Huang Siyu, Du Longhuan, Liu Song, Yang Qingcheng, Lei Changwei, Wang Hongning, Yang Liu, Yang Xin
Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Province, College of Life Sciences, Sichuan University, Chengdu 610064, China.
Animal Breeding and Genetics Key Laboratory of Sichuan Province, Sichuan Animal Science Academy, Chengdu 610066, China.
Animals (Basel). 2024 Nov 25;14(23):3387. doi: 10.3390/ani14233387.
Piglet diarrhea poses significant economic losses to the pig industry, posing a worldwide challenge that urgently needs to be addressed in pig breeding practices. Porcine rotavirus (PoRV) is an important viral diarrhea pathogen in piglets, with a high incidence rate and a tendency to cause growth retardation. To enhance the sensitivity and specificity of PoRV detection, we sequenced the NSP3 gene of G5 and G9 genotypes of rotavirus A (RVA), enabling simultaneous detection of the two serotypes. Subsequently, we developed a rapid PoRV detection method using a combination of recombinase-aided amplification (RAA) and CRISPR/Cas12a. In this method, Cas12a binds to RAA amplification products, guided by CRISPR-derived RNA (crRNA), which activates its cleavage activity and releases fluorescence by cutting FAM-BHQ-labeled single-stranded DNA (ssDNA). In the optimized reaction system, the recombinant plasmid PoRV can achieve a highly sensitive reaction within 30 min at 37 °C, with a detection limit as low as 2.43 copies/μL, which is ten times higher in sensitivity compared to the qPCR method. Results from specificity testing indicate that no cross-reactivity was observed between the RAA-CRISPR/Cas12a analysis of PoRV and other viral pathogens, including PoRV G3, PoRV G4, porcine epidemic diarrhea virus (PEDV), porcine epidemic diarrhea (PDCoV), and porcine reproductive and respiratory syndrome virus (PRRSV). In the clinical sample detection using the RAA-CRISPR/Cas12a method and qPCR, Cohen's Kappa value reached as high as 0.952. Furthermore, this approach eliminates the need for large-scale instrumentation, offering a visual result under an ultraviolet lamp through fluorescence signal output.
仔猪腹泻给养猪业带来了巨大的经济损失，这是一个全球性挑战，亟待在养猪实践中加以解决。猪轮状病毒（PoRV）是仔猪重要的病毒性腹泻病原体，发病率高，且有导致生长发育迟缓的趋势。为提高PoRV检测的灵敏度和特异性，我们对A组轮状病毒（RVA）G5和G9基因型的NSP3基因进行了测序，实现了两种血清型的同时检测。随后，我们开发了一种将重组酶辅助扩增（RAA）与CRISPR/Cas12a相结合的快速PoRV检测方法。在该方法中，Cas12a在CRISPR衍生RNA（crRNA）的引导下与RAA扩增产物结合，激活其切割活性，并通过切割FAM-BHQ标记的单链DNA（ssDNA）释放荧光。在优化的反应体系中，重组质粒PoRV在37℃下30分钟内即可实现高灵敏度反应，检测限低至2.43拷贝/μL，灵敏度是qPCR方法的10倍。特异性测试结果表明，PoRV的RAA-CRISPR/Cas12a分析与其他病毒病原体（包括PoRV G3、PoRV G4、猪流行性腹泻病毒（PEDV）、猪德尔塔冠状病毒（PDCoV）和猪繁殖与呼吸综合征病毒（PRRSV））之间未观察到交叉反应。使用RAA-CRISPR/Cas12a方法和qPCR对临床样本进行检测时，Cohen's Kappa值高达0.952。此外，该方法无需大型仪器设备，通过荧光信号输出在紫外灯下即可获得可视化结果。
