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

摘要:

【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10⁰ 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。

关键词:CRISPR;人腺病毒;等温扩增;核酸检测;ERASE试纸

Abstract:

[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.

Key words:CRISPR;human adenovirus;isothermal amplification;nucleic acid detection;ERASE strips