CN111206122A - 一种新型冠状病毒核酸检测试剂盒 - Google Patents
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Abstract
本发明公开了一种新型冠状病毒核酸检测试剂盒,包括用于扩增ORF1ab基因的核酸扩增试剂、对照品和超纯水。所述核酸扩增试剂包括SEQ NO.1~2所示的引物和SEQ NO.3所示的探针混合液,所述探针5’端修饰荧光基团FAM,3’端修饰淬灭基团BHQ‑1。本发明所述试剂盒通过特异性的引物及荧光探针,检测标本中是否存在ORF1ab基因,以此判断被检人是否患有COVID‑19。
Description
技术领域
本发明涉及一种病毒检测试剂盒,具体涉及一种新型冠状病毒核酸检测试剂盒。
背景技术
冠状病毒是自然界广泛存在的一大类病毒,为线性单股正链的RNA病毒,是许多家畜、宠物包括人类疾病的重要病原,可引起多种呼吸道、肠道疾病,如MERS和SARS等冠状病毒更会并引起严重的呼吸道疾病。2020年初爆发的新型冠状病毒(2019-nCoV)即属于冠状病毒科冠状病毒属,是目前已知的第7种可以感染人的冠状病毒。其病症被命名为COVID-19。
病毒都有其独特的基因序列,通过检测病人体内的病毒核酸,就可判断病人体内是否存在病毒。而人体内的病毒可能会随着患者的康复而消失,因此核酸检测是临床上判断患者是否感染与治愈的重要指标。因此,针对新型冠状病毒的快速检测是有效判断感染人群,预防病毒再次传播的重要手段,对于临床早发现、早诊断、早隔离、早治疗都至关重要,能做到有效防控新冠肺炎疫情。
目前,病毒核酸检测方法通常是采用荧光定量PCR法(qPCR)。qPCR即以病毒独特的基因序列为检测靶标,针对特定的基因序列设计特异性的寡核苷酸探针,并在其两侧分别添加荧光发光基团和猝灭基团。在扩增中,靶基因呈指数级增加,而DNA聚合酶利用核酸外切酶活性降解荧光探针,从而产生特定波长的荧光信号。目前的病毒检测试剂盒大多选择病毒的N基因、E基因、ORF1ab基因序列作为靶基因,E基因编码病毒包膜蛋白E,N基因编码病毒核衣壳蛋白N,ORF1ab基因编码多聚蛋白pp1ab。若样本中存在靶基因,则扩增出来的片段越多,累积得到的荧光信号就越强。而没有病毒的样本中,由于没有靶基因扩增,因此就检测不到荧光信号增强。
但核酸检测的准确与否,与多种因素有关,同一病人的不同检测样本,其检测结果也可能存在较大差别。此外,随着病程的变化,病人体内的病毒量也在动态变化。因此,开发高效灵敏的试剂盒是快速检测关键因素。
发明内容
本发明的目的在于提供一种新型冠状病毒核酸检测试剂盒,通过特异性的引物及荧光探针,检测标本中是否存在ORF1ab基因,以此判断被检人是否患有COVID-19。
本发明所述新型冠状病毒核酸检测试剂盒,包括用于扩增新型冠状病毒ORF1ab基因的核酸扩增试剂、对照品和超纯水。
所述核酸扩增试剂包括SEQ NO.1~2所示的引物和SEQ NO.3所示的探针混合液。
所述反应液的组成为:pH8.5、20mM Tris-HCl,40mM KCl,0.3%TritonX-100,4mMMgCl2,10%DMSO,0.4mM dATP,0.4mM dCTP,0.4mM dGTP,0.4mM dUTP,0.2mM dTTP,2M甜菜碱,0.04U/μL UDG酶,0.075U/μL Taq酶,0.05 U/μL逆转录酶,1μM Random primer,1μM。
所述SEQ ID NO.1和SEQ ID NO.2所示引物浓度及SEQ ID NO.3所示探针的浓度皆为2.5μM。
所述探针5’端修饰荧光基团FAM,3’端修饰淬灭基团BHQ-1。
所述对照品包括阴性对照品和阳性对照品。
所述新型冠状病毒核酸检测试剂盒的使用方法如下:
1)配置10μL反应体系:5μL反应液,2μL引物探针混合液,2μL模板RNA,1μL超纯水;
2)混匀后放入qPCR仪,设定荧光类型;
3)设定如下循环体系后开始扩增:50℃10min,95℃3min,95℃15s、58℃30s共40个循环,扩增结束后判读结果。
本试剂盒的有益效果如下:1)引入了dUTP/UDG防污染***,能够有效避免交叉反应造成的假阳性结果;2)针对新型冠状病毒ORF1ab基因设计的特异性引物探针,并配备特殊缓冲***能使DNA聚合酶发挥最大功效,提高反应效率。3)可得到更宽广的线性范围,对目的基因定量更准确,重复性好,可信度高,适合于RNA病毒等微量RNA的检测。
具体实施方式
实施例1新型冠状病毒核酸检测试剂盒的使用方法。
(1)将反应液、引物探针液、超纯水从-20℃冰箱中取出,室温下解冻后混匀。
(2)向qPCR反应专用管中配置表1所示的反应体系,震荡混匀后短暂离心。
(3)设置阴性对照、阳性对照的平行试验,阴性对照中以超纯水代替模板RNA,阳性对照使用试剂盒内阳性对照品代替模板RNA。
(4)于qPCR仪上设置反应体系体积、荧光类别等参数,并输入表2所示反应条件。
(5)结果判读:根据输出的每毫升拷贝数,判断样品中是否含有新型冠状病毒序列。
输出结果无Ct值或Ct值>40时为阴性;输出Ct值<37时可报告为阳性;输出Ct值在37-40之间时可重复实验,若Ct值<40,扩增曲线有明显起峰,该样本判断为阳性,否则为阴性。
实施例2 患者标本核酸检测。
按实施例1所述方法对山西医科大学肺科医院新冠确诊患者留存标本进行核酸检测,分别选取10例COVID-19患者标本和3例健康人标本,结果见表3,1-10号患者标本Ct值皆小于37,全部判断为阳性,11-13号健康人标本无Ct,检出率达100%。
序列表
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Claims (6)
1.一种新型冠状病毒核酸检测试剂盒,包括用于扩增新型冠状病毒ORF1ab基因的核酸扩增试剂,其特征是所述核酸扩增试剂包括SEQ ID NO.1~2所示引物和SEQ ID NO.3所示探针的混合液。
2.根据权利要求1所述的检测试剂盒,其特征是所述探针5’端修饰荧光基团FAM,3’端修饰淬灭基团BHQ-1。
3.根据权利要求1所述的检测试剂盒,其特征是所述核酸扩增试剂还包括反应液的组成为:pH8.5、20mM Tris-HCl,40mM KCl,0.3%TritonX-100,4mM MgCl2,10%DMSO,0.4mMdATP,0.4mM dCTP,0.4mM dGTP,0.4mM dUTP,0.4mM dTTP,2M甜菜碱,0.04U/μL UDG酶,0.075U/μL Taq酶,0.05 U/μL逆转录酶,1μM Random primer,1μM Oligo dT。
4.根据权利要求1所述的检测试剂盒,其特征是SEQ ID NO.1和SEQ ID NO.2所示引物浓度及SEQ ID NO.3所示探针的浓度皆为2.5μM。
5.根据权利要求1所述的检测试剂盒,其特征是还包括对照品和超纯水,所述对照品包括阴性对照品和阳性对照品。
6.一种权利要求1所述新型冠状病毒核酸检测试剂盒的使用方法,其特征在于,包括如下步骤:
1)配置10μL反应体系:5μL反应液,2μL引物探针混合液,2μL模板RNA,1μL超纯水;
2)混匀后放入qPCR仪,设定荧光类型;
3)设定如下循环体系后开始扩增:50℃10min,95℃3min,95℃15s、58℃30s共40个循环,扩增结束后判读结果。
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CN111690772A (zh) * | 2020-06-15 | 2020-09-22 | 桂林优利特医疗电子有限公司 | 一种新冠病毒核酸检测试剂盒、制备方法及应用 |
CN112553375A (zh) * | 2020-12-22 | 2021-03-26 | 武汉艾迪康医学检验所有限公司 | 一种呼吸道病毒检测试剂盒和方法 |
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