Liao Kai, Peng Wanqing, Qian Bingxu, Nan Wenlong, Shan Yuping, Zeng Dexin, Tang Fang, Wu Xiaodong, Chen Yiping, Xue Feng, Dai Jianjun
MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, 210095, China.
Laboratory of Diagnostics Development, China Animal Health and Epidemiology Center, 369 Nanjing Road, Qingdao, Shandong, 266032, China.
Anal Chim Acta. 2022 Aug 15;1221:340079. doi: 10.1016/j.aca.2022.340079. Epub 2022 Jun 27.
Lumpy skin disease (LSD) in cattle, a transboundary viral disease of cattle once restricted to Africa, has been spreading to many European and Asian countries in the past decade with huge economic losses. This emerging worldwide threat to cattle warrants the development of diagnostic methods for accurate disease screening of suspected samples to effectively control the spread of LSD. In this study, we integrated pre-amplification and three kinds of sensor systems with CRISPR and therefore established an LSD diagnosis platform with highly adaptable and ultra-sensitive advantages. It was the first CRISPR-powered platform that could identify lumpy skin disease virus from vaccine strains of goat pox virus and sheep pox virus. Its limit of detection (LOD) was one copy/reaction after introducing PCR or recombinase-aided amplification (RAA). Moreover, this platform achieved a satisfactory overall agreement in clinical diagnoses of 50 samples and its reproducibility and accuracy were superior to other qPCR methods we tested. The whole diagnostic procedure, from DNA extraction to the results, could complete in 5 h with a total cost of 1.7-9.6 $/test. Overall, this CRISPR-powered platform provided a novel diagnostic tool for portable, ultra-sensitive, rapid, and highly adaptable disease screening of LSD and may be an effective method to control this transboundary disease's spread.
牛结节性皮肤病（LSD）是一种曾局限于非洲的牛的跨界病毒性疾病，在过去十年中已蔓延至许多欧洲和亚洲国家，造成了巨大的经济损失。这种对牛群新出现的全球性威胁，需要开发诊断方法，以便对疑似样本进行准确的疾病筛查，从而有效控制牛结节性皮肤病的传播。在本研究中，我们将预扩增和三种传感器系统与CRISPR整合，从而建立了一个具有高度适应性和超灵敏优势的牛结节性皮肤病诊断平台。这是首个能够从山羊痘病毒和绵羊痘病毒疫苗株中识别牛结节性皮肤病病毒的CRISPR驱动平台。引入PCR或重组酶辅助扩增（RAA）后，其检测限（LOD）为一个拷贝/反应。此外，该平台在50份样本的临床诊断中总体一致性良好，其重复性和准确性优于我们测试的其他qPCR方法。从DNA提取到得出结果的整个诊断过程可在5小时内完成，每次检测总成本为1.7 - 9.6美元。总体而言，这个CRISPR驱动平台为牛结节性皮肤病的便携式、超灵敏、快速且高度适应性的疾病筛查提供了一种新型诊断工具，可能是控制这种跨界疾病传播的有效方法。
