Wang Haiyang, Qi Zhao, Wang Jiale, He Zhenjie, Lu Liting, Chen Zhe, Shao Ying, Wang Guijun, Wang Zhenyu, Tu Jian, Song Xiangjun
Anhui Province Key Laboratory of Veterinary Pathobiology and Disease Control, College of Animal Science and Technology, Anhui Agricultural University, Hefei, 230036, P.R. China.
Anhui Province Engineering Laboratory for Animal Food Quality and Bio-Safety, College of Animal Science and Technology, Anhui Agricultural University, Hefei, 230036, P.R. China.
BMC Vet Res. 2025 Apr 2;21(1):234. doi: 10.1186/s12917-025-04711-1.
Transmissible gastroenteritis (TGE) is a highly contagious intestinal disease caused by transmissible gastroenteritis virus (TGEV). The primary techniques for identifying TGEV involve enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and fluorescent quantitative PCR (qPCR). However, these approaches are complex, demanding specialized tools and significant time. Therefore, a precise, swift, and effective differential diagnosis method is crucial for TGEV prevention. In recent years, clustered regularly interspaced short palindromic repeats (CRISPR) and Cas-associated proteins have become popular for their high specificity, unique cleavage activity, and ease of detection. CRISPR-Cas12a, a novel RNA-guided nucleic acid endonuclease, is emerging as a powerful molecular scissor.
In this study, we designed three pairs of crRNA targeting the N gene of TGEV. Following the selection of the most appropriate crRNA, we established the loop-mediated isothermal (LAMP) amplification method with a sensitivity of 10 copies/µL. And based on this, we established the CRISPR-Cas12a fluorescence assay with a sensitivity of 10 copies/µL. Furthermore, we established a CRISPR/Cas12a lateral-flow dipstick assay with a sensitivity of 10 copies/µL. Importantly, none of these methods exhibited cross-reactivity with other related viruses, enabling quicker and more straightforward observation of experimental results.
We have successfully developed a CRISPR-Cas12a fluorescence assay and a CRISPR/Cas12a lateral-flow dipstick assay for clinical TGEV detection. Overall, we created a portable, quick, and sensitive TGEV assay with strong specificity utilizing the CRISPR-Cas12a system.
传染性胃肠炎（TGE）是由传染性胃肠炎病毒（TGEV）引起的一种高度传染性肠道疾病。鉴定TGEV的主要技术包括酶联免疫吸附测定（ELISA）、聚合酶链反应（PCR）和荧光定量PCR（qPCR）。然而，这些方法复杂，需要专门的工具且耗时较长。因此，一种精确、快速且有效的鉴别诊断方法对于TGEV的预防至关重要。近年来，成簇规律间隔短回文重复序列（CRISPR）及其相关蛋白因其高特异性、独特的切割活性和易于检测而受到关注。CRISPR-Cas12a是一种新型的RNA引导核酸内切酶，正成为一种强大的分子剪刀。
在本研究中，我们设计了三对靶向TGEV N基因的crRNA。在选择最合适的crRNA后，我们建立了环介导等温扩增（LAMP）方法，灵敏度为10拷贝/μL。在此基础上，我们建立了灵敏度为10拷贝/μL的CRISPR-Cas12a荧光检测方法。此外，我们还建立了灵敏度为10拷贝/μL的CRISPR/Cas12a侧流试纸条检测方法。重要的是，这些方法均未与其他相关病毒发生交叉反应，能够更快速、直观地观察实验结果。
我们成功开发了用于临床检测TGEV的CRISPR-Cas12a荧光检测方法和CRISPR/Cas12a侧流试纸条检测方法。总体而言，我们利用CRISPR-Cas12a系统创建了一种便携、快速、灵敏且特异性强的TGEV检测方法。
