WO2022095723A1 - 用于检测SARS-CoV-2的试剂盒及方法 - Google Patents

用于检测SARS-CoV-2的试剂盒及方法 Download PDF

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WO2022095723A1
WO2022095723A1 PCT/CN2021/125587 CN2021125587W WO2022095723A1 WO 2022095723 A1 WO2022095723 A1 WO 2022095723A1 CN 2021125587 W CN2021125587 W CN 2021125587W WO 2022095723 A1 WO2022095723 A1 WO 2022095723A1
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probe
primer
fluorescence
cov
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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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • 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
    • C12Q1/6858—Allele-specific amplification
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
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  • the present invention relates to kits and methods for detecting SARS-CoV-2.
  • Novel coronavirus (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) is a coronavirus newly discovered in humans in 2019, which is closely related to severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome MERS-CoV belongs to the genus beta coronavirus.
  • SARS-CoV-2 can cause a respiratory infection in humans, causing deadly inflammation of the lungs known as novel coronavirus pneumonia (COVID-19).
  • COVID-19 is an emerging acute respiratory infectious disease that is highly contagious in the population, mainly transmitted through respiratory droplets, respiratory secretions and direct contact.
  • the new crown pneumonia epidemic has now become a major global public health event. As of August 31, 2020, more than 25 million people worldwide have been infected with the new coronavirus, and the death toll has exceeded 840,000. Moreover, the number of infections and deaths continues to increase.
  • SARS-CoV-2 is a positive-strand RNA virus with 5 essential genes encoding 4 structural proteins: nucleoprotein (N), viral envelope (E), matrix protein (M) and spike protein (S), and RNA-dependent RNA polymerase (RdRp).
  • the nucleoprotein (N) wraps the RNA genome to form the nucleocapsid, which is surrounded by the viral envelope (E), which is embedded with proteins such as matrix protein (M) and spike protein (S).
  • E viral envelope
  • S RNA-dependent RNA polymerase
  • the name of the coronavirus comes from the crown protrusion composed of the spike protein (spike protein, S protein) exposed outside the virus lipid layer.
  • the S protein is used to bind to the specific receptor on the surface of the target cell and enter the cell to replicate. Infect.
  • SARS-CoV-2 uses the S protein to bind to the human receptor angiotensin converting enzyme 2 (ACE2) to invade host cells.
  • RNA viruses are highly susceptible to mutations, which may be related to their transmission and virulence.
  • SARS-CoV-2 has evolved into several different typical branches according to the mutation of its genome, and the most important mutation is the S protein D614G mutation.
  • the D614G mutation refers to the mutation of the 614th amino acid position D (aspartic acid) to G (glycine) of the S protein of the new coronavirus.
  • the change in the viral genome is the mutation of the base A at position 23403 to the base G (in the MN908947.3 genomic location, 23403A->G).
  • D614G mutant strains are usually accompanied by 214C->T, 3037C->T and 14408C->T mutations.
  • the D614G mutant strain first appeared in Europe and has now become the main circulating strain worldwide. According to the genome sequence of the new coronavirus published in the GISAID database, it was found that the virus strains carrying the mutation were mainly G, GR and GH types.
  • the new coronavirus S protein D614G mutation increases the number of spike proteins by 4-5 times and makes these proteins more stable, which in turn makes it easier for the virus to invade human cells.
  • the ability of the D614G mutation to infect human cells is increased by 9-10 times, and the sensitivity to individual convalescent serum can be reduced, but the impact of D614G mutation on the infectivity of the new coronavirus in the real environment is still to be determined Research.
  • the spike protein that assists SARS-CoV-2 entry into host cells is one of the main targets of vaccines and therapeutics, therefore, continuous monitoring of the D614G variant is key to understanding the infectivity and antigenicity of SARS-CoV-2.
  • the present invention develops a kit and method for detecting novel coronavirus based on nucleic acid variation analysis technology.
  • the kit and method of the present invention adopts asymmetric PCR and multi-color probe melting curve analysis technology, and can be used for novel coronaviruses (including S-D614/G614 strains and non-coronavirus) through fluorescence channel and two-dimensional label of melting point (Tm).
  • novel coronaviruses including S-D614/G614 strains and non-coronavirus
  • Tm two-dimensional label of melting point
  • the detection of S-D614/G614 strain) and the typing detection of S-D614 and S-G614 strains have the advantages of simple operation, short detection period and high sensitivity.
  • the present invention provides primers and probe sets for detecting SARS-CoV-2, which comprise a first primer whose sequence is shown in SEQ ID NO: 1, and whose sequence is shown in SEQ ID NO: 2
  • the 5' ends of the first probe and the second probe are each independently linked with a fluorescent group, and the 3' ends are each independently linked with a fluorescent quenching group, and the first probe
  • the fluorophore attached to one probe is different from the fluorophore attached to the second probe, and the fluorescence quencher attached to the first probe is the same as the fluorescence quencher attached to the second probe or different.
  • a ROX fluorophore is attached to the 5' end of the first probe, and a FAM fluorophore is attached to the 5' end of the second probe.
  • the first probe contains at least one locked nucleic acid modification (LNA modification).
  • LNA modification locked nucleic acid modification
  • the first probe is set forth in SEQ ID NO:5.
  • the second probe contains at least one locked nucleic acid modification (LNA modification).
  • LNA modification locked nucleic acid modification
  • the second probe is set forth in SEQ ID NO:6.
  • the present invention also provides a kit for detecting SARS-CoV-2, comprising the primers and probe sets as described above.
  • the present invention also provides a method for detecting SARS-CoV-2, comprising the steps of:
  • step 2) includes mixing the reagent for RT-PCR reaction with the sample to be tested, and performing RT-PCR reaction; wherein, the components and amount included in the reagent for RT-PCR reaction as follows:
  • the amplification and dissolution procedures of the RT-PCR reaction described in step 2) are as follows:
  • the samples to be tested include commercial products and samples in the environment.
  • the sample to be tested is a nucleic acid sample obtained by nucleic acid extraction or purification.
  • step 2) of the method the volume of the sample to be tested is 5 ⁇ 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 controls are D614 and G614 pseudoviruses in a volume of 5 ⁇ L.
  • the negative control is nucleic acid-free DEPC-H 2 O in a volume of 5 ⁇ L.
  • 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 first probe is attached and the second probe The second fluorescence corresponding to the fluorophore to which the needle is attached (eg, FAM, ROX fluorescence).
  • step 3 of the method if the negative quality control substance has no Tm value in both the first fluorescence and the second fluorescence channel, the negative quality control is qualified; if the positive quality control substance is in the first fluorescence and the second fluorescence channel Both fluorescence channels have melting curves, and the corresponding Tm value falls within the range of the determined reference value, the positive quality control is qualified.
  • the primer and probe set comprises a first primer set forth in SEQ ID NO:1, a second primer set forth in SEQ ID NO:2, and a second primer set forth in SEQ ID NO:5
  • the first probe shown and the second probe shown in SEQ ID NO: 6; the result judgment mode of the method is shown in Table 4-5.
  • the method is used to determine the presence or absence of a D614 strain, a G614 strain, and/or a non-D614/G614 strain in a test sample.
  • non-D614/G614 strain refers to the amino acid residue of the S protein at the position corresponding to position 614 of the wild-type S protein amino acid sequence (eg, the sequence shown in GenBank: QHD43416.1). SARS-CoV-2 strains that are not aspartic acid residues or glycine residues.
  • the expression "at the position corresponding to position 614 in the amino acid sequence of the wild-type S protein” means that when the target sequence is aligned with the amino acid sequence of the wild-type S protein to generate maximum identity, the target sequence is identical to the amino acid sequence of the wild-type S protein.
  • the 614th bit is in the equivalent position.
  • non-D614/G614 strain may be one non-D614/G614 strain, or may be multiple non-D614/G614 strains.
  • 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 also provides the use of the above-mentioned primers and probe sets or kits in the preparation of detection reagents for detecting SARS-CoV-2.
  • the detection reagent is used to determine the presence or absence of a D614 strain, a G614 strain, and/or a non-D614/G614 strain in a sample to be tested.
  • the present invention develops a kit and method for detecting novel coronavirus based on nucleic acid variation analysis technology.
  • the kit and method of the present invention adopts asymmetric PCR and multi-color probe melting curve analysis technology, and can be used for novel coronaviruses (including S-D614/G614 strains and non-coronavirus) through fluorescence channel and two-dimensional label of melting point (Tm).
  • novel coronaviruses including S-D614/G614 strains and non-coronavirus
  • Tm two-dimensional label of melting point
  • the detection of S-D614/G614 strains) and the typing detection of S-D614 and S-G614 strains have the advantages of simple operation, short detection period and high sensitivity:
  • the kit developed by the present invention for detecting novel coronavirus does not require PCR post-processing, and the operation is simple and fast.
  • the virus detection results and virus typing results can be obtained within 3 hours, and no special equipment is required, and it is suitable for real-time analysis. type work.
  • the primers and probes used in the present invention are all designed by themselves. Through the alignment of a large number of new coronavirus genome sequences, the conserved region covering the D614G mutation (23403A->G) on the S gene is selected for primer design. And by aligning the new coronavirus reference gene sequence (GenBank: MN908947.3) with the SARS-CoV (GenBank: NC_004718.3) and bat-SL-CoVZC45 (GenBank: MG772933.1) gene sequences released by the NCBI database, in the novel Amplification primers are designed for the conserved and specific region of coronavirus.
  • the kit of the present invention can not only be used for the typing detection of the new coronavirus S-D614 and S-G614 strains, but also can be directly used for the detection of other non-S-D614/G614 new coronavirus strains.
  • FIG. 1 is a typical result diagram of an embodiment of the present invention.
  • the present invention selects the conserved region covering the D614G mutation (23403A->G) on the S gene of the novel coronavirus to design primers and probes.
  • a detection probe was designed for D614 and G614 respectively.
  • the D614 detection probe was labeled with a ROX fluorophore at the 5' end and a quencher at the 3' end;
  • the G614 detection probe was labeled with a FAM fluorophore at the 5' end and the 3' end. Label the quencher group.
  • nasopharyngeal swabs Collect nasopharyngeal swabs, oropharyngeal swabs, anterior and middle nasal swabs, nasopharyngeal rinses/nasal aspirate, or bronchoalveolar lavage (BAL) samples from patients with suspected COVID-19.
  • 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.
  • RNA obtained after extraction should be used immediately or stored at -70°C.
  • PCR reaction solution components Dosage ( ⁇ L) DEPC-H 2 O 5.875
  • One step RT-PCR buffer 12.5 S-F (50 ⁇ M) (SEQ ID NO: 1) 0.025 S-R (50 ⁇ M) (SEQ ID NO: 2) 0.2 S-D614-P (50 ⁇ M) (SEQ ID NO:5) 0.2 S-G614-P (50 ⁇ M) (SEQ ID NO:6) 0.2 Enzyme mix 1 total 20
  • Enzyme mix comes from Shanghai Yisheng Biotechnology Co., Ltd., product number 13110ES60, and the main components are reverse transcriptase and hot-start DNA polymerase.
  • each type of strain will produce melting curves of FAM fluorescence and ROX fluorescence, that is, two corresponding Melting point Tm value.
  • the interpretation of the detection results is carried out by the combination of the fluorescence channel and the melting point Tm value.
  • the quality control of the negative control is qualified.
  • the quality control of the positive control is qualified.
  • test results should be performed after positive and negative controls have been tested and determined to be valid. If the control is not valid, the results for the test sample cannot be interpreted. When the above quality control meets the requirements, the test results of the samples can be evaluated according to Tables 4 and 5 below.
  • the probe with weak binding force may have lower sensitivity, so there may be only one melting peak for low concentrations.
  • Fig. 1 is a typical result diagram of an embodiment of the present invention, as shown in Fig. 1: Through the combined interpretation of fluorescence and melting point, the present invention can not only accurately identify the D614G mutation, but also identify the non-D614G mutation in the coverage area of the detection probe.

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Abstract

本发明公开了用于检测SARS-CoV-2的试剂盒及方法。本发明的用于检测SARS-CoV-2的试剂盒采用不对称PCR扩增，结合多色探针熔解曲线分析技术，可实现对SARS-CoV-2的检测以及S-D614和S-G614毒株的分型检测。

Description

用于检测SARS-CoV-2的试剂盒及方法 技术领域
本发明涉及用于检测SARS-CoV-2的试剂盒及方法。
背景技术
新型冠状病毒(严重急性呼吸综合征冠状病毒2，SARS-CoV-2)是2019年在人类中新发现的一种冠状病毒，与严重急性呼吸综合征冠状病毒(SARS-CoV)和中东呼吸综合征冠状病毒(MERS-CoV)同属于β属冠状病毒。SARS-CoV-2可引起人类呼吸道感染，造成致命的肺部炎症，称为新型冠状病毒肺炎(COVID-19)。COVID-19是一种新发的急性呼吸道传染病，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。新冠肺炎疫情目前已成为全球性重大公共卫生事件，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。
SARS-CoV-2属于正链RNA病毒，具有5个必需基因，分别编码4种结构蛋白：核蛋白(N)、病毒包膜(E)、基质蛋白(M)和刺突蛋白(S)，及RNA依赖性的RNA聚合酶(RdRp)。核蛋白(N)包裹RNA基因组构成核衣壳，外面围绕着病毒包膜(E)，病毒包膜包埋有基质蛋白(M)和刺突蛋白(S)等蛋白。冠状病毒的得名来自暴露于病毒脂质层外面的由棘突蛋白(spike protein，S蛋白)构成的冠状突起，利用S蛋白与靶细胞表面的特异性受体结合，进入细胞内复制而引起感染。经研究发现，SARS-CoV-2利用S蛋白与人类受体血管紧张素转换酶2(angiotensin converting enzyme 2，ACE2)结合进而侵入宿主细胞。RNA病毒极易发生突变，这可能与其传播和毒性相关。目前，SARS-CoV-2根据其基因组的突变情况已经进化出多个不同的典型分支，其中最重要的突变是S蛋白D614G突变。D614G突变指的是新型冠状病毒S蛋白第614氨基酸位点D(天冬氨酸)到G(甘氨酸)的突变，在病毒基因组上的改变是23403位的碱基A突变为碱基G(在MN908947.3基因组的位置，23403A->G)。D614G突变的毒株通常伴有214C->T、3037C->T和14408C->T变异。D614G变异毒株最早出现在欧洲，目前已成为世界范围内的主要流行毒株。根据GISAID数据库公布新冠病毒基因组序列，发现携带该突变的 病毒株主要是G型、GR型和GH型。
综合多项研究来看，新型冠状病毒S蛋白D614G突变使棘状蛋白数量增加4-5倍，并使这些蛋白更稳定，进而使病毒更容易侵入人体细胞。在实验室环境下，D614G突变的新冠病毒感染人细胞的能力提高9-10倍，并且能够降低对个体恢复期血清的敏感性，但是D614G突变在现实环境下对新冠病毒感染力的影响仍待研究。协助SARS-CoV-2进入宿主细胞的刺突蛋白是疫苗和治疗剂的主要靶标之一，因此，持续监测D614G变异是了解SARS-CoV-2感染性和抗原性的关键。
目前，D614G突变的监测依赖于全基因组测序技术，尚无关于D614G突变检测的分子诊断试剂。全基因组测序不仅耗时长、价格高，还需要专门的仪器设备和专业的生物信息分析人员，不利于大范围开展工作，特别是对于一些基层单位。
发明内容
本发明开发了一种基于核酸变异分析技术的用于检测新型冠状病毒的试剂盒及方法。本发明的试剂盒和方法采用不对称PCR和多色探针熔解曲线分析技术，通过荧光通道和熔点(Tm)的二维标签，可用于新型冠状病毒(包括S-D614/G614毒株以及非S-D614/G614毒株)的检测以及S-D614和S-G614毒株的分型检测，具有操作简单、检测周期短、灵敏度高等优点。
引物及探针组
因此，在一方面，本发明提供了用于检测SARS-CoV-2的引物及探针组，其包含序列如SEQ ID NO：1所示的第一引物，序列如SEQ ID NO：2所示的第二引物，序列如SEQ ID NO：3所示的第一探针和序列如SEQ ID NO：4所示的第二探针。
在某些实施方案中，所述第一探针和第二探针的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，所述第一探针连接的荧光基团与所述第二探针连接的荧光基团不同，所述第一探针连接的荧光淬灭基团与所述第二探针连接的荧光淬灭基团相同或不同。
在某些实施方案中，所述第一探针的5’端连接有ROX荧光基团，所述第二探针的5’端连接有FAM荧光基团。
在某些实施方案中，所述第一探针含有至少一个锁核酸修饰(LNA修饰)。
在某些实施方案中，所述第一探针如SEQ ID NO:5所示。
在某些实施方案中，所述第二探针含有至少一个锁核酸修饰(LNA修饰)。
在某些实施方案中，所述第二探针如SEQ ID NO:6所示。
试剂盒
在另一方面，本发明还提供了用于检测SARS-CoV-2的试剂盒，其包含如上所述的引物及探针组。
检测方法
在另一方面，本发明还提供了用于检测SARS-CoV-2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，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反应的试剂包含的成分及用量如下：
Figure PCTCN2021125587-appb-000001
在某些实施方案中，步骤2)中所述RT-PCR反应的扩增和溶解程序如下：
48-52℃，13-17min，1-3个循环；93-97℃，4-6min，1-3个循环；93-97℃,13-17s，53-57℃，13-17s,74-78℃，18-22s,45-55个循环；93-97℃，0.5-2min，1-3个循环；35-40℃，2-4min，1-3个循环；以0.02-0.06℃/s的升温速率从35-40℃升温至80-90℃，与此同时 采集荧光信号。
在某些实施方案中，所述方法步骤1)中，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，所述方法步骤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，所述方法步骤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)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D614和G614假病毒，体积为5μL。
在某些实施方案中，阴性质控品为无核酸DEPC-H 2O，体积为5μL。
在某些实施方案中，所述方法步骤2)中，在RT-PCR反应过程中采集荧光信号，所述荧光信号包括第一探针所连接的荧光基团对应的第一荧光以及第二探针所连接的荧光基团对应的第二荧光(例如，FAM、ROX荧光)。
在某些实施方案中，所述方法步骤3)中，若阴性质控品在第一荧光和第二荧光通道均无Tm值则阴性质控合格；若阳性质控品在第一荧光和第二荧光通道均有熔解曲线，且对应的Tm值落在已确定的参考值范围，则阳性质控合格。
在某些示例性实施方案中，所述引物及探针组包含如SEQ ID NO：1所示的第一引物，如SEQ ID NO：2所示的第二引物，如SEQ ID NO：5所示的第一探针和如SEQ ID NO：6所示的第二探针；所述方法的结果判定方式如表4-5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D614毒株、G614毒株和/或非D614/G614毒株。
如本文所使用的，术语“非D614/G614毒株”是指，S蛋白的与野生型S蛋白氨基酸序列(例如，GenBank:QHD43416.1所示的序列)第614位对应位置处的氨基酸残基不是天冬氨酸残基或甘氨酸残基的SARS-CoV-2病毒株。
表述“与野生型S蛋白氨基酸序列第614位对应位置处”是指，当将目标序列与野生型S蛋白氨基酸序列进行比对以产生最大同一性时，目标序列中与野生型S蛋白氨基 酸序列的第614位处于等同位置的位置。
易于理解，所述非D614/G614毒株可以是一种非D614/G614毒株，也可以是多种非D614/G614毒株。
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。
制备试剂用途
在另一方面，本发明还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SARS-CoV-2的检测试剂的用途。在某些实施方案中，所述检测试剂用于确定待测样品中是否存在D614毒株、G614毒株和/或非D614/G614毒株。
有益效果
本发明开发了一种基于核酸变异分析技术的用于检测新型冠状病毒的试剂盒及方法。本发明的试剂盒及方法采用不对称PCR和多色探针熔解曲线分析技术，通过荧光通道和熔点(Tm)的二维标签，可用于新型冠状病毒(包括S-D614/G614毒株以及非S-D614/G614毒株)的检测以及S-D614和S-G614毒株的分型检测，具有操作简单、检测周期短、灵敏度高等优点：
(1)本发明开发的用于检测新型冠状病毒的试剂盒无需PCR后处理，操作简便快速，3个小时内即可得到病毒检测结果和病毒分型结果，且无需特殊设备，适用于实时分型工作。
(2)本发明所使用的引物探针均为自行设计，通过大量新冠病毒基因组序列的比对，选择S基因上覆盖D614G突变(23403A->G)的保守区域进行引物设计。并且通过将新冠病毒参考基因序列(GenBank：MN908947.3)与NCBI数据库发布的SARS-CoV(GenBank：NC_004718.3)、bat-SL-CoVZC45(GenBank：MG772933.1)基因序列比对，在新型冠状病毒保守且特异的区域设计扩增引物。因此，本发明试剂盒不仅可用于新型冠状病毒S-D614和S-G614毒株的分型检测，还可直接用于其他非S-D614/G614新型冠状病毒毒株的检测。
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为本发明实施例的典型结果图。
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
本发明选取新型冠状病毒S基因上覆盖D614G突变(23403A->G)的保守区域进行引物、探针设计。针对D614和G614分别设计一条检测探针，其中D614检测探针5′端标记ROX荧光基团，3′端标记淬灭基团；G614检测探针5′端标记FAM荧光基团，3′端标记淬灭基团。经过引物探针组合大量筛选，最终确定的引物、探针序列如下表所示：
表1.SARS-CoV-2D614G突变检测试剂盒引物、探针序列
Figure PCTCN2021125587-appb-000002
注：“+”表示其3’相邻位置的核苷酸存在LNA修饰。
2.样品处理
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)样品。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，取1mL待测样品加入提取加样孔，然后按照提取试剂盒说明书中的操作方法进行提取。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。
3.本发明RT-PCR反应体系组分
针对RT-PCR反应中的上下游引物比例、引物用量、探针用量等进行了大量优化，最终确定的本发明的RT-PCR反应成分及用量如下表2所示。
表2.SARS-CoV-2D614G突变检测RT-PCR组分及用量
PCR反应液成分 用量(μL)
DEPC-H 2O 5.875
One step RT-PCR buffer 12.5
S-F(50μM)(SEQ ID NO:1) 0.025
S-R(50μM)(SEQ ID NO:2) 0.2
S-D614-P(50μM)(SEQ ID NO:5) 0.2
S-G614-P(50μM)(SEQ ID NO:6) 0.2
Enzyme mix 1
总量 20
注：1)向反应管中分别加入处理后的样品5μL，阳性质控品(D614和G614假病毒)5μL，阴性质控品(无核酸DEPC-H 2O)5μL；总反应体积为25μL。
2)Enzyme mix来源于上海翊圣生物科技有限公司，产品编号13110ES60，主要成 分为反转录酶和热启动DNA聚合酶。
4.本发明实时RT-PCR扩增和熔解分析反应程序
经过大量实验的对比优化，最终确定的反应条件见表3。
表3.RT-PCR扩增和熔解程序设置
Figure PCTCN2021125587-appb-000003
5.检测结果解释
由于D614和G614毒株的基因组在探针覆盖区域只有一个碱基的差异，因此通过熔解曲线分析，每种类型的毒株都会产生FAM荧光和ROX荧光的熔解曲线，即会产生两个对应的熔点Tm值。通过荧光通道和熔点Tm值的组合，来进行检测结果的判读。
若阴性对照在FAM和ROX荧光通道均无Tm值(没有熔解曲线)则阴性对照质控合格。
若阳性对照在FAM和ROX荧光通道均有熔解曲线，且对应的Tm值落在已确定的参考值范围(参照下表4)，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，则无法解释待测样品的结果。当上述质量控制符合要求时，可根据下表4、表5对样品的检测结果进行评价。
表4.阳性参考值范围
Figure PCTCN2021125587-appb-000004
表5.待测样品的检测结果可能情况
Figure PCTCN2021125587-appb-000005
Figure PCTCN2021125587-appb-000006
注：(1)*由于探针存在竞争关系，结合力弱的那条探针灵敏度可能较低，因此对于低浓度可能出现只有一个熔解峰的情况。
(2)“+”指熔解曲线分析产生的熔点落在该范围内；“-”指熔解曲线分析产生的熔点不落在该范围内。
(3)其他突变类型指在探针覆盖区域出现的非D614G单碱基突变。
(4)对于一种毒株，若在探针覆盖区域同时出现D614G突变和其他突变，该方法不能识别出这种共突变的情况，将其统一判断为其他突变类型。经序列分析，这种共突变的频率很低，因此不会影响该方法的准确性。
图1为本发明实施例的典型结果图，如图1所示：通过荧光-熔点的组合判读，本发明不仅能准确识别出D614G突变，还可识别出检测探针覆盖区域的非D614G突变。
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 (18)

  1. 用于检测SARS-CoV-2的引物及探针组，其包含序列如SEQ ID NO：1所示的第一引物，序列如SEQ ID NO：2所示的第二引物，序列如SEQ ID NO：3所示的第一探针和序列如SEQ ID NO：4所示的第二探针。
  2. 权利要求1的引物及探针组，其中，所述第一探针和第二探针的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，所述第一探针连接的荧光基团与所述第二探针连接的荧光基团不同；
    优选地，所述第一探针的5’端连接有ROX荧光基团，所述第二探针的5’端连接有FAM荧光基团。
  3. 权利要求1或2的引物及探针组，其中，所述第一探针含有至少一个锁核酸修饰(LNA修饰)。
  4. 权利要求1-3任一项的引物及探针组，其中，所述第一探针如SEQ ID NO:5所示。
  5. 权利要求1-4任一项的引物及探针组，其中，所述第二探针含有至少一个锁核酸修饰(LNA修饰)。
  6. 权利要求1-5任一项的引物及探针组，其中，所述第二探针如SEQ ID NO:6所示。
  7. 用于检测SARS-CoV-2的试剂盒，其包含权利要求1-6任一项所述的引物及探针组。
  8. 用于检测SARS-CoV-2的方法，其包括如下步骤：
    1)提供权利要求1-6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。
  9. 权利要求8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：
    Figure PCTCN2021125587-appb-100001
  10. 权利要求8或9所述的方法，其中，所述RT-PCR反应的扩增和溶解程序如下：
    48-52℃，13-17min，1-3个循环；93-97℃，4-6min，1-3个循环；93-97℃,13-17s，53-57℃，13-17s,74-78℃，18-22s,45-55个循环；93-97℃，0.5-2min，1-3个循环；35-40℃，2-4min，1-3个循环；以0.02-0.06℃/s的升温速率从35-40℃升温至80-90℃，与此同时采集荧光信号。
  11. 权利要求8-10任一项的方法，其中，步骤1)中所述的待测样品包括商品、环境中的样品。
  12. 权利要求8-10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8-12任一项的方法，其中，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。
  14. 权利要求8-13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D614和G614假病毒；
    优选地，阴性质控品为无核酸DEPC-H 2O。
  15. 权利要求8-14任一项的方法，其中，在RT-PCR反应过程中采集荧光信号，所述荧光信号包括第一探针所连接的荧光基团对应的第一荧光以及第二探针所连接的荧光基团对应的第二荧光。
  16. 权利要求15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通道均无Tm值则阴性质控合格；若阳性质控品在第一荧光和第二荧光通道均有熔解曲线，且对应的Tm值落在已确定的参考值范围，则阳性质控合格。
  17. 权利要求8-16任一项的方法，其中，所述方法用于确定待测样品中是否存在D614毒株、G614毒株和/或非D614/G614毒株。
  18. 权利要求1-6任一项的引物及探针组或权利要求7的试剂盒在制备用于检测SARS-CoV-2的检测试剂中的用途。
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