Evaluation of Extraction Methods for Efficient Detection of Enteric Viruses in Pork Meat Products

Abstract

We report an in-house protocol for extraction and purification of nucleic acids of enteric viruses, which gives more consistent results than representative commercial methods. The protocol uses 4 M guanidine thiocyanate, 0.5% N-lauroylsarcosine sodium salt, and 25 mM sodium citrate pH 7.0 supplemented with 0.14 M β-mercaptoethanol for lysis of virus particles. The addition of TRIzol followed by chloroform-based separation of the aqueous phase is used to purify nucleic acids from the lysate. RNA precipitation is performed using lithium chloride. This protocol was compared with QIAGEN's RNeasy Kit and bioMérieux's NucliSens method, by evaluating the ability of each to detect enteric viruses in a complex food matrix. Three different pork products, i.e., cooked ham, liver, and Spanish fermented sausage ("chorizo") were artificially contaminated with decreasing numbers of murine norovirus 1 and human adenovirus 2. The extracted and purified viral nucleic acids were detected by real-time polymerase chain reaction (PCR) assays. Whereas the two commercial extraction methods did not facilitate robust results (quantification was only possible with some viruses and/or some matrices), when coupled with the in-house protocol the linearity and the efficiency of the quantitative reverse transcription-PCR (qRT-PCR) assays were close to 1 in all the food matrices, independent of the virus. Scalability of the in-house method was evaluated by analysis of 1 and 2.5 g of spiked pig liver samples, and quantification was possible on 1 g samples contaminated with any of the two model viruses. Therefore, the in-house protocol facilitates robust qRT-PCR-based quantitative detection of viruses in pork products, and is moreover relatively cheap and simple to perform.

Detection of hepatitis E virus RNA in raw sausages and liver sausages from retail in Germany using an optimized method

“…The presence of guanidine thiocyanate which lyses the cells and inactivates RNases, as well as phenol, which denaturates proteins, within the TRI Reagent® Solution (Stals et al, 2011) may explain the efficient cell lysis. This finding is in accordance with other studies, which demonstrated that TRIzol® (a similar agent like TRI Reagent® Solution) is suitable for virus recovery especially from internally contaminated pork meat (Martinez-Martinez et al, 2011;Sair et al, 2002).…”

Public health risks associated with hepatitis E virus (HEV) as a food‐borne pathogen

“…For meat products like liver sausages, protocols using manual elimination of fat, disruption in phosphatebuffered saline (PBS), centrifugation and column-based RNA extraction (Colson et al, 2010), disruption using stomacher, centrifugation, polyethylene glycol (PEG) precipitation, chloroform-butanol treatment and bead-based RNA extraction (Martin-Latil et al, 2014), or disruption in TRI ® Reagent using stomacher, chloroform-butanol treatment and bead-based RNA extraction (Szabo et al, 2015) have been described. Systematic comparisons of the different methods by independent laboratories have not been published, although limited comparative studies on the efficiency of selected methods are available (Martinez-Martinez et al, 2011;Martin-Latil et al, 2014;Szabo et al, 2015). Reported detection limits of the methods are 2.9 9 10 3 HEV genome copies per 5 g raw sausage (Szabo et al, 2015), 5.3 9 10 4 HEV genome copies per 2 g liver sausage (Szabo et al, 2015) and 8.7 9 10 3 to 8.7 9 10 4 HEV genome copies per 3 g figatelli or liver sausage (Martinelli et al, 2015).…”

Propidium Monoazide Integrated with qPCR Enables the Detection and Enumeration of Infectious Enteric RNA and DNA Viruses in Clam and Fermented Sausages

“…Shellfish have been considered as a major source of food-borne viruses due to their filter-feeding mechanism that can concentrate virus from polluted waters (Carter, 2005). Pork meat products are also a significant route of zoonotic transmission, as enteric viruses can infect humans from the consumption of contaminated raw or undercooked pork meat (Martínez-Martínez et al, 2011). …”