重组酶聚合酶扩增结合Cas12a输血相关病原体核酸检测方法的可行性
Chinese Journal of Blood Transfusion(2020)
Abstract
目的 探讨重组酶聚合酶扩增(RPA)结合Cas12a检测输血相关病原体的可行性.方法 1)根据输血相关病原体HIV、HBV、HCV基因组保守区设计、筛选相应Cas12a crRNA.2)据crRNA靶序列区叠瓦状设计RPA巢式扩增内、外侧引物,凝胶电泳法和荧光探针法筛选巢式RPA内、外侧引物.3)通过质粒或病毒模拟标本梯度稀释,将含不同拷贝数或含量的靶序列RPA扩增,扩增产物以Cas12a检测评价RPA结合Cas12a的检测下限.4)用HIV、HBV、HCV相应近源病毒HTLV、WMHBV、HGBV的同源序列的重组质粒来评价所选crRNA的特异性.结果 建立起RPA结合Cas12a核酸检测系统,16份重组质粒的检测下限为10 cp,8份EcoHIV病毒模拟标本的检测下限为1fg,HIV、HBV、HCV 3种重组质粒及RPA扩增产物的交叉检测间未出现错误检出;10例HIV阳性确证标本的检测结果均为阳性.结论 RPA结合Cas12a检测HIV、HBV、HCV核酸是1种检出限和特异性均较好的核酸检测方法.
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