WO2022095731A1 - 用于检测新型冠状病毒的试剂盒及方法 - Google Patents

用于检测新型冠状病毒的试剂盒及方法 Download PDF

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WO2022095731A1
WO2022095731A1 PCT/CN2021/125665 CN2021125665W WO2022095731A1 WO 2022095731 A1 WO2022095731 A1 WO 2022095731A1 CN 2021125665 W CN2021125665 W CN 2021125665W WO 2022095731 A1 WO2022095731 A1 WO 2022095731A1
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probe
primer
nucleic acid
fluorescence
seq
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French (fr)
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张永有
宋娜杰
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厦门大学
厦门致善生物科技股份有限公司
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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
    • C12Q1/70—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
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes

Definitions

  • the present invention relates to kits and methods for detecting novel coronaviruses.
  • Coronavirus Disease 2019 (COVID-19) is an emerging acute respiratory infectious disease that has become a global pandemic.
  • the novel coronavirus is the pathogen that causes COVID-19.
  • Coronavirus is a class of RNA viruses with enveloped genomes and a linear single-stranded positive-stranded genome. Coronaviruses are divided into 4 genera, alpha, beta, gamma and delta, among which alpha and beta coronaviruses can infect mammals, while gamma and delta coronaviruses mainly infect birds. Six coronaviruses have previously been found to infect humans: alphacoronavirus (HCoV-229E, HCoV-NL63) and betacoronavirus (HCoV-HKU1, HCoV-OC43, MERS-CoV, SARS-CoV). Cold to severe lung infection.
  • alphacoronavirus HCV-229E, HCoV-NL63
  • betacoronavirus HCoV-HKU1, HCoV-OC43, MERS-CoV, SARS-CoV
  • Novel coronavirus is a beta-coronavirus that has not been previously found in humans, and is highly contagious in humans, mainly through respiratory droplets, Respiratory secretions and direct contact transmission.
  • Patients with COVID-19 often present with specific similar symptoms, such as fever, malaise and cough. Most patients present with mild flu-like symptoms and have a good prognosis. However, a small number of patients are critically ill and rapidly develop acute respiratory distress syndrome, respiratory failure, multiple organ failure and even death.
  • the new coronavirus is highly contagious, the population is generally susceptible, has an incubation period, and is contagious during the incubation period. Therefore, it is necessary to detect the infected person in time, control the source of infection, and cut off the transmission route in order to effectively control the spread of the epidemic.
  • the premise of taking all blocking measures is the need for rapid, accurate and sensitive diagnosis.
  • kits for the detection of new coronaviruses on the market at home and abroad, which are mainly based on two methods: immunological methods and molecular biological methods.
  • immunology-based methods mainly use colloidal gold method, chemiluminescence method and enzyme-linked immunosorbent method to detect novel coronavirus-specific IgM antibodies, IgG antibodies or antigens in the serum or plasma of patients.
  • clinical diagnosis is generally not based on serological testing alone.
  • the positive rates of new coronavirus IgM antibodies and IgG antibodies are low within one week of the onset of patients, so they are not suitable for early screening of new coronavirus infection.
  • false positives may occur in antibody detection: one is the setting of the positive judgment value of the kit, and some weak positive results near the positive judgment value are likely to be false positives; Whether there is color to judge positive and negative, there are individual differences in judgment; third, there are endogenous interfering substances in patient specimens that cause false positives in immunoassays, such as rheumatoid factor, complement, heterophilic antibodies, lysozyme, etc.; Fourth, in the pre-analytical process of specimen collection, transportation, and storage, specimen hemolysis, long specimen storage time, and incomplete specimen coagulation will also interfere with the test results.
  • the molecular biology-based method mainly uses real-time fluorescent PCR technology to specifically detect the new coronavirus nucleic acid in patients' throat swabs, sputum and other lower respiratory tract secretions, blood, feces, urine and other specimens.
  • Nucleic acid detection results are clinically used as one of the basis for diagnosis, but due to the sensitivity of the reagent itself and the mutation of the target sequence of the virus to be detected, false negative results will also occur in nucleic acid detection.
  • the kit for detecting novel coronavirus of the present invention adopts one-step reverse transcription polymerase chain reaction (One step RT-PCR) combined with Taqman probe method to perform dual-target detection of novel coronavirus ORF1ab and N gene.
  • the primers and probes targeting the ORF1ab gene and the N gene of the present invention have the lowest mutation rate in the coverage area, can minimize the false negative caused by the gene mutation of the virus target sequence to be detected, and have the advantages of high accuracy and high sensitivity .
  • the exogenous internal standard SUC2 gene can also be introduced to control the whole process from nucleic acid extraction to detection, which can avoid false negative results.
  • the present invention provides primers and probe sets for the detection of novel coronavirus, comprising primers I-F whose sequences are shown in SEQ ID NO: 1, and primers I-R whose sequences are shown in SEQ ID NO: 2 , the sequence is shown as probe I of SEQ ID NO: 3, the sequence is shown as primer II-F shown in SEQ ID NO: 4, the sequence is shown as primer II-R shown in SEQ ID NO: 5, and the sequence is shown as SEQ ID NO: : Probe II shown in 6.
  • the 5' ends of the probe I and the probe II are each independently attached with a fluorophore, and the 3' ends are each independently attached with a fluorescence quenching group, and the probe I
  • the fluorophore attached to the probe II and the fluorophore attached to the probe II are different from each other, and the fluorescence quencher group attached to the probe I and the fluorescence quencher group attached to the probe II may be the same or different.
  • a FAM fluorophore is attached to the 5' end of the probe I, and a ROX fluorophore is attached to the 5' end of the probe II.
  • the primer and probe set further comprises: primer III-F whose sequence is shown in SEQ ID NO: 7, primer III-R whose sequence is shown in SEQ ID NO: 8, and primer III-R whose sequence is shown in SEQ ID NO: 8; Probe III shown in SEQ ID NO:9.
  • a fluorophore is attached to the 5' end of the probe III, and a fluorescence quenching group is attached to the 3' end; and the fluorophore attached to the probe III is the same as the probe. I and the fluorophore attached to the probe II are different.
  • the fluorescence quenching group attached to the probe III, the fluorescence quenching group attached to the probe I and the fluorescence quenching group attached to the probe II may be the same, partially the same or different.
  • a HEX fluorophore is attached to the 5' end of the probe III.
  • the present invention provides a kit for detecting novel coronavirus, which includes the primers and probe sets as described above.
  • the present invention provides a method for detecting a novel coronavirus, comprising the steps of:
  • the set of primers and probes comprises the primers I-F, primers I-R, probe I, primer II-F, primer II-R, probe II, primer III-F, primer III-R , Probe III.
  • the test sample contains an internal standard nucleic acid comprising the SUC2 gene or a partial sequence thereof.
  • the internal standard nucleic acid is capable of annealing to the primer III-F or its complement, the primer III-R or its complement, and the probe III or its complement.
  • the internal standard nucleic acid is exogenously added to the test sample.
  • step 2) includes mixing the reagent for RT-PCR reaction with the sample to be tested, and performing RT-PCR reaction; wherein, the reagent for RT-PCR reaction includes: 2-5 ⁇ L Solution 1, 2-7 ⁇ L of OSTR buffer, 0.5-1.5 ⁇ L of mixed enzyme, primers and probe sets, and supplemented with DEPC-H 2 O to 20 ⁇ L; wherein, the primers and probe sets of each component of The concentration is 50 ⁇ M, and the amount is 0.05-3 ⁇ L.
  • the RT-PCR amplification procedure is as follows:
  • the samples to be tested in step 1) include commercial products and samples in the environment.
  • the sample to be tested in step 1) is a nucleic acid sample obtained by nucleic acid extraction or purification.
  • the reagent for RT-PCR reaction is a liquid reagent.
  • the volume of the sample to be tested is 5 ⁇ L, and the total reaction volume of the RT-PCR is 25 ⁇ L.
  • the reagents for RT-PCR reaction are dry reagents.
  • the volume of the sample to be tested is 25 ⁇ L, and the total reaction volume of the RT-PCR is 25 ⁇ L.
  • step 2) further comprises: mixing the primers and probe sets with the positive quality control product and the negative quality control product, respectively, and performing RT-PCR reaction.
  • the positive quality control product is a pseudovirus sample of novel coronavirus ORF1ab gene and N gene mixed with internal standard nucleic acid, wherein the internal standard nucleic acid comprises SUC2 gene or a partial sequence thereof.
  • the negative control substance is DEPC-H 2 O containing an internal standard nucleic acid, wherein the internal standard nucleic acid comprises the SUC2 gene or a partial sequence thereof.
  • a fluorescent signal is collected during the RT-PCR reaction, and the fluorescent signal includes the first fluorescence corresponding to the fluorophore to which the probe I is attached, and the first fluorescence that corresponds to the fluorophore attached to the probe II.
  • the second fluorescence corresponding to the fluorophore of , and the third fluorescence corresponding to the fluorophore to which the probe III is attached eg, FAM, ROX, HEX fluorescence).
  • step 3 if the negative quality control substance has no Ct value in the detection channel corresponding to the first fluorescence and the second fluorescence, and at the same time, the detection channel corresponding to the third fluorescence has a Ct value, the negative quality control Qualified; if the Ct value of the positive control substance in the first fluorescence corresponding detection channel is ⁇ 34, the Ct value in the second fluorescence corresponding detection channel is ⁇ 31, and the Ct value in the third fluorescence corresponding detection channel is ⁇ 32, the positive quality control qualified.
  • the Ct-free value means no amplification/fluorescence curve or Ct ⁇ 40.
  • results are interpreted as shown in Table 4 and Table 5.
  • the method is used to determine whether a novel coronavirus is present in a sample to be tested.
  • the methods are used for disease diagnostic or non-disease diagnostic purposes. In certain embodiments, the methods are used for non-disease diagnostic purposes.
  • the present invention provides the use of the above-mentioned primers and probe sets or kits in the preparation of detection reagents for the detection of novel coronavirus.
  • the present invention develops a kit for detecting novel coronavirus that can overcome the limitations of existing kits.
  • the present invention uses one-step reverse transcription polymerase chain reaction (One step RT-PCR) combined with Taqman probe method to qualitatively detect the ORF1ab gene and N gene specific region of SARS-CoV-2, and realize the sample through the change of fluorescent signal. Detection of RNA.
  • the exogenous internal standard SUC2 gene is introduced to control the whole process from nucleic acid extraction to detection, which can avoid false negative results.
  • the present invention adopts the dual target detection of novel coronavirus ORF1ab and N gene, so that the detection result is more accurate and reliable, and false negative results caused by mutation of one of the targets to be detected can be avoided.
  • the present invention can also use the exogenous internal standard SUC2 gene as the quality control system of the kit of the present invention to monitor the whole process from sample RNA extraction to nucleic acid detection, which can avoid to the greatest extent possible problems caused by improper experimental setup or operation and abnormal reagents. Or equipment failure and other reasons lead to inaccurate test results.
  • the primers and probes used in the present invention are all designed by themselves, and multiple primers and probes have been designed through the alignment of a large number of novel coronavirus genome sequences.
  • the mutation rate in the coverage area of the primer probe targeting ORF1ab gene of the kit of the present invention is the lowest, and the mutation rate in the coverage area of the primer probe targeting N gene is also the lowest among the current N gene targeting reactions. Therefore, the novel coronavirus nucleic acid detection kit of the present invention can minimize false negatives caused by gene mutation of the virus target sequence to be detected.
  • the kit of the present invention has higher detection sensitivity for ORF1ab gene and N gene.
  • Figure 1 shows the mutation of target regions of primers and probes of different SARS-CoV-2 detection kits.
  • FIG. 2 is a comparison between the embodiment of the present invention and the detection system recommended by the Chinese CDC.
  • FIG. 3 is a typical result diagram of an embodiment of the present invention.
  • FIG. 4 is a sample detection result diagram according to an embodiment of the present invention.
  • the amplification primers and detection probes of the novel coronavirus ORF1ab and N gene, and the exogenous internal standard SUC2 gene were designed by themselves (as shown in Table 1).
  • the detection probe of ORF1ab gene is labeled with a FAM fluorescent group at the 5' end and a quencher group at the 3' end;
  • the detection probe of N gene is labeled with a ROX fluorescent group at the 5' end and a quencher group at the 3' end;
  • SUC2 The 5' end of the gene detection probe is labeled with a HEX fluorescent group, and the 3' end is labeled with a quenching group.
  • nasopharyngeal swabs Collect nasopharyngeal swabs, oropharyngeal swabs, anterior and middle nasal swabs, nasopharyngeal rinses/nasal aspirate, or bronchoalveolar lavage (BAL) samples from suspected COVID-19 patients.
  • Nucleic acid extraction and purification were performed using the manual extraction kit "Viral RNA Extraction Kit (Cat. No.: 602101)" produced by Xiamen Zhishan Biotechnology Co., Ltd.
  • the extraction and purification process of the extraction kit consists of 4 steps: lysis, binding, washing and elution. During extraction, 20 ⁇ L of the internal control and 1 mL of the sample to be tested were added to the extraction wells, and then extracted according to the operation method in the extraction kit instructions.
  • 20 ⁇ L of the inner control and 20 ⁇ L of the new coronavirus positive control, 20 ⁇ L of the inner control and 20 ⁇ L of the negative control were added to separate extraction wells, and the extraction was performed simultaneously with the sample to be tested.
  • the internal control was added to each sample for simultaneous extraction, in order to control the extraction process of the sample.
  • the nucleic acid elution volume in the extraction kit is 60 ⁇ L, and the RNA obtained after extraction should be used immediately or stored at -70°C.
  • the internal control is the SUC2 pseudovirus
  • the negative control is the solution without nucleic acid
  • the positive control of the new coronavirus is the pseudovirus of the new coronavirus ORF1ab and N gene.
  • RT-PCR reaction components and amounts of the present invention are shown in Table 2 below.
  • Solution 1 comes from Feipeng Biological Co., Ltd. and is a component of the product AnstartOne-Step RT-PCR Mix, product number MD013.
  • Enzyme mix comes from Feipeng Biological Co., Ltd. It is a component of the product AnstartOne-Step RT-PCR Mix, product number MD013, the main components are Super MMLV Reverse Transcriptase and hot start Enzyme (AnstartTaq DNA Polymerase).
  • the negative control has no Ct value in the detection channel corresponding to ORF1ab and N genes (no amplification/fluorescence curve or Ct ⁇ 40), and at the same time, the internal control channel (SUC2) has a Ct value (Ct ⁇ 32), the negative control quality control is qualified .
  • SUC2 internal control
  • the quality control of the positive control is qualified.
  • SARS-CoV-2 positive result (ORF1ab+ or N+) does not require HEX detection channel to be detected, and high viral load can cause SUC2 signal to be undetectable or inhibited.
  • Figure 1 shows the mutation of target regions of primers and probes of different SARS-CoV-2 detection kits.
  • the results in Fig. 1 show that, compared with similar kits, the mutation rate (indicated by the arrow in the figure, "ZS-Orf1ab") in the coverage region of the primer probe targeting the ORF1ab gene of the kit of the present invention is the lowest, and the one targeting the N gene is the lowest.
  • the mutation rate in the region covered by the primer probe (indicated by the arrow in the figure, "ZS-N”) is also the lowest among the current N gene-targeting reactions.
  • FIG. 2 is a comparison between the embodiment of the present invention and the detection system recommended by the Chinese CDC. The results in FIG. 2 show that the present invention has higher detection sensitivity for ORF1ab gene and N gene.
  • FIG. 3 is a typical result diagram of an embodiment of the present invention.
  • Figure 3 shows that for the N gene, ORF1ab gene and internal control SUC2 gene of SARS-COV-2, there are characteristic amplification curves in the corresponding fluorescence channels without any cross-reaction.
  • FIG. 4 is a sample detection result diagram according to an embodiment of the present invention.
  • Figure 4 shows the positive control and negative control quality control qualified test results and the test results of the samples to be tested. Only when both the positive control and the negative control are qualified, the results of the samples to be tested will be interpreted. Refer to Table 4 and Table 5 for the interpretation of the results of the samples to be tested.

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Abstract

本发明公开了一种用于检测新型冠状病毒的试剂盒及方法。本发明的试剂盒采用一步法逆转录聚合酶链式反应(One step RT-PCR)结合Taqman探针法，定性检测新型冠状病毒的ORF1ab基因和N基因特异性区域，以及任选地一个非人源内标基因(SUC2基因) 。

Description

用于检测新型冠状病毒的试剂盒及方法 技术领域
本发明涉及用于检测新型冠状病毒的试剂盒及方法。
背景技术
新型冠状病毒肺炎(Coronavirus Disease 2019，COVID-19)为新发急性呼吸道传染病，目前已经成为全球性的大流行病。而新型冠状病毒是引起COVID-19的病原体。截至2020年8月31日，全球已经有超过2500万人感染新型冠状病毒，死亡人数超过84万。并且，感染人数和死亡人数还在不断的增加。
冠状病毒(Coronavirus，CoV)是一类具有囊膜，基因组为线性单股正链的RNA病毒，是自然界广泛存在的一大类病毒。冠状病毒分为α、β、γ和δ共4个属，其中α和β冠状病毒可以感染哺乳动物，而γ和δ冠状病毒主要感染鸟类。此前已经发现有6种冠状病毒可感染人类：α冠状病毒(HCoV-229E、HCoV-NL63)和β冠状病毒(HCoV-HKU1，HCoV-OC43、MERS-CoV、SARS-CoV)，患者表现从普通感冒到重症肺部感染。新型冠状病毒(严重急性呼吸综合征冠状病毒2，SARS-CoV-2)是一种先前尚未在人类中发现的β属冠状病毒，在人群中具有极强的传染性，主要通过呼吸道飞沫、呼吸道分泌物和直接接触传播。COVID-19患者通常表现出特定的相似症状，例如发烧、全身乏力和咳嗽。大多数患者都表现出轻度的流感样症状，预后良好。但少数患者病情危重，并迅速发展为急性呼吸窘迫综合征、呼吸衰竭、多器官衰竭甚至死亡。
新型冠状病毒具有极强的传染性、人群普遍易感、具有潜伏期，并且在潜伏期就具有传染性，因此需要及时地发现感染者，控制传染源，切断传播途径，才能有效地控制疫情的蔓延。而采取一切阻断措施的前提是需要有快速、准确、灵敏的诊断。
目前，国内外市面上有很多用于检测新型冠状病毒的试剂盒，其主要基于两种方法：免疫学方法和分子生物学方法。其中，基于免疫学的方法主要是采用胶体金方法、化学发光法和酶联免疫法，检测患者血清或血浆中的新型冠状病毒特异性IgM抗体、IgG抗体或抗原。但是，临床上一般不单独以血清学检测作为诊断依据。首先，新型冠状病毒IgM抗体、IgG抗体在患者发病一周内的阳性率均较低，因此不适合用于新型冠状病毒 感染的早期筛查。其次，抗体检测可能会出现假阳性：其一是试剂盒阳性判断值设置，一些处于阳性判断值附近的弱阳性结果，很可能是假阳性；其二，胶体金试纸条检测是通过肉眼观察颜色有否来判断阳性和阴性，存在个体判断的差异；其三，患者标本中存在导致免疫测定假阳性的内源性干扰物质，如类风湿因子、补体、嗜异性抗体、溶菌酶等；其四，标本采集、运送和保存等分析前过程中，标本溶血、标本贮存时间过长和标本凝固不全等也会对检测结果产生干扰。而基于分子生物学的方法主要是采用实时荧光PCR技术，特异性检测患者咽拭子、痰和其他下呼吸道分泌物、血液、粪便、尿液等标本中的新型冠状病毒核酸。临床上将核酸检测结果作为诊断的依据之一，但是由于试剂本身的灵敏度问题和病毒待测靶序列的变异问题，核酸检测也会出现假阴性的结果。
发明内容
本发明的用于检测新型冠状病毒的试剂盒采用一步法逆转录聚合酶链式反应(One step RT-PCR)结合Taqman探针法，进行新型冠状病毒ORF1ab和N基因的双靶标检测。本发明的靶向ORF1ab基因和N基因的引物及探针覆盖区域的突变率最低，可最大程度上减少由于病毒待测靶序列发生基因突变而导致的假阴性，具有准确性高、灵敏度高等优点。此外，还可引入外源性内标SUC2基因来质控从核酸提取到检测的整个过程，可以避免假阴性的结果。
引物及探针组
因此，在一方面，本发明提供了用于检测新型冠状病毒的引物及探针组，其包含序列如SEQ ID NO：1所示的引物I-F，序列如SEQ ID NO：2所示的引物I-R，序列如SEQ ID NO：3所示的探针I，序列如SEQ ID NO：4所示的引物II-F，序列如SEQ ID NO：5所示的引物II-R，序列如SEQ ID NO：6所示的探针II。
在某些实施方案中，所述探针I和探针II的5’端各自独立地连接有荧光基团，3’端各自独立地连接有荧光猝灭基团，并且，所述探针I连接的荧光基团与所述探针II连接的荧光基团彼此不同，所述探针I连接的荧光淬灭基团与所述探针II连接的荧光淬灭基团可以相同或不同。
在某些实施方案中，所述探针I的5’端连接有FAM荧光基团，所述探针II的5’端连接有ROX荧光基团。
在某些实施方案中，所述引物及探针组还包括：序列如SEQ ID NO：7所示的引物III-F，序列如SEQ ID NO：8所示的引物III-R，以及序列如SEQ ID NO：9所示的探针III。
在某些实施方案中，所述探针III的5’端连接有荧光基团，3’端连接有荧光猝灭基团；并且，所述探针III连接的荧光基团与所述探针I以及所述探针II连接的荧光基团各不相同。所述探针III连接的荧光淬灭基团、与所述探针I连接的荧光淬灭基团以及所述探针II连接的荧光淬灭基团可以相同、部分相同或各不相同。
在某些实施方案中，所述探针III的5’端连接有HEX荧光基团。
试剂盒
在另一方面，本发明提供了用于检测新型冠状病毒的试剂盒，其包括如上所述的引物及探针组。
检测方法
在另一方面，本发明提供了用于检测新型冠状病毒的方法，其包括如下步骤：
1)提供如上所述的引物及探针组，以及，待测样品；
2)将所述引物及探针组与待测样品混合，并进行RT-PCR反应；
3)结果判读。
在某些实施方案中，所述引物及探针组包含所述引物I-F、引物I-R、探针I、引物II-F、引物II-R、探针II、引物III-F、引物III-R、探针III。在此类实施方案中，所述待测样品含有内标核酸，所述内标核酸包含SUC2基因或其部分序列。在某些实施方案中，所述内标核酸能与所述引物III-F或其互补序列、所述引物III-R或其互补序列、以及所述探针III或其互补序列退火。
在某些实施方案中，所述内标核酸为外源添加至所述待测样品中。
在某些实施方案中，步骤2)包括将用于RT-PCR反应的试剂与待测样品混合，并进行RT-PCR反应；其中，所述用于RT-PCR反应的试剂包括：2-5μL的Solution 1、2-7μL的OSTR buffer、0.5-1.5μL混合酶、以及引物及探针组，并使用DEPC-H 2O补充至20μL；其中，所述引物及探针组中各组分的浓度为50μM，用量为0.05-3μL。
在某些实施方案中，所述RT-PCR的扩增程序如下：
48-52℃，13-17min，1-3个循环；93-97℃，2-5min，1-3个循环；93-97℃,13-17s，55-65℃，25-35s,40-50个循环，与此同时采集荧光信号。
在某些实施方案中，步骤1)所述的待测样品包括商品、环境中的样品。
在某些实施方案中，步骤1)中所述待测样品为通过核酸提取或纯化获得的核酸样品。
在某些实施方案中，步骤2)中，所述用于RT-PCR反应的试剂为液体试剂。在某些实施方案中，步骤2)中，所述待测样品体积为5μL，所述RT-PCR总反应体积为25μL。
在某些实施方案中，步骤2)中，所述用于RT-PCR反应的试剂为干试剂。在某些实施方案中，步骤2)中，所述待测样品体积为25μL，所述RT-PCR总反应体积为25μL。
在某些实施方案中，步骤2)还包括：将所述引物及探针组与阳性质控品和阴性质控品分别混合并进行RT-PCR反应。
在某些实施方案中，所述阳性质控品为混有内标核酸的新型冠状病毒ORF1ab基因和N基因的假病毒的样品，其中，所述内标核酸包含SUC2基因或其部分序列。
在某些实施方案中，所述阴性质控品为含有内标核酸的DEPC-H 2O，其中，所述内标核酸包含SUC2基因或其部分序列。
在某些实施方案中，在步骤2)中，在RT-PCR反应过程中采集荧光信号，所述的荧光信号包括探针I所连接的荧光基团对应的第一荧光，探针II所连接的荧光基团对应的第二荧光以及探针III所连接的荧光基团对应的第三荧光(例如，FAM、ROX、HEX荧光)。
在某些实施方案中，步骤3)中，若阴性质控品在第一荧光和第二荧光对应的检测通道无Ct值，同时，第三荧光对应的检测通道存在Ct值，则阴性质控合格；若阳性质控品在第一荧光对应检测通道的Ct≤34，在第二荧光对应检测通道的Ct≤31，且在第三荧光对应的检测通道的Ct值≤32，则阳性质控合格。
在某些实施方案中，所述的无Ct值是指没有扩增/荧光曲线或Ct≥40。
在某些实施方案中，所述结果判读方式如表4及表5所示。
在某些实施方案中，所述方法用于确定待测样品中是否存在新型冠状病毒。
在某些实施方案中，所述方法用于疾病诊断或非疾病诊断目的。在某些实施方案中，所述方法用于非疾病诊断目的。
试剂制备用途
在另一方面，本发明提供了如上所述的引物及探针组或试剂盒在制备用于检测新型冠状病毒的检测试剂中的用途。
有益效果
本发明开发了一种能够克服现有试剂盒局限性的用于检测新型冠状病毒的试剂盒。本发明使用一步法逆转录聚合酶链式反应(One step RT-PCR)结合Taqman探针法，定性检测SARS-CoV-2的ORF1ab基因和N基因特异性区域，通过荧光信号的变化来实现样本RNA的检测。同时引入外源性内标SUC2基因来质控从核酸提取到检测的整个过程，可以避免假阴性的结果。
本技术方案与现有技术相比，具有如下优点：
(1)本发明采用新型冠状病毒ORF1ab和N基因双靶标检测，使检测结果更加准确可靠，可以避免由于其中一个待测靶标发生突变而导致假阴性的结果。并且，本发明还可使用外源性的内标SUC2基因作为本发明试剂盒的质控系统，监测从样本RNA提取到核酸检测的全过程，可最大程度避免由于实验设置或操作不当、试剂异常或设备故障等原因导致检测结果不准确。
(2)本发明所使用的引物探针均为自行设计，通过大量新型冠状病毒基因组序列的比对，设计了多条引物和探针。与同类试剂盒比较，本发明试剂盒的靶向ORF1ab基因的引物探针覆盖区域的突变率最低，靶向N基因的引物探针覆盖区域的突变率也为目前靶向N基因反应中最低。因此，本发明的新型冠状病毒核酸检测试剂盒可以最大程度上减少由于病毒待测靶序列发生基因突变而导致的假阴性。
(3)相比于中国CDC推荐的检测体系，本发明试剂盒具有针对ORF1ab基因和N基因更高的检测灵敏度。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人 员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
附图说明
图1为不同SARS-CoV-2检测试剂盒的引物、探针靶区域的突变情况。
图2为本发明实施例与中国CDC推荐的检测体系的比较。
图3为本发明实施例的典型结果图。
图4为本发明实施例的样本检测结果图。
具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本申请所要求保护的范围。实施例中的实验方法，如无特殊说明，均为常规方法。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
1.本发明引物及探针
参照中国CDC新型冠状病毒核酸检测的引物探针设计区域，自行设计新型冠状病毒ORF1ab和N基因、外源性内标SUC2基因的扩增引物和检测探针(如表1所示)。其中ORF1ab基因的检测探针5′端标记FAM荧光基团，3′端标记淬灭基团；N基因的检测探针5′端标记ROX荧光基团，3′端标记淬灭基团；SUC2基因检测探针5′端标记HEX荧光基团，3′端标记淬灭基团。
表1.引物、探针序列
Figure PCTCN2021125665-appb-000001
Figure PCTCN2021125665-appb-000002
2.样本处理
采集疑似COVID-19患者的鼻咽拭子、口咽拭子、前鼻和中鼻拭子、鼻咽冲洗/鼻吸取物或支气管肺泡灌洗(BAL)样本。使用厦门致善生物科技股份有限公司生产的手工提取试剂盒“病毒RNA提取试剂盒(货号：602101)”进行核酸提取及纯化。提取试剂盒的提取纯化过程包括4个步骤：裂解、结合、洗涤和洗脱。提取时，分别取20μL内对照和1mL待测样本加入提取加样孔，然后按照提取试剂盒说明书中的操作方法进行提取。
为了对提取及检测过程进行质量控制，取20μL内对照与20μL的新型冠状病毒阳性对照、20μL内对照与20μL阴性对照分别加入另外的提取加样孔，与待测样本同步进行提取。每个样本中加入内对照同步进行提取，目的是为了质控样本的提取过程。提取试剂盒中核酸洗脱体积为60μL，提取后获得的RNA应立即使用或储存在-70℃保存。
注：内对照为SUC2假病毒、阴性对照为不含核酸的溶液、新型冠状病毒阳性对照为新型冠状病毒ORF1ab、N基因的假病毒。
3.本发明三重RT-PCR反应体系组分
本发明RT-PCR反应成分及用量如下表2所示。
表2.SARS-CoV-2RT-PCR组分及用量
PCR反应液成分 用量(μL)
DEPC-H 2O 10.1
Solution 1 2.5
OSTR buffer 5
ORF1ab-F(50μM) 0.3
ORF1ab-R(50μM) 0.3
ORF1ab-P(50μM) 0.05
N-F(50μM) 0.1
N-R(50μM) 0.1
N-P(50μM) 0.05
SUC2-F(50μM) 0.2
SUC2-R(50μM) 0.2
SUC2-P(50μM) 0.1
Enzyme mix 1
总量 20
注：1)若为液体试剂，则向反应管中分别加入处理后的样品5μL，阳性质控品5μL，阴性质控品5μL；若为干试剂，则向反应管中分别加入处理后的样品25μL，阳性质控品25μL，阴性质控品25μL，总反应体积为25μL。
2)Solution 1来源于菲鹏生物股份有限公司，是产品AnstartOne-Step RT-PCR Mix中的一种组成成分，产品编号MD013。
3)Enzyme mix来源于菲鹏生物股份有限公司，是产品AnstartOne-Step RT-PCR Mix中的一种组成成分，产品编号MD013，主要成分为超级反转录酶(Super MMLV Reverse Transcriptase)和热启动酶(AnstartTaq DNA Polymerase)。
4.本发明三重RT-PCR反应程序
经过大量实验的对比优化，最终确定的RT-PCR扩增反应条件见表3。
表3.RT-PCR程序设置
Figure PCTCN2021125665-appb-000003
Figure PCTCN2021125665-appb-000004
5.检测结果解释
若阴性对照在ORF1ab和N基因对应的检测通道无Ct值(没有扩增/荧光曲线或Ct≥40)，同时，内控通道(SUC2)存在Ct值(Ct≤32)，则阴性对照质控合格。阴性对照由于在提取前加入了SUC2(内对照)，因此检测结果中SUC2为阳性。
若阳性对照在ORF1ab和N基因对应的检测通道均有Ct值(分别为Ct≤34和Ct≤31)，且内控SUC2的Ct值≤32，则阳性对照质控合格。
临床标本检验结果的评估应在阳性和阴性对照已检测并确定有效后进行。如果对照品无效，则无法解释病人的结果。当上述质量控制符合要求时，可根据下表4对样本的ORF1ab、N和SUC2基因的Ct值进行评价。
表4.阳性参考值范围
Figure PCTCN2021125665-appb-000005
注：“+”指阳性；“-”指阴性。
表5.待测样品的检测结果可能情况
Figure PCTCN2021125665-appb-000006
注：“+”指阳性；“-”指阴性。
*由于PCR中存在竞争关系，SARS-CoV-2阳性结果(ORF1ab+或N+)不要求HEX检测通道必须为检出，病毒载量较高可引起SUC2信号无法检出或受到抑制的情况。
图1为不同SARS-CoV-2检测试剂盒的引物、探针靶区域的突变情况。图1的结果表明，与同类试剂盒比较，本发明试剂盒的靶向ORF1ab基因的引物探针覆盖区域的突变率(图中箭头所示，“ZS-Orf1ab”)最低，靶向N基因的引物探针覆盖区域的突变率(图中箭头所示，“ZS-N”)也为目前靶向N基因反应中最低。
图2为本发明实施例与中国CDC推荐的检测体系的比较。图2的结果表明，本发明具有针对ORF1ab基因和N基因更高的检测灵敏度。
图3为本发明实施例的典型结果图。图3中显示，对于SARS-COV-2的N基因、ORF1ab基因和内对照SUC2基因，在相对应的荧光通道均有特征扩增曲线，且无任何交叉反应。
图4为本发明实施例的样本检测结果图。图4显示了的阳性对照和阴性对照质控合格的检测结果以及待测样本的检测结果。在阳性对照、阴性对照均质控合格的情况下，才对待测样本的结果进行解释，待测样本的结果解释参照表4、表5。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。

Claims (17)

  1. 用于检测新型冠状病毒的引物及探针组，其包含序列如SEQ ID NO：1所示的引物I-F，序列如SEQ ID NO：2所示的引物I-R，序列如SEQ ID NO：3所示的探针I，序列如SEQ ID NO：4所示的引物II-F，序列如SEQ ID NO：5所示的引物II-R，序列如SEQ ID NO：6所示的探针II。
  2. 权利要求1的引物及探针组，其中，所述探针I和探针II的5’端各自独立地连接有荧光基团，3’端各自独立地连接有荧光猝灭基团，并且，所述探针I与所述探针II连接的荧光基团彼此不同；
    优选地，所述探针I的5’端连接有FAM荧光基团，所述探针II的5’端连接有ROX荧光基团。
  3. 权利要求1或2的引物及探针组，其还包含：序列如SEQ ID NO：7所示的引物III-F，序列如SEQ ID NO：8所示的引物III-R，以及序列如SEQ ID NO：9所示的探针III。
  4. 权利要求3的引物及探针组，其中，所述探针III的5’端连接有荧光基团，3’端连接有荧光猝灭基团；并且，所述探针III连接的荧光基团与所述探针I以及所述探针II连接的荧光基团各不相同；
    优选地，所述探针III的5’端连接有HEX荧光基团。
  5. 用于检测新型冠状病毒的试剂盒，其包括权利要求1-4任一项所述的引物及探针组。
  6. 用于检测新型冠状病毒的方法，其包括如下步骤：
    1)提供权利要求1-4任一项所述的引物及探针组，以及，待测样品；
    2)将所述引物及探针组与待测样品混合，并进行RT-PCR反应；
    3)结果判读。
  7. 权利要求6的方法，其中，步骤1)中所述引物及探针组如权利要求3或4所示，所述待测样品含有内标核酸，所述内标核酸包含SUC2基因或其部分序列。
  8. 权利要求7的方法，其中，所述内标核酸为外源添加至所述待测样品中。
  9. 权利要求6-8任一项所述的方法，其中，步骤2)包括将用于RT-PCR反应的试剂与待测样品混合，并进行RT-PCR反应；其中，所述用于RT-PCR反应的试剂包括：2-5μL的Solution 1、2-7μL的OSTR buffer、0.5-1.5μL混合酶、以及引物及探针组，并使用DEPC-H 2O补充至20μL；其中，所述引物及探针组中各组分的浓度为50μM，用量为0.05-3μL。
  10. 权利要求6-9任一项所述的方法，其中，所述RT-PCR的扩增程序如下：
    48-52℃，13-17min，1-3个循环；93-97℃，2-5min，1-3个循环；93-97℃,13-17s，55-65℃，25-35s,40-50个循环，与此同时采集荧光信号。
  11. 权利要求6-10任一项的方法，其中，步骤1)所述的待测样品包括商品、环境中的样品。
  12. 权利要求6-11任一项的方法，其中，步骤1)中所述待测样品为通过核酸提取或纯化获得的核酸样品。
  13. 权利要求6-12任一项的方法，其中，步骤2)还包括：将所述引物及探针组与阳性质控品和阴性质控品分别混合并进行RT-PCR反应；
    优选地，所述阳性质控品为混有内标核酸的新型冠状病毒ORF1ab基因和N基因的假病毒的样品，其中，所述内标核酸包含SUC2基因或其部分序列；
    优选地，所述阴性质控品为含有内标核酸的DEPC-H 2O，其中，所述内标核酸包含SUC2基因或其部分序列。
  14. 根据权利要求6-13任一项所述的方法，其中，在RT-PCR反应过程中采集荧光信号，所述荧光信号包括探针I所连接的荧光基团对应的第一荧光，探针II所连接的荧光基团对应的第二荧光以及探针III所连接的荧光基团对应的第三荧光。
  15. 权利要求14所述的方法，其中，步骤3)中，若阴性质控品在第一荧光和第二荧光对应的检测通道无Ct值，同时，第三荧光对应的检测通道存在Ct值，则阴性质控合格；若阳性质控品在第一荧光对应检测通道的Ct≤34，在第二荧光对应检测通道的Ct≤31，且在第三荧光对应的检测通道的Ct值≤32，则阳性质控合格。
  16. 权利要求15所述的方法，其中，所述的无Ct值是指没有扩增/荧光曲线或Ct≥40。
  17. 权利要求1-4任一项的引物及探针组或权利要求5的试剂盒在制备用于检测新型冠状病毒的检测试剂中的用途。
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