WO2021174674A1 - 检测冠状病毒并分型的组合物、试剂盒及方法 - Google Patents

检测冠状病毒并分型的组合物、试剂盒及方法 Download PDF

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WO2021174674A1
WO2021174674A1 PCT/CN2020/090054 CN2020090054W WO2021174674A1 WO 2021174674 A1 WO2021174674 A1 WO 2021174674A1 CN 2020090054 W CN2020090054 W CN 2020090054W WO 2021174674 A1 WO2021174674 A1 WO 2021174674A1
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coronavirus
seq
composition
cov
upstream primer
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French (fr)
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戴立忠
谭德勇
邓中平
刘佳
纪博知
程星
孙青芝
颜进
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圣湘生物科技股份有限公司
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Priority to EP20923345.1A priority Critical patent/EP4015654A4/en
Publication of WO2021174674A1 publication Critical patent/WO2021174674A1/zh
Priority to US17/891,291 priority patent/US20230193407A1/en

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    • C—CHEMISTRY; METALLURGY
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    • 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
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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
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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
    • C12Q1/6851—Quantitative amplification
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes

Definitions

  • the present invention belongs to the field of molecular biology detection, more specifically, the field of detection of coronaviruses. More specifically, the present invention can detect new coronaviruses 2019-nCoV, coronavirus 229E, coronavirus NL63, coronavirus OC43, and coronavirus HKU1. , Coronavirus MERSr-CoV, Coronavirus SARSr-CoV and typing.
  • Coronavirus is an unsegmented single-stranded positive-stranded RNA virus. It belongs to the Orthocoronavirinae family of Nidovirales (Coronaviridae). According to the serotype and genome characteristics, the coronavirus subfamily is divided into The four genera ⁇ , ⁇ , ⁇ and ⁇ can infect many animal species, including humans, bats, dogs, pigs, mice, birds, cows, whales, horses, goats, monkeys, etc.
  • coronaviruses There are 6 types of coronaviruses known to infect humans, including ⁇ 229E and NL63, ⁇ OC43 and HKU1, Middle East respiratory syndrome-associated coronavirus (MERSr-CoV) and severe acute respiratory syndrome-associated coronavirus (SARSr- CoV). Since December 2019, an unexplained pneumonia infection has occurred in Wuhan, Hubei province, and a new type of coronavirus belonging to the ⁇ genus has been isolated from the patient's lower respiratory tract. Subsequently, the World Health Organization named it "2019 Novel Coronavirus (2019-nCoV)".
  • 2019-nCoV Middle East respiratory syndrome-associated coronavirus
  • the new type of coronavirus belongs to the new type of coronavirus of the ⁇ genus. It has an envelope, and the particles are round or oval, often pleomorphic, with a diameter of 60-140nm. Its genetic characteristics are obviously different from SARSr-CoV and MERSr-CoV. According to the latest research results of researcher Shi Zhengli of Wuhan Institute of Virology, 2019-nCoV has very high similarity with a bat coronavirus strain RaTG13 isolated from the Chinese chrysanthemum bat before in the laboratory, and the overall genome similarity reaches 96.2%. In addition, studies have shown that 2019-nCoV and bat SARS-like coronaviruses (bat-SL-CoVZC45 and bat-SL-CoVZXC21) are more than 85% homologous.
  • coronavirus 229E coronavirus NL63, coronavirus OC43, and coronavirus HKU1 are common respiratory infections.
  • the symptoms after infection are mild and generally do not cause severe illness, but if it is a new type Coronavirus 2019-nCoV, coronavirus MERSr-CoV, coronavirus SARSr-CoV infection, the patient's infection symptoms are very serious, and it is very easy to produce severe illness.
  • coronavirus 2019- nCoV coronavirus MERSr-CoV
  • coronavirus SARSr-CoV coronavirus SARSr-CoV
  • the present invention provides a composition capable of detecting and typing coronaviruses, the composition comprising:
  • coronavirus NL63 upstream primer shown in SEQ ID NO: 4 The coronavirus NL63 downstream primer shown in SEQ ID NO: 5, and the coronavirus NL63 probe shown in SEQ ID NO: 6;
  • coronavirus HKU1 upstream primer shown in SEQ ID NO: 7 The coronavirus HKU1 downstream primer shown in SEQ ID NO: 8, and the coronavirus HKU1 probe shown in SEQ ID NO: 9;
  • the fluorescent groups in the first group are different from each other, and the fluorescent groups in the second group are different from each other.
  • the coronavirus includes: new coronavirus 2019-nCoV, coronavirus 229E, coronavirus NL63, coronavirus OC43, coronavirus HKU1, coronavirus MERSr-CoV, and coronavirus SARSr-CoV.
  • composition of the present invention combining the fluorescent probe method and the melting curve method, it is possible to use one tube to simultaneously detect and type seven coronaviruses in one test, with low cost, high throughput and less time-consuming. This allows a single test tube to give the information of 8 targets, which greatly improves the detection efficiency and is easy to operate.
  • the result of the reading process can be determined by the amplification curve and CT value. The whole detection process is carried out under a single-tube closed condition, which avoids false positives and environmental pollution caused by crossover between samples.
  • the fluorescent reporter group can be selected from FAM, HEX, ROX, VIC, CY5, 5-TAMRA, TET, CY3, and JOE, but is not limited thereto.
  • the fluorescent reporter group of the novel coronavirus 2019-nCoV probe shown in SEQ ID NO: 3 is FAM; the fluorescent reporter of the coronavirus NL63 probe shown in SEQ ID NO: 6 The group is HEX; the fluorescent reporter group of the coronavirus HKU1 probe shown in SEQ ID NO: 9 is ROX.
  • the fluorescent reporter group of the coronavirus 229E upstream primer shown in SEQ ID NO: 10 is FAM; the fluorescent reporter group of the coronavirus OC43 upstream primer shown in SEQ ID NO: 12 is HEX; the fluorescent reporter group of the upstream primer of the coronavirus MERSr-CoV shown in SEQ ID NO: 14 is ROX; the fluorescent reporter group of the coronavirus SARSr-CoV shown in SEQ ID NO: 16 is CY5.
  • the ingredients of the composition of the invention are present in the same package.
  • composition further includes: an internal standard upstream primer as shown in SEQ ID NO: 18, an internal standard downstream primer as shown in SEQ ID NO: 19, and an internal standard as shown in SEQ ID NO: 20 Probe.
  • the fluorescent reporter group of the internal standard probe shown in SEQ ID NO: 20 is CY5.
  • the amount of the primer in the composition is 50-150 nM; the amount of the probe in the composition is 25-75 nM.
  • the present invention provides the use of the above-mentioned composition of the present invention in preparing a kit for detecting and typing coronavirus.
  • the coronaviruses include: new coronavirus 2019-nCoV, coronavirus 229E, coronavirus NL63, coronavirus OC43, coronavirus HKU1, coronavirus MERSr-CoV, and coronavirus SARSr-CoV.
  • the present invention provides a kit for detecting and typing coronaviruses, the kit comprising the above-mentioned composition of the present invention.
  • the kit also includes at least one of nucleic acid release reagent, dNTP, reverse transcriptase, DNA polymerase, PCR buffer, and Mg 2+.
  • the amount of the primer in the composition is 50-150 nM; the amount of the probe in the composition is 25-75 nM; the amount of the dNTP is 0.2-0.3 mM.
  • the concentration of the reverse transcriptase is 5U/ ⁇ L-15U/ ⁇ L, for example, the reverse transcriptase may be murine leukemia reverse transcriptase (MMLV); the concentration of the DNA polymerase is 5U/ ⁇ L-15U/ ⁇ L, for example, the DNA polymerase may be Taq enzyme.
  • MMLV murine leukemia reverse transcriptase
  • the concentration of the DNA polymerase is 5U/ ⁇ L-15U/ ⁇ L
  • the DNA polymerase may be Taq enzyme.
  • the kit further includes the following components and amounts:
  • a method for detecting and typing a coronavirus includes the following steps:
  • step 2) Perform fluorescent quantitative PCR on the nucleic acid obtained in step 1) using the above-mentioned composition of the present invention or the above-mentioned kit of the present invention;
  • the sample used for detection may be a throat swab, sputum, alveolar lavage fluid, blood, etc., but is not limited thereto.
  • reaction conditions of the fluorescent quantitative PCR are:
  • the upstream primer of the novel coronavirus 2019-nCoV may be as shown in SEQ ID NO: 24, and the downstream primer of the novel coronavirus 2019-nCoV may be as shown in SEQ ID NO: 25.
  • the 2019-nCoV probe may be as shown in SEQ ID NO: 26;
  • the upstream primer of coronavirus NL63 may be as shown in SEQ ID NO: 30, and the downstream primer of coronavirus NL63 may be as shown in SEQ ID NO: 31.
  • the coronavirus NL63 probe It may be shown in SEQ ID NO: 32; the upstream primer of coronavirus HKU1 may be shown in SEQ ID NO: 36, the downstream primer of coronavirus HKU1 may be shown in SEQ ID NO: 37, and the coronavirus HKU1 probe may be shown in SEQ ID NO
  • the upstream primer of coronavirus 229E may be as shown in SEQ ID NO: 39, the downstream primer of coronavirus 229E may be as shown in SEQ ID NO: 40; the upstream primer of coronavirus OC43 may be as shown in SEQ ID NO: 45,
  • the downstream primer of coronavirus OC43 may be shown in SEQ ID NO: 46; the upstream primer of coronavirus MERSr-CoV may be shown in SEQ ID NO: 49, and the downstream primer of coronavirus MERSr-CoV may be shown in SEQ ID NO: 50; and /Or the upstream primer of the coronavirus SARSr-CoV may
  • Figure 1 A is the amplification curve of the composition of Table 1 of the present invention for detecting coronavirus 2019-nCoV; B is the amplification curve of the composition of Table 2 of the present invention for detecting coronavirus 2019-nCoV; C is the table of the present invention 3 The amplification curve of the composition for detecting the coronavirus 2019-nCoV;
  • Figure 2 A is the amplification curve of the composition of Table 1 of the present invention for detecting coronavirus NL63; B is the amplification curve of the composition of Table 2 of the present invention for detecting coronavirus NL63; C is the amplification curve of the composition of Table 3 of the present invention Amplification curve of coronavirus NL63;
  • Figure 3 A is the amplification curve of the composition of Table 1 of the present invention for detecting coronavirus HKU1; B is the amplification curve of the composition of Table 2 of the present invention for detecting coronavirus HKU1; C is the detection of the composition of Table 3 of the present invention Amplification curve of coronavirus HKU1;
  • Figure 4 A is the melting curve of the composition of Table 1 for detecting coronavirus 229E; B is the melting curve of the composition of Table 2 for detecting coronavirus 229E of the present invention; C is the melting curve of the composition of Table 3 for detecting coronavirus 229E of the present invention Melting curve graph;
  • A is the melting curve of the composition of Table 1 of the present invention for detecting coronavirus OC43
  • B is the melting curve of the composition of Table 2 of the present invention for detecting coronavirus OC43
  • C is the melting curve of the composition of Table 3 of the present invention for detecting coronavirus The melting curve of OC43;
  • A is the melting curve diagram of the composition of Table 1 of the present invention for detecting the coronavirus MERSr-CoV
  • B is the melting curve diagram of the composition of Table 2 of the present invention for detecting the coronavirus MERSr-CoV
  • C is the combination of Table 3 of the present invention The melting curve diagram of the coronavirus MERSr-CoV for the detection of substances;
  • Figure 7 A is the melting curve diagram of the composition of Table 1 of the present invention for detecting the coronavirus SARSr-CoV; B is the melting curve diagram of the composition of Table 2 of the present invention for detecting the coronavirus SARSr-CoV; C is the combination of Table 3 of the present invention The melting curve chart of the coronavirus SARSr-CoV;
  • Figure 8 A is the amplification curve diagram of the internal standard detection of the composition of Table 1 of the present invention; B is the amplification curve diagram of the internal standard detection of the composition of Table 2 of the present invention; C is the amplification curve of the internal standard detection of the composition of Table 3 of the present invention Graph.
  • the primers and probes used in the present invention are shown in Tables 1 to 3 below:
  • the fluorescent reporter group of the novel coronavirus 2019-nCoV probe is FAM; the fluorescent reporter group of the coronavirus NL63 probe is HEX; the fluorescent reporter group of the coronavirus HKU1 probe is ROX, and the 3'end of the probe It also has a BHQ1 or BHQ2 quenching group.
  • the fluorescent reporter group of the upstream primer of coronavirus 229E is FAM; the fluorescent reporter group of the upstream primer of coronavirus OC43 is HEX; the fluorescent reporter group of the upstream primer of coronavirus MERSr-CoV is ROX; the fluorescent reporter of coronavirus SARSr-CoV The group is CY5.
  • Example 2 Method for detecting and typing coronavirus
  • test samples of the present invention are throat swabs, sputum, alveolar lavage fluid, and blood.
  • the magnetic bead method is used to extract viral nucleic acid, and the following operations are performed in the sample processing room:
  • RNA extraction solution 1 Take an appropriate amount of 1.5mL sterile centrifuge tube, label the negative control, positive control and sample to be tested respectively, and add 300 ⁇ L RNA extraction solution 1 to each tube;
  • RNA extraction solution 2-mix 100 ⁇ L of RNA extraction solution 2-mix to each tube (mix thoroughly and pipette), shake and mix for 10 seconds, and let it stand at room temperature for 10 minutes;
  • the real-time fluorescent PCR reaction system is configured as follows:
  • the PCR amplification program is set as follows:
  • the target detection signal is FAM, HEX (or VIC), ROX and CY5, and the internal reference detection signal is CY5;
  • Baseline (baseline) setting Baseline is generally set to 3-15 cycles, which can be adjusted according to actual conditions. The adjustment principle is: select the area where the fluorescence signal is relatively stable before exponential amplification, the starting point (Start) avoids the signal fluctuations in the initial stage of fluorescence collection, and the end point (End) is 1-2 less than the first exponential amplification sample Ct cycle.
  • Threshold (threshold) setting The setting principle is that the threshold line just exceeds the highest point of the normal negative control substance.
  • the HEX channel detects a typical S-type amplification curve, and Ct ⁇ 40, it means that the coronavirus NL63 test result is positive; if the HEX channel detects a Tm (67.0 ⁇ 1.0°C) characteristic peak, it means that the coronavirus OC43 is positive ;
  • each target positive plasmid was detected to simulate clinical samples, and multiple PCR detection was performed on the Hongshi fluorescence quantitative PCR instrument.
  • the results are shown in the figure Shown in 1 ⁇ 8. It can be seen from the figure:
  • the Ct value is about 25; when the composition in Table 2 is detected, the Ct value is about 28; when the composition in Table 3 is detected, the Ct value is Around 32, the amplification curves of A, B, and C are relatively straight and straight.
  • the Ct value is about 30; when tested with the composition in Table 2, the Ct value is about 29, but the curve of B is not as tall and straight as A; with the composition in Table 3 At the time of detection, the Ct value was around 34, and there was basically no amplification curve.
  • the Ct value is about 25; when the composition in Table 2 is detected, the Ct value is about 30; when the composition in Table 3 is detected, the Ct value is about 34 ,
  • the amplification curves of A and B are relatively straight and straight, and C basically has no amplification curve.
  • the characteristic peak is obvious; when the composition in Table 2 is detected, the characteristic peak is more obvious; when the composition in Table 3 is detected, the characteristic peak is not obvious.
  • the characteristic peak is obvious; when the composition in Table 2 is detected, the characteristic peak is more obvious; when the composition in Table 3 is detected, the characteristic peak is not obvious.
  • the characteristic peak is obvious; when the composition in Table 2 is used, the characteristic peak is more obvious; when the composition in Table 3 is detected, the characteristic peak is not obvious.
  • the characteristic peak is obvious; when the composition in Table 2 is used, the characteristic peak is more obvious; when the composition in Table 3 is detected, the characteristic peak is not obvious.
  • the Ct value is about 27; when the composition in Table 2 is tested, the Ct value is about 30; when the composition in Table 3 is tested, the Ct value is about 31.
  • the amplification curves of A and B are relatively straight, and the amplification curve of C is not straight.

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Abstract

本发明提供了一种组合物，其能够检测新型冠状病毒2019-nCoV、冠状病毒229E、冠状病毒NL63、冠状病毒OC43、冠状病毒HKU1、冠状病毒MERSr-CoV、冠状病毒SARSr-CoV并分型；与此同时，还提供了包含所述组合物的试剂盒以及用于检测冠状病毒并分型的方法。本发明的组合物结合荧光探针法和熔解曲线法，能够在一管同时检测七个冠状病毒并进行分型。

Description

检测冠状病毒并分型的组合物、试剂盒及方法 技术领域
本发明属于分子生物学检测领域，更具体地，属于冠状病毒的检测领域，更具体地，本发明能够检测新型冠状病毒2019-nCoV、冠状病毒229E、冠状病毒NL63、冠状病毒OC43、冠状病毒HKU1、冠状病毒MERSr-CoV、冠状病毒SARSr-CoV并分型。
背景技术
冠状病毒为不分节段的单股正链RNA病毒，属于巢病毒目(Nidovirales)冠状病毒科(Coronaviridae)正冠状病毒亚科(Orthocoronavirinae)，根据血清型和基因组特点冠状病毒亚科被分为α、β、γ和δ四个属，可以感染许多动物物种，包括人、蝙蝠、狗、猪、老鼠、鸟、牛、鲸、马、山羊、猴子等。已知感染人的冠状病毒有6种，包括α属的229E和NL63，β属的OC43和HKU1、中东呼吸综合征相关冠状病毒(MERSr-CoV)和严重急性呼吸综合征相关冠状病毒(SARSr-CoV)。2019年12月以来，湖北省武汉市发生不明原因肺炎感染事件，从患者下呼吸道分离出一种属于β属的新型冠状病毒。随后，世界卫生组织将其命名为“2019新型冠状病毒(2019-nCoV)”。
新型冠状病毒属于β属的新型冠状病毒，有包膜，颗粒呈圆形或椭圆形，常为多形性，直径60-140nm。其基因特征与SARSr-CoV和MERSr-CoV有明显区别。根据武汉病毒所石正丽研究员最新研究成果发现，2019-nCoV与其实验室之前从中华菊头蝠中分离到的一个蝙蝠冠状病毒毒株RaTG13相似度非常高，整体基因组相似度达96.2％。另外，研究显示2019-nCoV与蝙蝠SARS样冠状病毒(bat-SL-CoVZC45和bat-SL-CoVZXC21)同源性也达85％以上。
在可以感染人的7种冠状病毒中，冠状病毒229E、冠状病毒NL63、冠状 病毒OC43、冠状病毒HKU1为常见的呼吸道感染病毒，感染人后病症较轻，一般不会引起重症，但如果是新型冠状病毒2019-nCoV、冠状病毒MERSr-CoV、冠状病毒SARSr-CoV感染，则患者感染症状就非常严重，且极易产生重症。如果能够在一次测试中就对各种冠状病毒进行明确的分型，将会极大方便后续的诊疗措施，做到精准治疗和防控，尤其针对传染性和危害性大的新型冠状病毒2019-nCoV、冠状病毒MERSr-CoV、冠状病毒SARSr-CoV，可以做到及时控制传染源，阻断病毒大流行和大爆发。
因此，本领域需求同时并快速鉴定多个冠状病毒并分型的相关产品。
发明内容
有鉴于此，本发明提供了一种能够检测冠状病毒并分型的组合物，所述组合物包括：
第一组：
如SEQ ID NO:1所示的新型冠状病毒2019-nCoV上游引物、如SEQ ID NO:2所示的新型冠状病毒2019-nCoV下游引物，和如SEQ ID NO:3所示的新型冠状病毒2019-nCoV探针；
如SEQ ID NO:4所示的冠状病毒NL63上游引物、如SEQ ID NO:5所示的冠状病毒NL63下游引物，和如SEQ ID NO:6所示的冠状病毒NL63探针；
如SEQ ID NO:7所示的冠状病毒HKU1上游引物、如SEQ ID NO:8所示的冠状病毒HKU1下游引物，和如SEQ ID NO:9所示的冠状病毒HKU1探针；
第二组：
如SEQ ID NO:10所示的冠状病毒229E上游引物和如SEQ ID NO:11所示的冠状病毒229E下游引物；
如SEQ ID NO:12所示的冠状病毒OC43上游引物和如SEQ ID NO:13所示的冠状病毒OC43下游引物；
如SEQ ID NO:14所示的冠状病毒MERSr-CoV上游引物和如SEQ ID NO:15所示的冠状病毒MERSr-CoV下游引物；
如SEQ ID NO:16所示的冠状病毒SARSr-CoV上游引物和如SEQ ID NO:17所示的冠状病毒SARSr-CoV下游引物；
其中，第一组内的荧光基团彼此互不相同，并且第二组内的荧光基团彼此互不相同。
所述冠状病毒包括：新型冠状病毒2019-nCoV、冠状病毒229E、冠状病毒NL63、冠状病毒OC43、冠状病毒HKU1、冠状病毒MERSr-CoV、冠状病毒SARSr-CoV。
使用本发明的组合物，结合荧光探针法和熔解曲线法，能够使用一个管在一次试验中同时检测七个冠状病毒并进行分型，其成本低，通量高，耗时少。使得单次试验一管能给出8个靶点的信息，极大的提升了检测效率，并且操作简便，结果读取过程通过扩增曲线和CT值即可以判定。检测全过程均在单管封闭条件下进行，避免了由于样本间交叉引起的假阳性和环境污染。
在本发明中，荧光报告基团可以选自FAM、HEX、ROX、VIC、CY5、5-TAMRA、TET、CY3和JOE，但不限于此。
在一个具体的实施方案中，如SEQ ID NO:3所示的新型冠状病毒2019-nCoV探针的荧光报告基团为FAM；如SEQ ID NO:6所示的冠状病毒NL63探针的荧光报告基团为HEX；如SEQ ID NO:9所示的冠状病毒HKU1探针的荧光报告基团为ROX。
在一个具体的实施方案中，如SEQ ID NO:10所示的冠状病毒229E上游引物的荧光报告基团为FAM；如SEQ ID NO:12所示的冠状病毒OC43上游引物的荧光报告基团为HEX；如SEQ ID NO:14所示的冠状病毒MERSr-CoV上游引物的荧光报告基团为ROX；如SEQ ID NO:16所示的冠状病毒SARSr-CoV的荧光报告基团为CY5。
在一个实施方案中，本发明的组合物的各成分存在于同一个包装中。
进一步地，所述组合物还包括：如SEQ ID NO:18所示的内标上游引物、如SEQ ID NO:19所示的内标下游引物，和如SEQ ID NO:20所示的内标探针。
在一个具体的实施方案中，如SEQ ID NO:20所示的内标探针的荧光报告 基团为CY5。
进一步地，所述组合物中引物的用量为50～150nM；所述组合物中探针的用量为25～75nM。
第二方面，本发明提供了上述本发明的组合物在制备检测冠状病毒并分型的试剂盒中的用途。
所述冠状病毒包括：新型冠状病毒2019-nCoV，冠状病毒229E、冠状病毒NL63、冠状病毒OC43、冠状病毒HKU1、冠状病毒MERSr-CoV、冠状病毒SARSr-CoV。
第三方面，本发明提供了一种检测冠状病毒并分型的试剂盒，所述试剂盒包括上述本发明的组合物。
进一步地，所述试剂盒还包括核酸释放试剂、dNTP、逆转录酶、DNA聚合酶、PCR缓冲液以及Mg 2+中的至少一种。
进一步地，所述组合物中引物的用量为50～150nM；所述组合物中探针的用量为25～75nM；所述dNTP的用量为0.2～0.3mM。
进一步地，所述逆转录酶的浓度为5U/μL～15U/μL，例如逆转录酶可以是鼠白血病逆转录酶(MMLV)；所述DNA聚合酶的浓度为5U/μL～15U/μL，例如DNA聚合酶可以是Taq酶。
在一个具体的实施方案中，所述试剂盒除上述的本发明的组合物之外，还进一步包括以下组份和用量：
Figure PCTCN2020090054-appb-000001
第四方面，提供了一种用于检测冠状病毒并分型的方法，所述方法包括以下步骤：
1)释放待测样本的核酸；
2)使用如上述本发明的组合物或上述本发明的试剂盒对步骤1)获得的核酸进行荧光定量PCR；
3)获得并分析结果。
在本发明中，用于检测的样本可以是咽拭子、痰液、肺泡灌洗液、血液等，但不限于此。
进一步地，所述荧光定量PCR的反应条件为：
Figure PCTCN2020090054-appb-000002
在本发明的变型中，可选地，新型冠状病毒2019-nCoV上游引物可以如SEQ ID NO:24所示，新型冠状病毒2019-nCoV下游引物可以如SEQ ID NO:25所示，新型冠状病毒2019-nCoV探针可以如SEQ ID NO:26所示；冠状病毒NL63上游引物可以如SEQ ID NO:30所示，冠状病毒NL63下游引物可以如SEQ ID NO:31所示，冠状病毒NL63探针可以如SEQ ID NO:32所示；冠状病毒HKU1上游引物可以如SEQ ID NO:36所示，冠状病毒HKU1下游引物可以如SEQ ID NO:37所示，冠状病毒HKU1探针可以如SEQ ID NO:38所示；冠状病毒229E上游引物可以如SEQ ID NO:39所示，冠状病毒229E下游引物可以如SEQ ID  NO:40所示；冠状病毒OC43上游引物可以如SEQ ID NO:45所示，冠状病毒OC43下游引物可以如SEQ ID NO:46所示；冠状病毒MERSr-CoV上游引物可以如SEQ ID NO:49所示，冠状病毒MERSr-CoV下游引物可以如SEQ ID NO:50所示；和/或冠状病毒SARSr-CoV上游引物可以如SEQ ID NO:51所示，SARSr-CoV下游引物可以如SEQ ID NO:52所示。
附图说明
图1：A为本发明表1组合物检测冠状病毒2019-nCoV的扩增曲线图；B为本发明的表2组合物检测冠状病毒2019-nCoV的扩增曲线图；C为本发明的表3组合物检测冠状病毒2019-nCoV的扩增曲线图；
图2：A为本发明表1组合物检测冠状病毒NL63的扩增曲线图；B为本发明的表2组合物检测冠状病毒NL63的扩增曲线图；C为本发明的表3组合物检测冠状病毒NL63的扩增曲线图；
图3：A为本发明表1组合物检测冠状病毒HKU1的扩增曲线图；B为本发明的表2组合物检测冠状病毒HKU1的扩增曲线图；C为本发明的表3组合物检测冠状病毒HKU1的扩增曲线图；
图4：A为表1组合物检测冠状病毒229E的熔解曲线图；B为本发明的表2组合物检测冠状病毒229E的熔解曲线图；C为本发明的表3组合物检测冠状病毒229E的熔解曲线图；
图5：A为本发明表1组合物检测冠状病毒OC43的熔解曲线图；B为本发明的表2组合物检测冠状病毒OC43的熔解曲线图；C为本发明的表3组合物检测冠状病毒OC43的熔解曲线图；
图6：A为本发明表1组合物检测冠状病毒MERSr-CoV的熔解曲线图；B为本发明的表2组合物检测冠状病毒MERSr-CoV的熔解曲线图；C为本发明的表3组合物检测冠状病毒MERSr-CoV的熔解曲线图；
图7：A为本发明表1组合物检测冠状病毒SARSr-CoV的熔解曲线图；B为本发明的表2组合物检测冠状病毒SARSr-CoV的熔解曲线图；C为本发明的 表3组合物检测冠状病毒SARSr-CoV的熔解曲线图；
图8：A为本发明表1组合物检测内标的扩增曲线图；B为本发明的表2组合物检测内标的扩增曲线图；C为本发明的表3组合物检测内标的扩增曲线图。
具体实施方式
下文将结合具体实施方式和实施例，具体阐述本发明，本发明的优点和各种效果将由此更加清楚地呈现。本领域技术人员应理解，这些具体实施方式和实施例是用于说明本发明，而非限制本发明。
实施例1、本发明所使用的引物及探针
本发明所使用的引物及探针如下表1～3所示：
表1
Figure PCTCN2020090054-appb-000003
Figure PCTCN2020090054-appb-000004
表2
Figure PCTCN2020090054-appb-000005
Figure PCTCN2020090054-appb-000006
表3
Figure PCTCN2020090054-appb-000007
Figure PCTCN2020090054-appb-000008
Figure PCTCN2020090054-appb-000009
其中，新型冠状病毒2019-nCoV探针的荧光报告基团为FAM；冠状病毒NL63探针的荧光报告基团为HEX；冠状病毒HKU1探针的荧光报告基团为ROX，探针的3’末端还具有BHQ1或BHQ2猝灭基团。
冠状病毒229E上游引物的荧光报告基团为FAM；冠状病毒OC43上游引物的荧光报告基团为HEX；冠状病毒MERSr-CoV上游引物的荧光报告基团为ROX；冠状病毒SARSr-CoV的荧光报告基团为CY5。
实施例2、检测冠状病毒并分型的方法
本发明检测样本为咽拭子、痰液、肺泡灌洗液、血液。磁珠法提取病毒核酸，在样本处理室进行如下操作：
2.1取适量1.5mL灭菌离心管，分别标记阴性对照、阳性对照及待测样本，每管加入300μL RNA提取溶液1；
2.2每管加入200μL待测样本或阴性对照、阳性对照；盖上管盖，震荡混匀10秒钟，瞬时离心；
2.3每管加入100μL RNA提取溶液2-mix(充分混匀后吸取)，震荡混匀10秒钟后，室温静置10分钟；
2.4瞬时离心后将离心管置于分离器上，3分钟后缓慢将溶液吸出(注意不要碰到吸附于管壁的棕色物)；
2.5每管加入600μL RNA提取溶液3和200μL RNA提取溶液4，震荡混匀5秒钟,瞬时离心后将离心管再次置于分离器上；
2.6约3分钟后，上清液分为两层，将吸头***离心管底部，从底部开始缓慢将液体完全吸出丢弃，静置1分钟后将管底残余液体完全吸出丢弃。
2.7每管加入50μL PCR-mix，用吸头吸取PCR-mix洗脱吸附于离心管壁的棕色残留物，反复几次尽量将其完全洗脱，然后将洗脱后的全部棕色混合液转移至0.2mL PCR反应管中，盖上管盖，转移到扩增检测区。
实时荧光PCR反应体系按照如下进行配置：
Figure PCTCN2020090054-appb-000010
PCR扩增程序如下进行设置：
Figure PCTCN2020090054-appb-000011
结果分析：
1)目的检测信号为FAM、HEX(或VIC)、ROX及CY5，内参检测信号 为CY5；
2)Baseline(基线)的设置：Baseline一般设置为3-15个循环，具体可根据实际情况进行调整。其调整原则为：选择指数扩增前荧光信号较稳定的区域，起点(Start)避开荧光采集起始阶段的信号波动，终点(End)比最早出现指数扩增的样本Ct减少1-2个循环。Threshold(阈值)的设置：设定原则以阈值线刚好超过正常阴性对照品的最高点。
3)先分析内标在CY5通道是否检测扩增曲线，且Ct≤39，若有，表示本次检测有效，继续进行后续分析:
A)若FAM通道检测到典型的S型扩增曲线，且Ct＜39，表示新型冠状病毒2019-nCoV检测结果为阳性；若FAM通道检测到Tm(69.5±1.0℃)特征峰，表示冠状病毒229E检测结果为阳性；
B)若HEX通道检测到典型的S型扩增曲线，且Ct＜40，表示冠状病毒NL63检测结果为阳性；若HEX通道检测到Tm(67.0±1.0℃)特征峰，表示冠状病毒OC43为阳性；
C)若ROX通道检测到典型的S型扩增曲线，且Ct＜40，表示冠状病毒HKU1检测结果为阳性；若ROX通道检测到Tm(70.5±1.0℃特征峰，表示冠状病毒MERSr-CoV检测结果为阳性；
D)若CY5通道检测到Tm(68.0±1.0℃)特征峰，表示冠状病毒SARSr-CoV检测结果为阳性；
4)若内标在CY5通道没有检测到Ct或Ct＞39，表示本次检测样本浓度太低或者有干扰物质抑制反应，需重新准备实验。
实施例3、本发明组合物测试阳性对照的检测结果
使用本发明表1～表3的组合物，按照实施例2所述的方法，对各个靶标阳性质粒进行检测，以模拟临床样本，在宏石荧光定量PCR仪器上进行多重PCR检测，结果如图1～8所示。从图中可以看出：
冠状病毒2019-nCoV用表1中的组合物检测时，Ct值在25左右；用表2 中的组合物检测时，Ct值在28左右；用表3中的组合物检测时，Ct值在32左右，A、B和C的扩增曲线均较为挺拔。
冠状病毒NL63用表1中的组合物检测时，Ct值在30左右；用表2中的组合物检测时，Ct值在29左右，但B的曲线不如A挺拔；用表3中的组合物检测时，Ct值在34左右，基本上没有扩增曲线。
冠状病毒HKU1用表1中的组合物检测时，Ct值在25左右；用表2中的组合物检测时，Ct值在30左右；用表3中的组合物检测时，Ct值在34左右，A、B的扩增曲线均较为挺拔，C基本上没有扩增曲线。
冠状病毒229E用表1中的组合物检测时，特征峰明显；用表2中的组合物检测时，特征峰较为明显；用表3中的组合物检测时，特征峰不明显。
冠状病毒OC43用表1中的组合物检测时，特征峰明显；用表2中的组合物检测时，特征峰较为明显；用表3中的组合物检测时，特征峰不明显。
冠状病毒MERSr-CoV用表1中的组合物检测时，特征峰明显；用表2中的组合物检测时，特征峰较为明显；用表3中的组合物检测时，特征峰不明显。
冠状病毒SARSr-CoV用表1中的组合物检测时，特征峰明显；用表2中的组合物检测时，特征峰较为明显；用表3中的组合物检测时，特征峰不明显。
内标用表1中的组合物检测时，Ct值在27左右；用表2中的组合物检测时，Ct值在30左右；用表3中的组合物检测时，Ct值在31左右，A、B的扩增曲线均较为挺拔，C的扩增曲线不挺拔。

Claims (10)

  1. 一种能够检测冠状病毒并分型的组合物，所述组合物包括：
    第一组：
    如SEQ ID NO:1所示的新型冠状病毒2019-nCoV上游引物、如SEQ ID NO:2所示的新型冠状病毒2019-nCoV下游引物，和如SEQ ID NO:3所示的新型冠状病毒2019-nCoV探针；
    如SEQ ID NO:4所示的冠状病毒NL63上游引物、如SEQ ID NO:5所示的冠状病毒NL63下游引物，和如SEQ ID NO:6所示的冠状病毒NL63探针；
    如SEQ ID NO:7所示的冠状病毒HKU1上游引物、如SEQ ID NO:8所示的冠状病毒HKU1下游引物，和如SEQ ID NO:9所示的冠状病毒HKU1探针；
    第二组：
    如SEQ ID NO:10所示的冠状病毒229E上游引物和如SEQ ID NO:11所示的冠状病毒229E下游引物；
    如SEQ ID NO:12所示的冠状病毒OC43上游引物和如SEQ ID NO:13所示的冠状病毒OC43下游引物；
    如SEQ ID NO:14所示的冠状病毒MERSr-CoV上游引物和如SEQ ID NO:15所示的冠状病毒MERSr-CoV下游引物；
    如SEQ ID NO:16所示的冠状病毒SARSr-CoV上游引物和如SEQ ID NO:17所示的冠状病毒SARSr-CoV下游引物；
    其中，第一组内的荧光基团彼此互不相同，并且第二组内的荧光基团彼此互不相同。
  2. 根据权利要求1所述的组合物，其中，所述组合物进一步包括如SEQ ID NO:18所示的内标上游引物、如SEQ ID NO:19所示的内标下游引物，和如SEQ ID NO:20所示的内标探针。
  3. 根据权利要求1所述的组合物，其中，SEQ ID NO:3所示的新型冠状病毒2019-nCoV探针的荧光报告基团为FAM；如SEQ ID NO:6所示的冠状病毒 NL63探针的荧光报告基团为HEX；如SEQ ID NO:9所示的冠状病毒HKU1探针的荧光报告基团为ROX。
  4. 根据权利要求1所述的组合物，其中，如SEQ ID NO:10所示的冠状病毒229E上游引物的荧光报告基团为FAM；如SEQ ID NO:12所示的冠状病毒OC43上游引物的荧光报告基团为HEX；如SEQ ID NO:14所示的冠状病毒MERSr-CoV上游引物的荧光报告基团为ROX；如SEQ ID NO:16所示的冠状病毒SARSr-CoV的荧光报告基团为CY5。
  5. 权利要求1～4中任一项所述的组合物在制备检测冠状病毒并分型的试剂盒中的用途。
  6. 一种用于检测冠状病毒并分型的试剂盒，所述试剂盒包括权利要求1～4中任一项所述的组合物。
  7. 根据权利要求6所述的试剂盒，其中，所述试剂盒还包括核酸释放试剂、dNTP、逆转录酶、DNA聚合酶、PCR缓冲液中的至少一种。
  8. 根据权利要求6所述的试剂盒，其中，所述组合物中引物的用量为50～150nM；所述组合物中探针的用量为25～75nM。
  9. 一种用于检测冠状病毒并分型的方法，所述方法包括以下步骤：
    1)释放待测样本的核酸；优选地，所述样本为咽拭子、痰液、肺泡灌洗液、血液；
    2)使用如权利要求1～4中任一项所述的组合物对步骤1)获得的核酸进行荧光定量PCR；
    3)获得并分析结果。
  10. 根据权利要求9所述的方法，其中，所述荧光定量PCR的反应条件为：
    Figure PCTCN2020090054-appb-100001
    Figure PCTCN2020090054-appb-100002
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CN115044704A (zh) * 2021-11-22 2022-09-13 江汉大学 一种人冠状病毒HCoV-229E的MNP标记位点、引物组合物、试剂盒及其应用
CN115044704B (zh) * 2021-11-22 2023-06-16 江汉大学 一种人冠状病毒HCoV-229E的MNP标记位点、引物组合物、试剂盒及其应用
CN113981152A (zh) * 2021-12-28 2022-01-28 深圳联合医学科技有限公司 检测SARS-CoV-2变异株的组合物、试剂盒、方法及其用途
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