Specific High-Sensitivity Enzymatic Molecular Detection System Termed RPA-Based CRISPR-Cas13a for Duck Tembusu Virus Diagnostics
Specific High-Sensitivity Enzymatic Molecular Detection System Termed RPA-Based CRISPR-Cas13a for Duck Tembusu Virus Diagnostics
复制标题
用于鸭天宝苏病毒诊断的基于 RPA 的 CRISPR-Cas13a 的特定高灵敏度酶分子检测系统
DOI：
10.1021/acs.bioconjchem.2c00200
复制
发表时间：
2022-06-15
影响因子：
4.7
通讯作者：
Tang, Yi
中科院分区：
文献类型：
--
作者：
He, Dalin;Liu, Gang;Tang, Yi
在中国，造成蛋落综合征的病原体坦布苏病毒（TMUV）给养鸭业造成了巨大的经济损失。为了成功地预防和控制感染，必须升级现有技术以实现快速检测病毒。这项工作是首次尝试开发一种基于重组酶聚合酶扩增（RPA）的簇状规则间隔短回文重复(CRISPR)-Cas13a方法用于TMUV感染诊断，其中CRISPR-Cas13a系统被利用，即CRISPR RNA （crRNA）的可编程性和混杂的RNase在识别目标RNA时对Cas13a进行侧切。利用原核表达系统表达LwCas13a可溶性蛋白，用Ni-NTA琼脂糖进行纯化。在特定crRNA的设计中，使用的靶标是体外转录的TMUV NS3 RNA。用于Cas13a活性验证的信号是rna结合荧光基团（单链）和猝灭荧光团。本研究将Cas13a与T7转录和RPA相结合，建立了一种特异性的高灵敏度酶促分子检测系统——基于RPA的CRISPR-Cas13a，用于室温下TMUV的灵敏检测。该系统可在50分钟内检测出10(2)份目标TMUV DNA标准拷贝/ μ L。结果表明，该病毒的特异性优于其他禽流感病毒。此外，基于rpa的CRISPR-Cas13a检测系统已成功应用于临床样品，其性能可与逆转录酶实时定量聚合酶链反应（RT-qPCR）相媲美。我们基于rpa的CRISPR-Cas13a检测系统可靠、简单、特异、灵敏，可以扩展和推广到其他病毒的鉴定中，可以在现场使用便携式横向流量试纸进行快速检测。
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 10(2) copies of the target TMUV DNA standard/mu L 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.