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

Feasibility of transfusion-related pathogen nucleic acid detection method by Recombinase polymerase amplification combined with Cas12a

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1 fg,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。

Objective To study the feasibility of recombinase polymerase amplification(RPA) combined with Cas12 a in detection of transfusion related pathogens. Methods 1) Cas12 a crRNA was designed and screened in the conserved region of HIV, HBV and HCV genome. 2) Nested RPA primers of crRNA targeted sequence were designed imbricately, then the nested RPA primers were selected by gel electrophoresis and fluorescence probe. 3) The target sequences with different copy numbers or contents by gradient dilution of plasmid or virus simulated samples were amplified by RPA. Then RPA products were detected by Cas12 a to evaluate the detection limit of RPA combined with Cas12 a. 4) The specificity of the selected crRNA was evaluated by recombinant plasmids of homologous sequences of HTLV, WMHBV and HGBV of HIV, HBV and HCV. Results The RPA combined with Cas12 a nucleic acid detection system was established. The detection limit of the system was 10 cp for 16 ecombinant plasmids′ samples, and that of 8 lentiviral nucleic acid samples of Eco-HIV was 1 fg. No error was found in the cross detection of recombinant plasmids of HIV, HBV, HCV and RPA amplification products. The detection results of 10 HIV positive samples were consistent with the confirmation results. Conclusion RPA combined with cas12 a is a specific nucleic acid detection method with a low detection limit for HIV, HBV and HCV.