Xia Yongbo, Li Yue, He Yihong, Wang Xiaowei, Qiu Wenjing, Diao Xiaoyuan, Li Yunfei, Gao Junfeng, Shen Hanqin, Xue Chunyi, Cao Yongchang, Li Peng, Xu Zhichao
State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510006, China.
Department of Immunology and Genomic Medicine, National Jewish Health, Denver, CO, 80206, USA.
Adv Biotechnol (Singap). 2024 Feb 27;2(1):7. doi: 10.1007/s44307-024-00015-x.
Porcine epidemic diarrhea virus (PEDV), Transmissible gastroenteritis virus (TGEV), Porcine deltacoronavirus (PDCoV) and Swine acute diarrhea syndrome coronavirus (SADS-CoV) rank among the most frequently encountered swine enteric coronaviruses (SECoVs), leading to substantial economic losses to the swine industry. The availability of a rapid and highly sensitive detection method proves beneficial for the monitoring and surveillance of SECoVs. Based on the N genes of four distinct SECoVs, a novel detection method was developed in this study by combining recombinant enzyme polymerase isothermal amplification (RPA) with clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins (Cas) 12a. Results showed that the cut-off value of CRISPR-Cas12a assay for SADS-CoV, PEDV, PDCoV and TGEV was 2.19 × 10 Relative Fluorescence Units (RFU), 1.57 × 10 RFU, 3.07 × 10 RFU and 1.64 × 10 RFU, respectively. The coefficient of variation (CV) of within and between runs by CRISPR-Cas12a assay for 6 clinical diarrhea samples were both less than 10%. The CRISPR-Cas12a assay demonstrated high specificity for TGEV, PEDV, PDCoV, and SADS-CoV with no cross-reactivity to other common swine viruses. This method also exhibited a low limit of detection of 2 copies for each virus. Additionally, the results demonstrated a perfect agreement (100%) between the CRISPR-Cas12a assay and the RT-qPCR assay. Finally, a total of 494 pig samples from the field tested by CRISPR-Cas12a assay showed that positive rate for SADS-CoV, TGEV, PDCoV and PEDV was 0, 0, 1.2% and 48.6%, respectively. The results suggested the great potential of CRISPR-Cas12a assay to detect SECoVs in the field.
猪流行性腹泻病毒(PEDV)、传染性胃肠炎病毒(TGEV)、猪三角洲冠状病毒(PDCoV)和猪急性腹泻综合征冠状病毒(SADS-CoV)是最常见的猪肠道冠状病毒(SECoV),给养猪业造成了巨大经济损失。快速且高度灵敏的检测方法对于SECoV的监测和 surveillance 很有帮助。基于四种不同SECoV的N基因,本研究通过将重组酶聚合酶等温扩增(RPA)与成簇规律间隔短回文重复序列(CRISPR)相关蛋白(Cas)12a相结合,开发了一种新型检测方法。结果表明,CRISPR-Cas12a检测法对SADS-CoV、PEDV、PDCoV和TGEV的截断值分别为2.19×10相对荧光单位(RFU)、1.57×10 RFU、3.07×10 RFU和1.64×10 RFU。CRISPR-Cas12a检测法对6份临床腹泻样本进行批内和批间检测的变异系数(CV)均小于10%。CRISPR-Cas12a检测法对TGEV、PEDV、PDCoV和SADS-CoV具有高度特异性,与其他常见猪病毒无交叉反应。该方法对每种病毒的检测限也低至2个拷贝。此外,结果表明CRISPR-Cas12a检测法与RT-qPCR检测法完全一致(100%)。最后,通过CRISPR-Cas12a检测法对总共494份田间猪样本进行检测,结果显示SADS-CoV、TGEV、PDCoV和PEDV的阳性率分别为0、0、1.2%和48.6%。结果表明CRISPR-Cas12a检测法在田间检测SECoV方面具有巨大潜力。