Specific High-Sensitivity Enzymatic Molecular Detection System Termed RPA-Based CRISPR-Cas13a for Duck Tembusu Virus...

In China, drastic losses in the economy have been caused by the Tembusu virus (TMUV), the causative agent of the egg-drop syndrome, to the duck-raising industry. To succeed in preventing and controlling infections, extant techniques must be upgraded to achieve fast detection of viruses. This work is the first attempt to present the development of a recombinase polymerase amplification (RPA)-based clustered regularly interspaced short palindromic repeats (CRISPRs)-Cas13a approach for the TMUV infection diagnosis, where the CRISPR-Cas13a system is exploited, i.e., the programmability of CRISPR RNA (crRNA) and the promiscuous RNase collateral cleavage of Cas13a upon recognition of target RNAs. A prokaryotic expression system was utilized for the expression of LwCas13a soluble protein, while its purification was accomplished by nickel-nitrilotriacetic acid (Ni-NTA) agarose. In the design of a particular crRNA, the target used was the TMUV NS3 RNA transcribed in vitro. The signals used for the Cas13a activity validation were an RNA-bound fluorescent group (single-stranded) and a quenching fluorophore. In the present work, a specific high-sensitivity enzymatic molecular detection system termed RPA-based CRISPR-Cas13a was established by combining Cas13a with T7 transcription and RPA for sensitive detection of TMUV at room temperature. This system can detect 10² copies of the target TMUV DNA standard/μL within 50 min. A comparison revealed that the specificity was superior to that for other avian viruses. Furthermore, the RPA-based CRISPR-Cas13a detection system was successfully applied for clinical samples, and its performance is comparable to the reverse-transcriptase real-time quantitative polymerase chain reaction (RT-qPCR). Being satisfyingly reliable, simple, specific, and sensitive, our RPA-based CRISPR-Cas13a detection system could be expanded and universalized for identifying other viruses, enabling quick detection in the field with a portable lateral flow dipstick.