Ossio Axel, Merino-Mascorro Angel, Leon Juan S, Heredia Norma, Garcia Santos
Laboratorio de Bioquímica y Genética de Microorganismos, Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León, 066455, San Nicolás de los Garza, N.L., Mexico.
Hubert Department of Global Health, Rollins School of Public Health, Emory University, Atlanta, GA, USA.
Food Environ Virol. 2025 Jun 21;17(3):35. doi: 10.1007/s12560-025-09651-5.
Standard food detection methods do not distinguish between infectious and non-infectious human norovirus leading to uncertainty in the management of a norovirus positive food sample. These methods also require expensive RT-qPCR-based equipment and reagents. In contrast, CRISPR-based, compared to RT-qPCR-based, detection methods are generally less expensive and yield similar sensitivity and specificity. Our goal was to detect norovirus with an intact capsid, a proxy for infectivity, through a CRISPR-Cas13a-based detection method together with an RNase-capsid integrity assay. We termed this assay: Foodborne RNA-virus Enzymatic Sensing for High-throughput on fresh produce (CRISPR FRESH) reflecting its potential to detect infectious or potentially infectious virus particles. Our CRISPR FRESH method detected murine norovirus (MNV-1), with an intact capsid, at a limit of detection of 2.59 log10 gc/25 g (5 gc/rx). This method did not cross-react with other targets (synthetic DNA targets for hepatitis A virus; human norovirus GI, GII; rotavirus). Compared with RT-qPCR, CRISPR FRESH showed an increased sensitivity when detecting low copy numbers of RNase-pre-treated MNV-1 in lettuce and blueberries samples. Viral detection with the RT-qPCR assay is quantifiable while the CRISPR assay is present/absent. This report describes a CRISPR-based detection of potentially infectious viruses in food samples.
标准的食品检测方法无法区分感染性和非感染性人类诺如病毒，这导致诺如病毒呈阳性的食品样本管理存在不确定性。这些方法还需要昂贵的基于逆转录定量聚合酶链反应（RT-qPCR）的设备和试剂。相比之下，与基于RT-qPCR的检测方法相比，基于成簇规律间隔短回文重复序列（CRISPR）的检测方法通常成本更低，且灵敏度和特异性相当。我们的目标是通过基于CRISPR-Cas13a的检测方法以及核糖核酸酶-衣壳完整性检测，来检测具有完整衣壳的诺如病毒（感染性的替代指标）。我们将此检测方法命名为：新鲜农产品上高通量食源性RNA病毒酶促传感检测法（CRISPR FRESH），以反映其检测感染性或潜在感染性病毒颗粒的潜力。我们的CRISPR FRESH方法检测到具有完整衣壳的鼠诺如病毒（MNV-1），检测限为2.59 log10基因组拷贝数/25克（5个基因组拷贝数/反应）。该方法与其他靶标（甲型肝炎病毒的合成DNA靶标；人类诺如病毒GI、GII；轮状病毒）无交叉反应。与RT-qPCR相比，在检测生菜和蓝莓样本中经核糖核酸酶预处理的低拷贝数MNV-1时，CRISPR FRESH显示出更高的灵敏度。RT-qPCR检测法可对病毒进行定量，而CRISPR检测法只能判断病毒是否存在。本报告描述了基于CRISPR的食品样本中潜在感染性病毒的检测方法。
