Yang Zhihui, Leonard Susan R, Mammel Mark K, Elkins Christopher A, Kulka Michael
Division of Molecular Biology, Office of Applied research and Safety Assessment, Center for Food Safety and Applied Nutrition, Food and Drug Administration, Laurel, MD, 20708, USA.
Division of Molecular Biology, Office of Applied research and Safety Assessment, Center for Food Safety and Applied Nutrition, Food and Drug Administration, Laurel, MD, 20708, USA.
J Virol Methods. 2016 Oct;236:221-230. doi: 10.1016/j.jviromet.2016.07.013. Epub 2016 Jul 18.
Detection and identification of viruses in food samples are technically challenging due largely to the low viral copy number in contaminated food items, and the lack of effective culture enrichment methods that are amenable to regulatory applications for many of the common foodborne viruses. Using an Illumina MiSeq platform and two hepatitis A virus (HAV) cell-culture adapted strains as a representative enteric virus species, this study examined the limits of single-stranded RNA (ssRNA) viral detection following next-generation sequencing without pre-amplification of the viral genome. Complete viral genome sequences were obtained from HAV samples of varying purities and with an input as low as 2ng total RNA containing 1.4×10(5) copies of viral RNA. In addition, single nucleotide variations were reproducibly detected over the range of concentrations examined, and their identity confirmed by alternate sequencing technology. In summary, next-generation sequencing technology has the potential for sensitive detection/identification of a viral genome at a low copy number. This study provides a benchmark for metagenomic sequencing application as is required for virus detection in complex food matrices using a culture-independent diagnostic approach.
食品样本中病毒的检测和鉴定在技术上具有挑战性，这主要是由于受污染食品中的病毒拷贝数较低，而且对于许多常见的食源性病毒而言，缺乏适用于监管应用的有效培养富集方法。本研究使用Illumina MiSeq平台以及两种甲型肝炎病毒（HAV）细胞培养适应株作为代表性肠道病毒物种，在不预先扩增病毒基因组的情况下，研究了下一代测序后单链RNA（ssRNA）病毒检测的极限。从不同纯度且输入低至2ng总RNA（含1.4×10⁵个病毒RNA拷贝）的HAV样本中获得了完整的病毒基因组序列。此外，可以在检测的浓度范围内重复检测到单核苷酸变异，并通过替代测序技术确认了它们的身份。总之，下一代测序技术具有在低拷贝数下灵敏检测/鉴定病毒基因组的潜力。本研究为使用非培养诊断方法在复杂食品基质中进行病毒检测所需的宏基因组测序应用提供了一个基准。
