Table 1_Sensitive, specific, and rapid on-site detection of calf diarrhea pathogens using the RPA-CRISPR/Cas 12a assay.docx

Calf diarrhea is a common gastrointestinal disease that usually occurs within one month of birth. The disease causes the greatest economic losses to the cattle industry. Currently, a variety of diagnostic methods have been developed for calf diarrhea infections. However, existing methods are still unsatisfactory in terms of sensitivity, specificity, simplicity, cost, and speed.To provide a more sensitive, specific, simpler, and faster detection method, we recently developed an RPA-CRISPR/Cas12a assay that can detect BVDV, BCoV, BRV, and ETEC infections in cattle on-site. Testing for each pathogen is performed in a single test tube, without the need to open the tube in the middle, and can be completed in under 50 minutes.The RPA-CRISPR/Cas12a assay can detect BVDV, BCoV, BRV, and ETEC at concentrations of at least 10 copies/μL. The RPA-CRISPR/Cas12a assay does not produce false-positive results due to the presence of other pathogens. The sensitivity of BCoV, BRV, and ETEC in the RPA-CRISPR/Cas12a quadruple assay is equivalent to that of single qPCR. The sensitivity of BVDV in the quadruple assay is slightly lower than that of the single qPCR method.Due to its sensitivity, specificity, simplicity, and rapidity, the RPA-CRISPR/Cas12a assay is more practical for on-site detection of cattle diarrhea pathogens than any existing detection method.

犊牛腹泻（Calf diarrhea）是一种常见胃肠道疾病，通常发生于出生后1个月内，该疾病给养牛业造成的经济损失最为严重。目前已针对犊牛腹泻感染开发出多种诊断方法，但现有方法在灵敏度、特异性、操作简便性、检测成本与速度方面仍不尽如人意。为开发一种灵敏度更高、特异性更强、操作更简便且检测更快的检测手段，本研究近期开发了RPA-CRISPR/Cas12a检测体系，可现场检测牛体内的牛病毒性腹泻病毒（BVDV）、牛冠状病毒（BCoV）、牛轮状病毒（BRV）以及产肠毒素大肠杆菌（ETEC）感染。该检测可在单管内完成所有靶标病原体的检测，全程无需中途开盖，且可在50分钟内完成全部操作。该RPA-CRISPR/Cas12a检测体系可检出最低浓度为10 copies/μL的上述四种病原体，且不会因其他病原体的存在而产生假阳性结果。在四重组检测体系中，BCoV、BRV与ETEC的检测灵敏度与单重实时荧光定量PCR（qPCR）相当，而BVDV的检测灵敏度略低于单重qPCR方法。凭借其优异的灵敏度、特异性、简便性与快速性，该RPA-CRISPR/Cas12a检测体系相较于现有各类检测方法，更适用于牛腹泻病原体的现场检测。