The development and application of multiplex real-time reverse transcriptase polymerase chain reaction assays for the detection of enteric viruses on berry fruits and in water samples

Abstract

The transmission of enteric viruses by food, food products and water remains a wellrecognised, largely underestimated widespread public health problem. Outbreaks of gastroenteritis and hepatitis A due to the consumption of contaminated berry fruits have become a growing phenomenon worldwide. Contamination of fresh produce and other minimally processed foods can be attributed to pre- and post-harvest irrigation and washing water and food handlers. The prevention of such outbreaks relies on the optimisation of adequate methods for the recovery and detection of enteric viruses from food matrices and irrigation water. The aim of this study was to develop and apply optimised multiplex real-time reverse transcriptase-polymerase chain reaction (rt RT-PCR) assays for the detection of selected enteric viruses on berry fruits and in paired associated irrigation waters. In this study quality control measures were implemented by the development and optimisation of an internal amplification control (IAC) for norovirus (NoV) GII to monitor for the success of the amplification process. Mengovirus was used as a process control to validate the recovery and nucleic acid extraction of selected enteric viruses from strawberries and in associated irrigation waters. Three multiplex rt RT-PCR assays for the detection of NoV GI, NoV GII, sapovirus , hepatitis A virus, human astrovirus, human rotavirus and mengovirus were optimised with the IAC. Blackberries and strawberries were used to assess the efficiency of three nucleic acid extraction kits with the most efficient used in further investigations. Three elution buffers based on protein concentration, pH, Tris and elution period were assessed for the recovery of the viruses from the berry fruits. The pH more so than the protein concentration proved to be more effective in the recovery of the selected enteric viruses from the strawberries with no analytical significant differences noted for the two 3% glycine-beef extract (GBE) buffers assessed, irrespective of the parameters considered. During the period September 2010 to August 2011, strawberries and associated irrigation water were collected from which NoV GII, NoV GI and HAV could be recovered using a 3% tris-GBE pH 9.5 elution buffer and a glass-wool absorption elution method, respectively, and detected using optimised singleplex rt RT-PCR assays. The irrigation water samples together with eight surface and three groundwater samples collected from the Limpopo area was retested using the optimised multiplex rt RT-PCR assays. The multiplex rt RT-PCR assays proved to be more efficient in the detection of NoVs than the commercial environmental rt RT-PCR assays with lower detection efficiencies noted for HAV. Commercially obtained strawberries were dipped in polluted surface water, the viruses recovered from both and detected using the optimised multiplex rt RT-PCR assays resulted in the detection of similar viruses on both the strawberries and polluted irrigation water. Norovirus GII.7 and swine NoV GII.18 were identified on the strawberries and in the associated irrigation water, respectively. This is the first report of swine NoVs in South Africa, and begs the question as to the possibility of zoonotic NoV infection. This link between the viruses detected on the surface of the strawberries and in the irrigation water could not be confirmed by typing data. From this study, a functional AC was developed and used in the development and optimisation of three multiplex rt RT-PCR assays which made the gathering of new data of the role of irrigation water as a source of contamination of irrigated berry fruits in SA possible.