Khan Haroon, Khan Adeel, Liu Yufeng, Wang Su, Bibi Sumaira, Xu Hongpan, Liu Yuan, Durrani Samran, Jin Lian, He Nongyue, Xiong Tao
State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China.
Department of Clinical Laboratory, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing 210008, China.
Chin Chem Lett. 2019 Dec;30(12):2201-2204. doi: 10.1016/j.cclet.2019.10.032. Epub 2019 Oct 31.
Canine parvovirus type 2 (CPV-2) infection is the most lethal disease of dogs with higher mortality in puppies worldwide. In today's world, dogs are an integral part of our communities as well as dogs breeding and rearing has become a lucrative business. Therefore, a fast, accurate, portable, and cost-effective CPV-2 detection method with the ability for on-site detection is highly desired. In this study, we for the first time proposed a nanosystem for CPV-2 DNA detection with RNA-guided RNA endonuclease Cas13a, which upon activation results in collateral RNA degradation. We expressed LwCas13a in prokaryotic expression system and purified it through nickel column. Activity of Cas13a was verified by RNA-bound fluorescent group while using a quenched fluorescent probe as signals. Further Cas13a was combined with Recombinase polymerase amplification (RPA) and T7 transcription to establish molecular detection system termed specific high-sensitivity enzymatic reporter un-locking (SHERLOCK) for sensitive detection of CPV-2 DNA. This nanosystem can detect 100 amol/L CPV-2 DNA within 30 min. The proposed nanosystem exhibited high specificity when tested for CPV-2 and other dog viruses. This CRISPR-Cas13a mediated sensitive detection approach can be of formidable advantage during CPV-2 outbreaks because it is time-efficient, less laborious and does not involve the use of sophisticated instruments.
犬细小病毒2型（CPV-2）感染是犬类最致命的疾病，在全球范围内幼犬的死亡率更高。在当今世界，犬类是我们社区不可或缺的一部分，而且犬类繁殖和饲养已成为一项利润丰厚的业务。因此，迫切需要一种快速、准确、便携且经济高效的能够进行现场检测的CPV-2检测方法。在本研究中，我们首次提出了一种利用RNA引导的RNA内切酶Cas13a检测CPV-2 DNA的纳米系统，该系统激活后会导致附带的RNA降解。我们在原核表达系统中表达了LwCas13a，并通过镍柱进行纯化。使用淬灭荧光探针作为信号，通过与RNA结合的荧光基团验证了Cas13a的活性。进一步将Cas13a与重组酶聚合酶扩增（RPA）和T7转录相结合，建立了名为特异性高灵敏度酶促报告分子解锁（SHERLOCK）的分子检测系统，用于灵敏检测CPV-2 DNA。该纳米系统可在30分钟内检测到100 amol/L的CPV-2 DNA。当对CPV-2和其他犬类病毒进行检测时，所提出的纳米系统表现出高特异性。这种CRISPR-Cas13a介导的灵敏检测方法在CPV-2爆发期间可能具有巨大优势，因为它省时、省力且不涉及使用复杂仪器。
