Performance of Two Real-Time RT-PCR Assays for the Quantification of GI and GII Noroviruses and Hepatitis A Virus in Env
- PDF / 265,126 Bytes
- 8 Pages / 595.276 x 790.866 pts Page_size
- 76 Downloads / 132 Views
Performance of Two Real-Time RT-PCR Assays for the Quantification of GI and GII Noroviruses and Hepatitis A Virus in Environmental Water Samples Ann De Keuckelaere & Ambroos Stals & Leen Baert & Mieke Uyttendaele
Received: 28 January 2013 / Accepted: 20 May 2013 / Published online: 18 June 2013 # Springer Science+Business Media New York 2013
Abstract In this study, the performance of two real-time reverse transcription polymerase chain reaction (RT-qPCR) assays for the detection of hepatitis A viruses (HAV) and GI and GII noroviruses (NoV) was tested in the presence of an environmental matrix by analyzing 15 inoculated environmental water samples. For the detection of HAV, an in-house two-step RT-qPCR from literature was compared with a commercial one-step real-time RT-PCR of Ceeram (La Chapelle-sur-Erdre, France). For the detection of GI and GII NoV, an in-house duplex two-step RT-qPCR assay was used and compared with the results obtained using two commercial singleplex one-step RT-qPCR assays of Ceeram (France). The performance of the two RT-qPCR assays was determined by comparing (1) standard curves, (2) the number of detected genomic copies, and (3) the influence of inhibition by RNA dilution. Both assays for the detection of GI and GII NoV performed likewise. For the detection of HAV, the differences in genomic copies detected were to some extent more apparent and in favor of the commercial one-step assay. When the HAV RT-qPCR assays were compared in terms of inhibition, the performance of the commercial one-step RT-qPCR kit was less affected for the detection of HAV in undiluted RNA in comparison to the in-house two-step RT-qPCR assay. On the other hand, inhibition had only a marginal influence on the performance of both assays for detection of HAV in the 1/10 diluted RNA. In conclusion, only minor differences were observed between the in-house RT-qPCR assays and the commercial one-step assays for the detection of HAV and NoV in environmental water samples. A. De Keuckelaere (*) : A. Stals : L. Baert : M. Uyttendaele Department of Food Safety and Food Quality, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, 9000 Ghent, Belgium e-mail: [email protected]
Keywords Norovirus . Hepatitis Avirus . Real-time RT-PCR . Water
Introduction Food-borne viruses are recognized as a major cause of foodborne outbreaks throughout the world. Estimates say that these viruses are responsible for 59 % of all domestically acquired food-borne illnesses in the USA. Noroviruses (NoV) are responsible for the major part (99 %) of these events causing annually an estimated 5.5 million food-borne illness cases (Scallan et al. 2011). Likewise, food-borne viruses are an important causative agent of food-borne outbreaks in Europe, causing 15.0 % of all outbreaks. In the latter outbreaks, NoV were the most important food-borne viruses (EFSA 2012). Next to NoV, hepatitis A viruses (HAV) are considered as a great risk for food-borne outbreaks (Koopmans and Duizer 2004). According to the FAO/WHO, NoV and HAV ar
Data Loading...
