CRISPR/Cas12a-Based Diagnostic Platform Accurately Detects Nocardia farcinica Targeting a Novel Species-Specific Gene

作者：Xiaotong Qiu, Shuai Xu, Xueping Liu, Hongtao Ren, Lichao Han, Zhenjun Li · 发表于：Frontiers in Cellular and Infection Microbiology · 年份：2022 · DOI：10.3389/fcimb.2022.884411 · 被引用次数：14 · 研究领域：Actinomycetales infections and treatment、CRISPR and Genetic Engineering、Advanced biosensing and bioanalysis techniques

Under the COVID-19 pandemic background, nucleic acid detection has become the gold standard to rapidly diagnose the infectious disease. A rapid, low cost, reliable nucleic acid detection platform will be the key to control next potential pandemic. In this study, a nucleic acid detection platform, which combined CRISPR/Cas12a-based detection with loop-mediated isothermal amplification (LAMP), was developed and termed CRISPR-CLA. In the CRISPR-CLA system, LAMP preamplification was employed, and CRISPR/Cas12a-based detection was used to monitor the preamplicons. The forward inner primer (FIP) was engineered with a protospacer adjacent motif (PAM) site TTTA of Cas12a effector at the linker region; thus, the CRISPR-CLA platform can detect any sequence as long as the primer design meets the requirement of LAMP. To demonstrate the validity of the CRISPR-CLA system, it was applied for the molecular diagnosis of nocardiosis caused by Nocardia farcinica ( N. farcinica ). A highly conserved and species-specific gene pbr1 of N. farcinica , which was first reported in this study, was used as the target of detection. A set of LAMP primers targeting a fragment of pbr1 of the N. farcinica reference strain IFM 10152 was designed according to the principle of CRISPR-CLA. Three CRISPR RNAs (crRNAs) with different lengths were designed, and the most efficient crRNA was screened out. Additionally, three single-strand DNA (ssDNA) probes were tested to further optimize the detection system. As a re...