The development of LENTICULES™ as reference materials for noroviruses

Aims: To investigate the potential for LENTICULES (TM) to act as reference materials (RMs) for noroviruses (NoV) [genogroups I (GI) and II (GII)] by determining their homogeneity and stability characteristics. Methods and Results: NoV used in this study originated from human faecal material, screened for the absence of other faecally transmitted pathogens. The norovirus strains present in the faecal material were characterized by sequencing, and samples containing GI and GII strains representative of genotypes commonly circulating in the community were selected. RMs were produced utilizing modified lenticulating technology. A batch comprising 500 LENTICULES (TM) containing both norovirus genogroups was produced according to ISO Guide 34. The batch was tested and quantified using an ISO 17025 accredited quantitative real-time RT-PCR assay. Sufficient homogeneity was established using procedures described by Fearn and Thompson (2010), while stability at less than -15 degrees C and ambient temperature (17-22 degrees C) was assessed over 52 weeks and 7 days, respectively. Conclusions: Lenticulation was shown to be an effective means of preservation of detectable NoV. LENTICULES (TM) were sufficiently homogeneous and stable throughout medium-term frozen and short-term storage at room temperature to serve as RMs. Virus LENTICULES (TM) have the advantages of being easy to manipulate, provide assigned values and do not require the manipulation of high titre clinical material. Significance and Impact of the Study: The results of this study show that norovirus LENTICULES (TM) can be used as stable RMs for quantitative real-time RT-PCR assays. They can be utilized as in-run positive extraction controls and potentially for method calibration and to enable more easy comparison of data generated by the variety of differing norovirus determination methods that have emerged in recent years. LENTICULES (TM) have the potential to provide essential elements of laboratory quality assurance systems for laboratories implementing these new methods for virus testing in foodstuffs and for those running routine analyses.