Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata, Via Manfredonia 20, 71121 Foggia, Italy.
Department of Food Safety Nutrition and Veterinary Public Health, Istituto Superiore di Sanità, 00161 Rome, Italy.
Viruses. 2024 Mar 7;16(3):413. doi: 10.3390/v16030413.
Hepatitis E virus (HEV) represents an emerging risk in industrialized countries where the consumption of contaminated food plays a pivotal role. Quantitative real-time RT-PCR (RT-qPCR) is one of the most suitable methods for the detection and quantification of viruses in food. Nevertheless, quantification using RT-qPCR has limitations. Droplet digital PCR (ddPCR) provides the precise quantification of nucleic acids without the need for a standard curve and a reduction in the effect on virus quantification due to the presence of inhibitors. The objectives of the present work were (i) to develop a method for the absolute quantification of HEV in swine tissues based on ddPCR technology and provide internal process control for recovery assessment and (ii) to evaluate the performance of the method by analyzing a selection of naturally contaminated wild boar muscle samples previously tested using RT-qPCR. The method was optimized using a set of in vitro synthesized HEV RNA and quantified dsDNA. The limit of detection of the developed ddPCR assay was 0.34 genome copies/µL. The analysis of the wild boar samples confirmed the validity of the ddPCR assay. The duplex ddPCR method showed no reduction in efficiency compared to individual assays. The method developed in the present study could represent a sensitive assay for the detection and absolute quantification of HEV RNA in food samples with the advantage of presenting the co-amplification of internal process control.
戊型肝炎病毒（HEV）是工业化国家的一种新兴风险，其在这些国家中，污染食物的消费起着关键作用。实时荧光定量 RT-PCR（RT-qPCR）是检测和定量食品中病毒的最适宜方法之一。然而，RT-qPCR 的定量存在一定局限性。微滴数字 PCR（ddPCR）可提供无需标准曲线的核酸精确定量，并减少因存在抑制剂而对病毒定量的影响。本研究的目的是（i）基于 ddPCR 技术开发一种用于猪组织中 HEV 绝对定量的方法，并提供内部过程控制以评估回收率，以及（ii）通过分析先前使用 RT-qPCR 测试的一组天然污染野猪肌肉样本来评估该方法的性能。该方法使用一组体外合成的 HEV RNA 和定量 dsDNA 进行优化。所开发的 ddPCR 检测方法的检测限为 0.34 基因组拷贝/µL。对野猪样本的分析证实了 ddPCR 检测方法的有效性。与单独检测相比，双重 ddPCR 方法的效率没有降低。本研究中开发的方法可作为一种灵敏的检测和定量食品样本中 HEV RNA 的方法，其优势在于可同时进行内部过程控制的扩增。
