和/或本发明第二方面所述的探针组的用途，用于制备PCR检测试剂盒，所述PCR检测试剂盒用于检测新型冠状病毒2019-nCoV核酸。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1：优化检测体系的扩增曲线；
图2：灵敏度检测结果；
图3：特异性测试结果；
图4：重复性测试结果；
图5：典型临床样本检测结果；
图6：对照引物对E-NCOV-F2和E-NCOV-R2的检测结果；
图7：对照引物对E-NCOV-F3和E-NCOV-R3的检测结果。
具体实施方式
本发明人通过广泛而深入的研究，获得一种多重检测新型冠状病毒2019-nCoV核酸的试剂盒及方法，通过针对性检测新型冠状病毒2019-nCoV的膜蛋白核酸靶标，能够高效率、高特异性、低成本的对新型冠状病毒2019-nCoV感染患者进行检测，并预防可能因病毒变异性而出现的假阴性，对可能因突变而引起的漏检进行确认，显著提高了病毒鉴定的准确性。
在描述本发明之前，应当理解本发明不限于所述的具体方法和实验条件，因为这类方法和条件可以变动。还应当理解本文所用的术语其目的仅在于描述具体实施方案，并且不意图是限制性的，本发明的范围将仅由所附的权利要求书限制。
除非另外定义，否则本文中所用的全部技术与科学术语均具有如本发明所属领域的普通技术人员通常理解的相同含义。如本文所用，在提到具体列举的数值中使用时，术语“约”意指该值可以从列举的值变动不多于1％。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
虽然在本发明的实施或测试中可以使用与本发明中所述相似或等价的任何方法和材料，本文在此处例举优选的方法和材料。
多重PCR
多重PCR(multiplex PCR)，又称多重引物PCR或复合PCR，它是在同一PCR反应体系里加上二对以上引物，同时扩增出多个核酸片段的PCR反应，其反应原理，反应试剂和操作过程与一般PCR相同。
影响多重PCR反应的因素有很多，比如：
(1)反应体系不平衡，反应体系的不平衡导致在前期的几轮反应中某些优势引物及其模板迅速扩增，获得大量的扩增产物，而这些扩增产物同时又是DNA聚合酶的良好抑制剂。所以，随着扩增产物的大量出现，聚合酶的聚合能力被越来越强烈的抑制，因此，前期处于劣势的引物及其模板，这时就更加难以反应，最终导致扩增产物量非常之小，以至于无法检测。
(2)引物特异性，如果引物与体系中其他非目的基因片段结合力更强，那么目的基因结合引物的能力就会受到竟争，从而导致扩增效率下降。
(3)最佳退火温度不一致，将多对引物放置入一个体系中扩增，由于进行PCR反应的退火温度相同，所以要求每一对引物的最佳退火温度接近。
(4)引物二聚体，包括引物间的二聚体以及引物自身所形成的发卡结构，还有一类是第三方DNA介导的聚体，这些二聚体和非特异引物一样都会干扰引物与目标结合位点的竟争，影响扩增效率。
虽然上述提及了影响扩增效率的几个因素，但更多的因素尚不清楚。到目前为止，还没有一个可以明确预测扩增效率的有效方法。
本发明涉及生物检测技术和医学诊断领域，特别是涉及一种用于新型冠状病毒(2019-nCoV)的实时荧光RT-PCR检测方法。本试剂盒主要包括RT-PCR反应液、引物探针混合液、RT-PCR反应酶系、DEPC H 2O，以及分隔并集中包装这些试剂瓶或管的包装盒。
按照新型冠状病毒的诊疗指南，新型冠状病毒核酸检测是确诊的金标准。本发明开发了一种检测新型冠状病毒的E基因的检测试剂盒，本试剂盒同时包括内源性内标检测系统，用于对标本采集、核酸提取过程及PCR扩增过程的监控，可减少假阴性结果的出现。本试剂盒为双重荧光检测试剂盒，具有敏度高、特异性强、重复性好等特点。
本发明提供一种特异性检测样品中2019新型冠状病毒E基因的试剂盒，其中用于检测E基因的引物序列为：
SEQ ID NO.1：TTTTCTTGCTTTCGTGGTA和SEQ ID NO.2:GGTTTTACAAGACTCACGTT，
对应的检测探针的序列为SEQ ID NO.3:ATTGCAGCAGTACGCACACA。
在一个实施例中，所述试剂盒还包括内标质控及扩增引物和检测探针；内标质控扩增引物序列分别为：
SEQ ID NO.4:CAAGTAAGTTTCTCCGAATCCC和SEQ ID NO.5:GCTGAAGTCCCATGACCGT，
对应的检测探针的序列为SEQ ID NO.6：CAACTGGAGGTAGAGACGGACTGCG。
在其中一个实施例中，所述试剂盒包括含阳性质控品(含有E基因扩增靶序列的假病毒)，以及阴性质控品(hela细胞核酸)。
在其中一个实施例中，所述试剂盒包括RT-PCR反应液、引物探针混合液、RT-PCR反应酶系、和DEPC H 2O。优选地，RT-PCR反应液包括：pH8.8的Tris-HCl、Mg 2+、Tween-20。优选地，引物探针混合液包括：SEQ ID NO.:1-6所示的引物探针序列。优选地，RT-PCR反应酶系包括：热启动Taq酶、逆转录酶和dNTPs。
本发明还提供了一种新型冠状病毒(2019-nCoV)的检测方法，包括如下步骤：提取待测样本(提取试剂采用中山大学达安基因股份有限公司生产的核酸提取或纯化试剂(粤穗械备20170583号)，得到核酸样本；和RT-PCR反应液、引物探针混合液、RT-PCR反应酶系以及DEPC H 2O混合，在实时荧光PCR仪中进行扩增反应，PCR扩增程序如下；
50℃，15min，1个循环
95℃，15min；1个循环
94℃，15sec，55℃，45sec(搜集荧光)；45个循环。
PCR结束后通过不同荧光通道曲线及Ct值判断对应病原体核酸的阴、阳性，检测结果可用于新型冠状病毒感染的辅助诊断以及药物疗效的观察，为研究提供可靠的依据。
本发明中新型冠状病毒2019-nCoV的基因序列参见GISAID：BetaCov/Wuhan/WH01/2019|EPI_ISL_406798；关于其E基因的寡核苷酸序列信息 可参考文献(Roujian Lu,Xiang Zhao,Juan Li,et.al,Genomic characterisation and epidemiology of 2019novel coronavirus:implications for virus origins and receptor binding.Lancet.2020 Jan 30)。
本试剂盒组成详见表1和表2，可针对性检测新型冠状病毒2019-nCoV的E基因，对可能因突变而引起的漏检进行确认，能够显著提高病毒鉴定的准确性。
表1试剂盒组成
Figure PCTCN2020141570-appb-000001
试剂盒所需引物、探针序列如表2：
表2引物、探针及序列号
引物探针名称 引物/探针序列 SEQ ID NO.
E-NCOV-F TTTTCTTGCTTTCGTGGTA 1
E-NCOV-R GGTTTTACAAGACTCACGTT 2
E-NCOV-P ATTGCAGCAGTACGCACACA 3
RP30-F CAAGTAAGTTTCTCCGAATCCC 4
RP30-R GCTGAAGTCCCATGACCGT 5
RP30-P CAACTGGAGGTAGAGACGGACTGCG 6
优选地，所述荧光基团选自下组：FAM、VIC、HEX、NED、ROX、TET、JOE、TAMRA、CY3、CY5。
优选地，所述淬灭基因选自下组：MGB、BHQ-1、BHQ-2、BHQ-3。
本发明的引物设计中，对特异性引物及探针经大量试验筛选，并对其进行组合、优化、验证，最终优选出组合后不会互相干扰、扩增效率高、特异性好的双重检测最优引探组合。
本发明所述试剂盒用于判定检测有效标准为：
每次检测均同时检测阴性质控品、阳性质控品，当检测结果的阳性质控品为阳性，且阴性质控品为阴性时，表明实验结果有效。
本发明所述试剂盒的使用方法包括以下步骤：
(1)提取检测样本中的总核酸，获得核酸样本。
(2)将所述核酸样本与RT-PCR反应液、引物探针混合液、RT-PCR反应酶系、和DEPC H 2O混合，制得PCR反应体系。
(3)实时荧光PCR反应，程序如下：
第一阶段：50℃2-15min，95℃10-15min，1个循环；
第二阶段：94℃10-15s，55-60℃45s，45个循环。
PCR结束后通过不同荧光通道曲线及Ct值判断对应病原体核酸的阴、阳性给出检测结果。
本发明的有益效果在于：
(1)本发明通过针对性检测新型冠状病毒2019-nCoV的膜蛋白核酸靶标，能够高效率、高特异性、低成本的对新型冠状病毒2019-nCoV感染患者进行检测，并预防可能因病毒变异性而出现的假阴性，对可能因突变而引起的漏检进行确认，显著提高了病毒鉴定的准确性。
(2)本发明的检测试剂盒，同时包括新型冠状病毒2019-nCoV的膜蛋白核酸靶标检测系统和内源性内标检测系统，为双重荧光检测试剂盒，可以对标本采集、核酸提取过程及PCR扩增过程进行监控，减少假阴性结果的出现。
(3)本发明经过多轮筛选验证，从大量引物探针组合中获得了灵敏度高、特异性强、重复性好，并且适用于多重检测的引物探针组合。
本发明适用于对新型冠状病毒2019-nCoV核酸的检测，为病毒鉴定和防控提供可靠的依据，值得推广应用。另外，本发明的方法同样适用于非诊断的目的，例如，在疫情防控过程中，使用本发明的检测方法对环境中的病毒核酸进行检测，这些病毒核酸信息可以作为公众健康管理之需。
下面结合具体实施例，进一步详陈本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明详细条件的实验方法，通常按照常规条件如美国Sambrook.J等著《分子克隆实验室指南》(黄培堂等译，北京：科学出版社，2002年)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。以下实施例中所用的实验材料和试剂如无特别说明均可从市售渠道获得。
实施例1引物和探针的设计
根据新型冠状病毒的序列，分析其基因组的保守区域。在这些保守区域中设计出数十组针对2019新型冠状病毒E基因检测的特异性引物和探针序列。另外为了对标本采集、核酸提取过程及PCR扩增过程进行监控，选取人类基因组DNA中核糖核酸酶P(RPP30)设计内标引物和探针。
在上述引物、探针的设计过程中，尽量避免发卡结构、引物内部二聚体、引物间二聚体以及错配的形成。
此外，通过对NCBI Blast在线数据库(https://blast.ncbi.nlm.nih.gov/Blast.cgi)对上述设计的新型冠状病毒特异性引物和探针序列进行比对分析避免与其他病毒或人类基因发生非特异结核。通过多轮筛选和优化，最终确定了一套灵敏度和特异性最优的引物、探针序列。
引物探针序列：
序列名称 序列 SEQ ID NO.:
E-NCOV-F TTTTCTTGCTTTCGTGGTA 1
E-NCOV-R GGTTTTACAAGACTCACGTT 2
E-NCOV-P ATTGCAGCAGTACGCACACA 3
RP30-F CAAGTAAGTTTCTCCGAATCCC 4
RP30-R GCTGAAGTCCCATGACCGT 5
RP30-P CAACTGGAGGTAGAGACGGACTGCG 6
其中，E-NCOV-P的5’端荧光基团为FAM，3’端猝灭基团为BHQ1；RP30-P的5’端荧光基团为VIC，3’端猝灭基团为BHQ1。
实施例2检验方法及体系
采用的Taqman探针检测新型冠状病毒RNA的原理是：先利用加入的引物序列E-NCOV-F与E-NCOV-R作为逆转录引物将提取出的病毒RNA逆转录为带有锚定序列的cDNA，然后再利用E-NCOV-F与E-NCOV-R作为实时荧光PCR检测的正向和反向引物进行扩增。
本方法为一步扩增法，即将病毒RNA逆转录为cDNA与DNA扩增两个步骤结合在一起，无需换管进行操作。
为了确定引物和探针的检测体系，分别尝试了不同浓度的引物和探针对荧光PCR反应的影响。结果显示，当E-NCOV-F，E-NCOV-R引物浓度为10pmol，E-NCOV-P探针的浓度为5pmol，RP30-F，RP30-R引物浓度为8pmol，RP30-P探针的浓度为3pmol，扩增曲线最好。
检测结果如图1所示，图1中由左至右分别为引物浓度为15pmol、10pmol、8pmol，探针浓度为8pmol、5pmol、3pmol时假病毒梯度检测结果。
最终确定的反应体系(25μL)如下：
Figure PCTCN2020141570-appb-000002
其中RT-PCR反应酶系由热启动Taq酶、逆转录酶、dNTPs组成。逆转录酶可选择mMLV等常用逆转录酶。热启动Taq酶、逆转录酶、dNTPs均可采用市售产品，如Qiagen公司的产品,其中每人份RT-PCR反应酶系中热启动Taq酶的用量为8U，逆转录酶用量为3U，dNTPs用量为10mmol。
通过对不同退火温度进行测试，最终确定在ABI7500荧光PCR仪上的反应程序为：50℃15分钟，1个循环；95℃15分钟，1个循环；94℃15秒→55℃45秒(收集荧光)，45个循环。
实施例3灵敏度检测
将目的片段连接到构建好的pET28a-MS2载体中并转化表达宿主菌BL21感受态细胞，挑取单克隆并测序验证后进行诱导表达,将破碎完全的表达产物以RNaseA和DNaseⅠ消化处理后即为含有目的片段的病毒样颗粒。
将测定浓度后的假病毒稀释到合适浓度后再进行10倍倍比稀释，浓度分别为1.00E+06、1.00E+05、1.00E+04、1.00E+03、1.00E+02copies/ml。
用上述确定的检测体系和循环参数对上述假病毒进行检测(图2)。结果显示，此种检测方法具有较高的灵敏度，灵敏度为1.00E+02copies/ml。
实施例4特异性检测
将甲型/乙型流感病毒、冠状病毒229E、冠状病毒NL63、冠状病毒OC43、 冠状病毒HKU1、呼吸道合胞病毒、腺病毒、肺炎支原体、肺炎衣原体作为特异性样本进行检测(图3)。
检测结果表明，检测特异性参考品(甲型/乙型流感病毒、冠状病毒229E、冠状病毒NL63、冠状病毒OC43、冠状病毒HKU1、呼吸道合胞病毒、腺病毒、肺炎支原体、肺炎衣原体)，检测结果均为阴性，表明本发明的试剂盒特异性良好。
实施例5重复性检测
选取浓度为5.00E+04copies/ml和1.00E+03copies/ml的假病毒进行检测，采用上述确定的检测体系和循环参数，各重复10次(图4)。
结果表明本发明的试剂盒和检测方法重复性较好。
实施例6临床样本检测
检测样本核酸的提取：
(1)临床待测样本核酸模板提取
采集22例疑似咽拭子临床样本，提取待测样本(提取试剂采用中山大学达安基因股份有限公司生产的核酸提取或纯化试剂(粤穗械备20170583号)，得到核酸样本(阳性质控和阴性质控同步参与提取)；取5μL核酸样本配制PCR反应体系，在实时荧光PCR仪中进行扩增反应，荧光通道依次选择VIC、FAM,PCR扩增程序如下；
50℃，15min，95℃，15min；1个循环
94℃，15sec，55℃，45sec(收集荧光)；45个循环。
PCR结束后通过不同荧光通道曲线及Ct值判断对应病原体核酸的阴、阳性。
在所检测的22例疑似临床样本中，共检出17例新型冠状病毒2019-nCoV核酸阳性临床样本。典型检测结果如图5所示。
测序验证结果表明本发明检测体系检测准确率达到了100％，进一步证明了本发明体系检测的临床检测准确性。
对比例1
本发明人在研究过程中，针对新型冠状病毒2019-nCoV目标核酸序列筛选了数十组PCR引物和探针，经过大量测试，最终获得了灵敏度和特异性能够满足临床检测需求，且能够进行多重检测的引物、探针组合。
针对新型冠状病毒2019-nCoV的E基因检测靶标，本发明人经过了大量的筛选、组合。设计的部分典型引物序列如下：
对照上游引物E-NCOV-F2：CTTTCGTGGTATTCTTGCTA(SEQ ID NO.7)
对照下游引物E-NCOV-R2：CTCACGTTAACAATATTGCAG(SEQ ID NO.8)
对照上游引物E-NCOV-F3：AGTTACACTAGCCATCCTT(SEQ ID NO.9)
对照下游引物E-NCOV-R3：TTTAACACGAGAGTAAACGTA(SEQ ID NO.10)
具体检测步骤、检测条件、探针序列同以上实施例，进行PCR检测测试。
使用E-NCOV-F2和E-NCOV-R2的检测结果如图6所示，检测结果表明该引物对特异性较差。
使用E-NCOV-F3和E-NCOV-R3的检测结果表明该引物对在单一检测体系中对E基因靶核酸的特异性和灵敏度较佳，但是在多重检测体系中E基因靶低浓度核酸扩增受到明显抑制，单重和多重体系检测结果如图7所示。表明对照引物对E-NCOV-F3和E-NCOV-R3无法应用于多重检测体系中。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种用于检测新型冠状病毒2019-nCoV核酸的试剂盒，其特征在于，所述试剂盒包括引物对集和探针组；
    其中，所述引物对集包括第一引物对和内标引物对，
    所述第一引物对包括：
    如SEQ ID NO.1所示的正向引物和如SEQ ID NO.2所示的反向引物，
    所述内标引物对包括：
    如SEQ ID NO.4所示的正向引物和如SEQ ID NO.5所示的反向引物；
    所述探针组包括：
    核苷酸序列如SEQ ID NO.3所示的第一探针和核苷酸序列如SEQ ID NO.6所示的内控探针。
  2. 如权利要求1所述的试剂盒，其特征在于，所述试剂盒包括第一容器，所述第一容器内包含引物探针混合液，所述引物探针混合液中包含所述引物对集和所述探针组。
  3. 如权利要求2所述的试剂盒，其特征在于，所述试剂盒还包括第二容器，所述第二容器内包含RT-PCR反应酶系，所述RT-PCR反应酶系包括热启动Taq酶和逆转录酶。
  4. 如权利要求3所述的试剂盒，其特征在于，所述第二容器内还包含dNTPs。
  5. 如权利要求2所述的试剂盒，其特征在于，所述试剂盒还包括第三容器，所述第三容器内包含RT-PCR反应液，所述RT-PCR反应液包括Tris-HCl缓冲液、Mg2+和Tween-20。
  6. 如权利要求2所述的试剂盒，其特征在于，所述试剂盒还包括第四容器，所述第四容器内包含DEPC H 2O。
  7. 如权利要求1所述的试剂盒，其特征在于，所述第一探针的5’端包含有荧光报告基团，所述第一探针的3’端包含有荧光淬灭基团；所述内控探针的5’端包含有荧光报告基团，所述内控探针的3’端包含有荧光淬灭基团；并且，所述第一探针的5’端荧光报告基团和所述内控探针的5’端荧光报告基团不同。
  8. 如权利要求7所述的试剂盒，其特征在于，所述第一探针的5’端荧光报告基团为FAM，所述第一探针的3’端荧光猝灭基团为BHQ1；所述内控探针的5’端荧光报告基团为VIC，所述内控探针的3’端荧光猝灭基团为BHQ1。
  9. 一种非诊断目的的多重检测新型冠状病毒2019-nCoV核酸的方法，所述方法包括步骤：
    (1)提供待检测对象的核酸样本；
    (2)制备RT-PCR反应体系并进行RT-PCR检测：
    其中，所述RT-PCR反应体系包括：步骤(1)提供的所述核酸样本、引物对集和探针组；
    所述引物对集包括第一引物对和内标引物对，
    所述第一引物对包括：
    如SEQ ID NO.1所示的正向引物和如SEQ ID NO.2所示的反向引物，
    所述内标引物对包括：
    如SEQ ID NO.4所示的正向引物和如SEQ ID NO.5所示的反向引物；
    所述探针组包括：
    核苷酸序列如SEQ ID NO.3所示的第一探针和核苷酸序列如SEQ ID NO.6所示的内控探针。
  10. 权利要求9所述的方法，其特征在于，所述核酸样本来自环境样本。
PCT/CN2020/141570 2020-03-03 2020-12-30 一种新型冠状病毒rt-pcr检测方法及试剂盒 WO2021174984A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US17/905,662 US20230114907A1 (en) 2020-03-03 2020-12-30 Rt-pcr detection method and kit for novel coronavirus
EP20922897.2A EP4116440A4 (en) 2020-03-03 2020-12-30 RT-PCR DETECTION METHODS AND KIT FOR NEW CORONAVIRUS

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010137482.0 2020-03-03
CN202010137482.0A CN110982943B (zh) 2020-03-03 2020-03-03 一种新型冠状病毒rt-pcr检测方法及试剂盒

Publications (1)

Publication Number Publication Date
WO2021174984A1 true WO2021174984A1 (zh) 2021-09-10

Family

ID=70081329

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/141570 WO2021174984A1 (zh) 2020-03-03 2020-12-30 一种新型冠状病毒rt-pcr检测方法及试剂盒

Country Status (4)

Country Link
US (1) US20230114907A1 (zh)
EP (1) EP4116440A4 (zh)
CN (1) CN110982943B (zh)
WO (1) WO2021174984A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113832262A (zh) * 2021-11-10 2021-12-24 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感染性的寡核苷酸,以及含有该寡核苷酸的试剂盒及应用
CN113881812A (zh) * 2021-12-03 2022-01-04 深圳联合医学科技有限公司 检测SARS-CoV-2突变株的组合物、试剂盒、方法及其用途

Families Citing this family (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021156882A1 (en) * 2020-02-04 2021-08-12 Indian Institute Of Technology Delhi Primer sets, biomarkers, kit and applications thereof
CN110982943B (zh) * 2020-03-03 2020-06-30 中山大学达安基因股份有限公司 一种新型冠状病毒rt-pcr检测方法及试剂盒
CN111394519B (zh) * 2020-04-13 2023-03-21 南京实践医学检验有限公司 基于数字pcr的新型冠状病毒的核酸定量检测试剂盒及应用
CN111363851A (zh) * 2020-04-16 2020-07-03 大连晶泰生物技术有限公司 一种检测新型冠状病毒的方法和试剂盒
CN111321251B (zh) * 2020-04-16 2020-08-14 圣湘生物科技股份有限公司 检测引起呼吸道感染的病原体并分型的组合物、试剂盒、方法及用途
CN111621595A (zh) * 2020-04-21 2020-09-04 贵州医科大学 新型冠状病毒qRT-PCR一步法试剂盒反应液及其试剂盒和方法
CN111676315A (zh) * 2020-04-21 2020-09-18 中国贵州茅台酒厂(集团)有限责任公司职工医院 检测新型冠状病毒ORF1ab基因的引物和探针及其试剂盒和方法
CN113549708A (zh) * 2020-04-24 2021-10-26 首都医科大学附属北京天坛医院 基于poct模式的新型冠状病毒covid-19快速检测试剂盒
CN111549176B (zh) * 2020-04-27 2024-05-28 生物岛实验室 用于检测SARS-CoV-2的LAMP引物组及试剂盒
CN112111597B (zh) * 2020-04-28 2023-07-21 安徽同科生物科技有限公司 一种免核酸提取快速检测新型冠状病毒2019-nCoV核酸的荧光PCR试剂盒
CN111304372B (zh) * 2020-04-29 2020-08-14 圣湘生物科技股份有限公司 检测2019新型冠状病毒突变的组合物、用途及试剂盒
CN111500777A (zh) * 2020-04-30 2020-08-07 嘉兴实践医学科技有限公司 一种基于荧光rt-pcr方法检测新型冠状病毒核酸的试剂盒
CN111549182B (zh) * 2020-05-26 2023-09-29 生物岛实验室 用于检测SARS-CoV-2的探针及试剂盒
CN111471803A (zh) * 2020-05-29 2020-07-31 武汉生命之美科技有限公司 一种新型冠状病毒covid-19感染检测试剂盒
CN111621604A (zh) * 2020-06-24 2020-09-04 上海五色石医学研究股份有限公司 一种新型冠状病毒核酸检测引物探针组合物、试剂盒及方法
CN113684316A (zh) * 2020-09-07 2021-11-23 四川大学华西医院 基于数字pcr的新型冠状病毒检测试剂盒及应用
CN111926121A (zh) * 2020-09-27 2020-11-13 南京黎明生物制品有限公司 一种用于2019-nCoV E基因检测的核酸组合物及其试剂盒与生产方法
CN113151608B (zh) * 2021-05-26 2021-11-05 华中农业大学 检测具有感染性SARS-CoV-2的PCR靶序列、引物和探针及应用
US20230203603A1 (en) * 2021-06-08 2023-06-29 Shanghai Focusgen Biotech Ltd Rt-pcr detection reagent for detecting novel coronavirus, kit and detection method thereof
CN113481324B (zh) * 2021-07-06 2023-12-05 广州达安基因股份有限公司 检测新型冠状病毒及其d614g突变株的方法和试剂盒
CN117210615B (zh) * 2023-09-25 2024-04-02 江苏省农业科学院 猪曼那角病毒TaqMan荧光定量RT-PCR检测方法与应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050233314A1 (en) * 2003-06-30 2005-10-20 National Health Research Institutes Sensitive and quantitative detection of pathogens by real-time nested PCR
CN1906306A (zh) * 2003-08-22 2007-01-31 博驰生物医疗研究有限公司 Sars相关冠状病毒的多等位分子检测
WO2011133433A2 (en) * 2010-04-16 2011-10-27 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services, Centers For Disease Control And Prevention Real time pcr assay for detection of bacterial respiratory pathogens
CN105624335A (zh) * 2016-03-10 2016-06-01 中山大学达安基因股份有限公司 一种基因检测中东呼吸综合征冠状病毒的试剂盒
CN110982943A (zh) * 2020-03-03 2020-04-10 中山大学达安基因股份有限公司 一种新型冠状病毒rt-pcr检测方法及试剂盒

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111500768B (zh) * 2020-03-23 2022-11-25 中国检验检疫科学研究院 鉴定新型冠状病毒的引物探针及在双重数字pcr的用途
CN111926121A (zh) * 2020-09-27 2020-11-13 南京黎明生物制品有限公司 一种用于2019-nCoV E基因检测的核酸组合物及其试剂盒与生产方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050233314A1 (en) * 2003-06-30 2005-10-20 National Health Research Institutes Sensitive and quantitative detection of pathogens by real-time nested PCR
CN1906306A (zh) * 2003-08-22 2007-01-31 博驰生物医疗研究有限公司 Sars相关冠状病毒的多等位分子检测
WO2011133433A2 (en) * 2010-04-16 2011-10-27 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services, Centers For Disease Control And Prevention Real time pcr assay for detection of bacterial respiratory pathogens
CN105624335A (zh) * 2016-03-10 2016-06-01 中山大学达安基因股份有限公司 一种基因检测中东呼吸综合征冠状病毒的试剂盒
CN110982943A (zh) * 2020-03-03 2020-04-10 中山大学达安基因股份有限公司 一种新型冠状病毒rt-pcr检测方法及试剂盒

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
GUO XIAOBO, LIN JINGLAI CHUNYAN ZHANG YANGMIN GONG WEIFENG SHI MIN YANG RUILI: "New Coronavirus and Real-Time Fluorescent RT-PCR Nucleic Acid Test", SHAANXI MEDICAL JOURNAL, vol. 49, no. 3, 26 February 2020 (2020-02-26), pages 264 - 266, XP055842211, DOI: 10.3969/j.issn.1000-7377.2020.03.002 *
HUANG CHAOLIN; WANG YEMING; LI XINGWANG; REN LILI; ZHAO JIANPING; HU YI; ZHANG LI; FAN GUOHUI; XU JIUYANG; GU XIAOYING; CHENG ZHEN: "Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China", THE LANCET, ELSEVIER, AMSTERDAM, NL, vol. 395, no. 10223, 24 January 2020 (2020-01-24), AMSTERDAM, NL, pages 497 - 506, XP086050317, ISSN: 0140-6736, DOI: 10.1016/S0140-6736(20)30183-5 *
KAM KAI-QIAN, YUNG CHEE FU, CUI LIN, TZER PIN LIN RAYMOND, MAK TZE MINN, MAIWALD MATTHIAS, LI JIAHUI, CHONG CHIA YIN, NADUA KAREN,: "A Well Infant With Coronavirus Disease 2019 (COVID-19) With High Viral Load", CLINICAL INFECTIOUS DISEASES, THE UNIVERSITY OF CHICAGO PRESS, CHICAGO, IL., US, vol. 71, no. 15, 28 July 2020 (2020-07-28), US, pages 847 - 849, XP055842208, ISSN: 1058-4838, DOI: 10.1093/cid/ciaa201 *
ROUJIAN LUXIANG ZHAOJUAN LI: "Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding", LANCET, 30 January 2020 (2020-01-30)
SAMBROOK. J ET AL.: "Guide to Molecular Cloning Laboratory", 2002, SCIENCE PRESS
See also references of EP4116440A4

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113832262A (zh) * 2021-11-10 2021-12-24 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感染性的寡核苷酸,以及含有该寡核苷酸的试剂盒及应用
CN113881812A (zh) * 2021-12-03 2022-01-04 深圳联合医学科技有限公司 检测SARS-CoV-2突变株的组合物、试剂盒、方法及其用途
CN113881812B (zh) * 2021-12-03 2022-02-15 深圳联合医学科技有限公司 检测SARS-CoV-2突变株的组合物、试剂盒、方法及其用途

Also Published As

Publication number Publication date
EP4116440A4 (en) 2024-05-01
CN110982943B (zh) 2020-06-30
US20230114907A1 (en) 2023-04-13
EP4116440A1 (en) 2023-01-11
CN110982943A (zh) 2020-04-10

Similar Documents

Publication Publication Date Title
WO2021174984A1 (zh) 一种新型冠状病毒rt-pcr检测方法及试剂盒
CN111020064B (zh) 新型冠状病毒ORF1ab基因核酸检测试剂盒
CN111004870B (zh) 新型冠状病毒n基因核酸检测试剂盒
US11649511B2 (en) Multiplex PCR method for the detection of SARS-CoV-2
WO2022057060A1 (zh) 多重检测呼吸道病毒核酸的方法及试剂盒
WO2021238087A1 (zh) 基于热对流pcr的新型冠状病毒快速检测试剂盒
US20230070496A1 (en) Nucleic Acid Detection Kit For Novel Coronavirus 2019-nCoV
CN113652505B (zh) 检测新型冠状病毒及其voc-202012/01突变株的方法和试剂盒
WO2022257002A1 (en) Rt-pcr detection reagent for detecting novel coronavirus, kit and detection method thereof
CN111471800B (zh) 检测新型冠状病毒的试剂盒及其扩增引物组合物
CN113481325A (zh) 检测新型冠状病毒b.1.1.7突变株的方法和试剂盒
CN111676316B (zh) 一种快速区分非洲猪瘟病毒基因ii型与其它基因型的引物、探针及其检测方法
CN112662809A (zh) 一种用于新型冠状病毒covid-19检测的核酸组合物及其应用
CN113481324B (zh) 检测新型冠状病毒及其d614g突变株的方法和试剂盒
CN111500777A (zh) 一种基于荧光rt-pcr方法检测新型冠状病毒核酸的试剂盒
CN114262758B (zh) 检测新型冠状病毒突变株的试剂盒及检测方法
CN114807432B (zh) 快速检测新型冠状病毒及其Delta突变株的试剂盒和方法
CN117344062B (zh) 一种用于快速检测新型冠状病毒核酸的试剂盒和使用方法
CN116179760A (zh) 一种基于rpa的流感病毒h10n3快速检测试剂、试剂盒及使用方法
CN117737309A (zh) 琅琊病毒、墨江病毒、尼帕病毒和雪松病毒的四重qRT-PCR方法
CN115717177A (zh) 一种基于环介导等温扩增的流感病毒h10n3快速检测试剂、试剂盒及使用方法
CN116855639A (zh) 一种快速检测人腺病毒4型的引物、探针及试剂盒
CN116516073A (zh) 一种快速检测人腺病毒14型的引物、探针及试剂盒

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20922897

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2020922897

Country of ref document: EP

Effective date: 20221004