Abstract：
The mixed infection of duck hepatitis A virus 3 (DHAV-3) and novel duck reovirus (NDRV) has caused significant losses to the global duck farming industry. On-site point-of-care testing of viruses plays a crucial role in the early diagnosis, prevention, and disease control. Here, we proposed an RPA-CRISPR Cas12a/Cas13a one-pot strategy (DRCFS) for rapid and simultaneous detection of DHAV-3 and NDRV. This method integrated the reaction of RPA and CRISPR Cas12a/Cas13a in a single tube, eliminating the need to open the lid during the intermediate processes and thereby avoiding aerosol contamination. On this basis, we proposed a dual RPA-CRISPR strategy coupled with a lateral flow analysis platform (DRC-LFA). This circumvented the necessity for complex instruments, enabling direct visual interpretation of results, making the test more accessible and user-friendly. Our findings demonstrated that the DRCFS method could detect DHAV-3 and NDRV at concentrations as low as 100 copy/μL, while DRC-LFA achieved limit of 101 copies/μL within 35 min. Furthermore, when DRCFS, DRC-LFA, and qPCR were employed collectively for clinical samples analysis, all three methods yielded consistent results. The specificity, sensitivity, and user-friendly of these methods rendered them invaluable for on-site virus detection.
摘要：
鸭甲型肝炎病毒3型(DHAV-3)和新型鸭呼肠孤病毒(NDRV)的混合感染给全球鸭养殖业造成了重大损失。病毒的现场即时检测在早期诊断中起着至关重要的作用,预防,和疾病控制。这里,我们提出了一种RPA-CRISPRCas12a/Cas13a一锅法(DRCFS),用于DHAV-3和NDRV的快速同时检测.该方法将RPA和CRISPRCas12a/Cas13a的反应整合在单个管中，消除了在中间过程中打开盖子的需要，从而避免了气溶胶污染。在此基础上,我们提出了一种双RPA-CRISPR策略和横向流动分析平台（DRC-LFA）。这避免了复杂仪器的必要性，能够直接直观地解释结果，使测试更易于访问和用户友好。我们的发现表明，DRCFS方法可以在低至100拷贝/μL的浓度下检测DHAV-3和NDRV，而DRC-LFA在35分钟内达到101拷贝/μL的极限。此外，当DRCFS，DRC-LFA,和qPCR共同用于临床样品分析，这三种方法均获得了一致的结果.特异性，灵敏度,这些方法的用户友好性使得它们对于现场病毒检测非常宝贵。
