Authors: Mónica MartínezMartínez Marta DiezValcarce Nigel Cook Marta Hernández David RodríguezLázaro
Publish Date: 2010/09/30
Volume: 4, Issue: 1, Pages: 13-22
Abstract
We report an inhouse 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 05 Nlauroylsarcosine sodium salt and 25 mM sodium citrate pH 70 supplemented with 014 M βmercaptoethanol for lysis of virus particles The addition of TRIzol followed by chloroformbased 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 ie 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 realtime 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 inhouse protocol the linearity and the efficiency of the quantitative reverse transcriptionPCR qRTPCR assays were close to 1 in all the food matrices independent of the virus Scalability of the inhouse method was evaluated by analysis of 1 and 25 g of spiked pig liver samples and quantification was possible on 1 g samples contaminated with any of the two model viruses Therefore the inhouse protocol facilitates robust qRTPCRbased quantitative detection of viruses in pork products and is moreover relatively cheap and simple to performThe research leading to these results has received funding from the European Community’s Seventh Framework Programme FP7/20072013 under grant agreement n° KBBE213178 VITAL The authors thank Dr Franco Ruggeri and Dr Ilaria Dibartolo Istituto Superiore di Sanità Rome for kindly providing the MNV1 and HAdV2 viruses
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