Perrin A, Loutreul J, Boudaud N, Bertrand I, Gantzer C
Université de Lorraine, LCPME (Laboratoire de Chimie Physique et Microbiologie pour l'Environnement), UMR 7564, Nancy, France; CNRS, LCPME, UMR 7564, Nancy, France.
ACTALIA, Food Safety Department, Villers-Bocage F-14310, France.
J Virol Methods. 2015 Nov;224:95-101. doi: 10.1016/j.jviromet.2015.08.005. Epub 2015 Aug 28.
In recent years, foodborne viruses, especially human noroviruses (NoV) and hepatitis A virus (HAV), have been increasingly reported as the causes of foodborne disease outbreaks. Soft red fruits, especially raspberries, have a high incidence among the types of food concerned. Due to low infectious doses and low concentrations of enteric viruses in food samples, it is necessary to have an efficient and rapid detection method to implement prevention measures. A standard method for virus detection and quantification in food, including raspberries (XP CEN ISO/TS 15216-1 and -2, 2013) is currently available. This method proposes a consensus detection approach by RT-real time PCR (RT-qPCR) but also a virus extraction procedure based on the elution-concentration principle. In this study, an alternative method of extraction in which RNAs are directly extracted from food matrices (based on direct RNA extraction) has been optimized. First, each step was improved to make it a highly rapid, specific and simple method. Second, the standard virus concentration method was compared with the optimized direct RNA extraction one. Human enteric viral surrogates, Murine Norovirus (MNV) and F-specific RNA bacteriophage GA, were selected according to their adhesion properties and resistance to pH close to our main targets (NoV and HAV). Raspberries were artificially contaminated using two different techniques (immersion and spotting) in order to define a recovery rate and the amounts of virus recovered. Results showed that the direct RNA extraction method revealed significantly higher viral extraction efficiency (46.2%) than the elution-concentration method (20.3%), with similar proportions of inhibitors for both. In the same way with inoculation by spotting, the best recovery rate of GA phage (39.7% against 0.7%) and MNV (42.8% against 0.5%) was observed by direct RNA extraction. For the lowest concentrations of phage and virus in the immersion bath, only the direct RNA extraction method allowed their recovery. Direct RNA extraction proved to be more effective (best recovery rate), faster (<8h) and simpler (fewer steps) than the one proposed in the CEN ISO standard method when it came to detecting enteric viruses on raspberries.
近年来，食源性病毒，尤其是人诺如病毒（NoV）和甲型肝炎病毒（HAV），作为食源性疾病暴发的病因越来越多地被报道。在相关食品类型中，软质红色水果，尤其是树莓，发病率较高。由于食品样本中肠道病毒的感染剂量低且浓度低，因此有必要采用高效快速的检测方法来实施预防措施。目前有一种用于食品（包括树莓）中病毒检测和定量的标准方法（XP CEN ISO/TS 15216-1和-2，2013）。该方法提出了一种通过逆转录实时聚合酶链反应（RT-qPCR）的共识检测方法，还有一种基于洗脱-浓缩原理的病毒提取程序。在本研究中，对一种替代提取方法进行了优化，即直接从食品基质中提取RNA（基于直接RNA提取）。首先，对每个步骤进行改进，使其成为一种高度快速、特异且简单的方法。其次，将标准病毒浓缩方法与优化后的直接RNA提取方法进行比较。根据人肠道病毒替代物小鼠诺如病毒（MNV）和F特异性RNA噬菌体GA的粘附特性和对接近我们主要目标（NoV和HAV）的pH的抗性来选择它们。使用两种不同技术（浸泡和点滴）对树莓进行人工污染，以确定回收率和回收的病毒量。结果表明，直接RNA提取方法显示出比洗脱-浓缩方法（20.3%）显著更高的病毒提取效率（46.2%），两者的抑制剂比例相似。同样，在点滴接种时，通过直接RNA提取观察到GA噬菌体的最佳回收率（39.7%，而洗脱-浓缩方法为0.7%）和MNV的最佳回收率（42.8%，而洗脱-浓缩方法为0.5%）。对于浸泡液中最低浓度的噬菌体和病毒，只有直接RNA提取方法能够回收它们。在检测树莓上的肠道病毒时，直接RNA提取方法被证明比CEN ISO标准方法中提出的方法更有效（最佳回收率）、更快（<八小时）且更简单（步骤更少）。
