基于CRISPR-Cas13a的人腺病毒B亚属检测方法的建立

Establishment of human adenovirus species B detection method based on CRISPR-Cas13a

【背景】人腺病毒B亚属(human adenovirus species B,HAdV-B)可引起呼吸道感染和重症肺炎,患者死亡率高,目前缺乏快速、准确的检测方法。【目的】开发一种高特异、高灵敏、操作简便的HAdV-B检测方法。【方法】在人腺病毒E4基因区筛选出HAdV-B的保守序列并选取了特异性重组酶辅助扩增(recombinase-aided amplification,RAA)引物和CRISPR RNA(crRNA),建立结合RAA技术、CRISPR-Cas13a系统和消线法核酸检测试纸技术(easy-readout and sensitive enhanced,ERASE)的针对HAdV-B的核酸快速检测方法。【结果】该方法在35 min的检测时间里,可检测到低至10~0 copy/μL的HAdV-B DNA,该方法的灵敏度与实时荧光定量PCR相当,与其他呼吸道病原体无交叉反应。模拟样本检测结果显示能检测到C_t值为36.19的弱阳性样本。【结论】本研究建立的针对HAdV-B试纸的检测方法能够简单、快速、准确地检测目标核酸,无需专业核酸检测设备,为HAdV-B的检测提供了新的方法。

[Background]Human adenovirus species B(HAdV-B)can cause respiratory tract infections and severe pneumonia with high mortality rates.Currently,there is a lack of rapid and accurate detection methods for HAdV-B.[Objective]To develop a highly specific,sensitive,and user-friendly detection method for HAdV-B.[Methods]Conserved sequences of HAdV-B were screened in the human adenovirus E4 gene region,and specific recombinase-aided amplification(RAA)primers and CRISPR RNA(crRNA)were selected.We then established a rapid nucleic acid detection method targeting HAdV-B by combining RAA,CRISPR-Cas13a system,and easy-readout and sensitive enhanced(ERASE)nucleic acid test strip.[Results]The established method could detect HAdV-B DNA as low as 100 copy/μL within 35 min,with the sensitivity comparable to real-time PCR.No cross-reactivity with other respiratory pathogens was observed.The testing results with simulated samples showed that the method can detect the weak positive samples with a Ct value of 36.19.[Conclusion]The test strip assay established in this study for HAdV-B can detect the target nucleic acids in a simple,rapid,and accurate manner without the need for specialized nucleic acid detection equipment,which provides a new method for detecting HAdV-B.