WO2021226754A1 - 一种病毒重组酶-聚合酶扩增检测方法 - Google Patents
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Abstract
Description
COVID-19 鉴定试剂盒以荧光定量PCR方法为主,实践显示其检测特异性高,成本低,操作方便。但是,该类试剂盒是根据已知病毒序列设计特异性引物和探针,因此只能鉴别已知病毒类型,不能鉴定未知新型病毒;此外,由于病毒基因序列变异,会导致引物和探针扩增失败,检测敏感性降低,因此需要定期更换引物和探针,并重新进行实际评估,而且根据目前临床实际使用情况表明现有的核酸检测存在假阴性,其原因可能由于实时荧光检测时检测灵敏度不够,从而导致其检测下限较高,检测结果存在部分误差。其次使用的较多的是胶体金检测方法,其是根据IgM、IgG类抗体是人体感染新冠病毒后产生的免疫防御蛋白,IgG是感染14天后出现的抗体,产生后持续存在,可以作为既往感染的指标,此法检测的IgG在感染后持续存在导致不能很准确的对预后患者的感染情况进行有效评估和精确健康阶段检测。而酶联免疫法、恒温扩增-实时荧光法、杂交捕获免疫荧光法等方法均需要多个连续步骤进行,操作复杂程度高,耗时长,无法适应紧急检测的需要。
在COVID-19检测中,使用RPA技术能够实现快速高灵敏度的检测,然而临床实践中,样品来源复杂,数量巨大,经常面临急需拿到准确的检测结果的情形,如何获得更加明显的检测信号是急需解决的问题。
Claims (19)
- 一种病毒核酸扩增方法,其特征在于,所述方法包括第一步RPA或RT-RPA反应和第二步RPA反应,所述第二步RPA反应的引物对位于所述第一步RPA或RT-RPA反应引物对扩增获得的模板内,且与第一步RPA或RT-RPA反应引物对不重叠或小于10bp的重叠。
- 根据权利要求1的扩增方法,其特征在于,所述方法还包括,在第一步RPA或RT-RPA反应体系中加入RNA模板进行反应,取1%-100%第一步RPA或RT-RPA反应产物加入第二步RPA反应体系进行第二步RPA反应。
- 根据权利要求1或2的扩增方法,其特征在于,所述第一步RPA或RT-RPA反应体系中RNA模板为4个copy到1E 6个copy。
- 根据权利要求1或2的扩增方法,其特征在于,所述第一步RPA或RT-RPA反应或第二步RPA反应的反应时间分别为5-30min。
- 根据权利要求1或2的扩增方法,其特征在于,所述第一步RPA或RT-RPA反应或第二步RPA反应的反应温度为35℃到45℃,优选的是37℃-42℃,更优选的是37℃。
- 一种用于病毒检测的引物对,其特征在于,所述引物对包括第一对RPA或RT-RPA引物对,和/或第二对RPA引物对,分别依次用于第一步RPA或RT-RPA反应和第二步RPA反应,所述第二步RPA反应的引物对位于所述第一步RPA或RT-RPA反应引物对扩增获得的模板内,且与第一步RPA或RT-RPA反应引物对不重叠或小于10bp的重叠。
- 一种与权利要求6所述的引物对配套使用的病毒检测的探针,其特征在于,所述探针在第二步RPA反应时使用,所述探针添加可以识别/thf/或者/idSp/的核酸内切酶。
- 一种用于病毒检测的序列组合,其特征在于,包括权利要求6所述的引物对和权利要求7所述的探针。
- 一种试剂盒,其特征在于,其包含权利要求6的引物对、权利要求7的探针和/或权利要求8的序列组合。
- 权利要求6的引物对、权利要求7的探针和/或权利要求8的序列组合在制备检测病毒试剂盒中的用途。
- 一种用于病毒检测的重组酶-聚合酶扩增方法,其特征在于,具体步骤包括:(1)将病毒样本与核酸释放剂混合,裂解病毒衣壳并释放病毒核酸;(2)两步RPA反应:包括第一步RPA或RT-RPA反应和第二步RPA反应,所述第二步RPA反应的引物对位于所述第一步RPA或RT-RPA反应引物对扩增获得的模板内,且与第一步RPA反应引物对不重叠或小于10bp的重叠。
- 根据权利要求11的扩增方法,其特征在于,所述方法还包括,在第一步RPA或RT-RPA反应体系中加入RNA模板进行反应,取第一步RPA或RT-RPA反应产物加入第二步RPA反应体系进行第二步RPA反应。
- 根据权利要求11或12的所述扩增方法,其特征在于,其中所述步骤(1)的病毒RNA原液样本来源于鼻咽拭子、深咳痰液、肺泡灌洗液、唾液样品或肺组织活检标本。
- 根据权利要求11或12的所述扩增方法,其特征在于,所述第一步RPA或RT-RPA反应体系中RNA模板为4个copy到1E 6个copy。
- 根据权利要求11或12的扩增方法,其特征在于,所述第一步RPA或RT-RPA反应或第二步RPA反应的反应时间分别为5-30min。
- 根据权利要求11或12的扩增方法,其特征在于,所述第一步RPA或RT-RPA反应或第二步RPA反应的反应温度为35℃到45℃,优选的是37℃-42℃,更优选的是37℃。
- 根据权利要求11或12的扩增方法,其特征在于,所述在第一步RPA或RT-RPA反应体系中加入RNA模板进行反应,取1%-100%第一步RPA或RT-RPA反应产物加入第二步RPA反应体系进行第二步RPA反应。
- 根据权利要求11或12的扩增方法,其特征在于,所述方法还包括胶体金层析显色步骤,取第二步RPA反应产物加入到DEPC水中混合均匀进行稀释,吸取稀释产物用于胶体金层析显色。
- 根据权利要求11或12的扩增方法,其特征在于,所述方法还包括荧光探针检测步骤。
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CN116622805A (zh) * | 2023-07-18 | 2023-08-22 | 九天览月生物科技(天津)有限公司 | 一种用于核酸检测的试剂盒 |
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