Purpose: This study compared the detection sensitivity of previously described real-time RT-PCR to RT-LAMP assays using clinical outbreak samples.
Methods: Human norovirus GII clinical isolates (total of 9) were assayed by heat release or RNA extracts obtained by the TRIzol™ method using real-time SYBR Green I based RT-PCR kits and RT-LAMP assays in triplicate. Serially diluted heat-released samples were used in 50 µl reaction volumes. For the RT-PCR assay, reaction conditions included reverse transcription at 50 °C for 40 min, 94 °C for 3 min, and 42 cycles at 94 °C/45 s, 56 °C/45 s, 58 °C/30 s, and final extension at 72 °C/7 min, while the RT-LAMP assay was carried out at 63 °C for 90 min. Confirmation of product size (98 bp and Tm of 84 °C for RT-PCR) or ladder patterns for the RT-LAMP assay was obtained by agarose gel electrophoresis.
Results: The detection limit study indicated that viral RNA extracted by the TRIzol™ method consistently detected one-log RT-PCR unit (-1 dilution) using the real-time-RT-PCR assay for all 9 tested clinical samples. However, the detection limit improved by one log using the RT-LAMP assay compared to the RT-PCR assay. Heat-released samples showed one-log higher detection than RNA extracts by the RT-PCR assay (2-log RT-PCR units), but was less sensitive by RT-LAMP for some samples, due to stool sample inhibition.
Significance: The RT-LAMP assay can be completed within 1.5 h followed by agarose gel electrophoresis, without requiring expensive thermocyclers. The RT-LAMP assay is more sensitive than the RT-PCR assay for extracted RNA. This assay shows promise for the rapid screening of hNoV GII in clinical samples and has potential application for food testing.