Development and application of a loop-mediated isothermal amplification assay on rapid and sensitive detection of rotavirus in fecal samples and artificially seeded oysters

- The RT-LAMP primers were designed and the reaction conditions were optimized.
- The specificity and sensitivity of the RT-LAMP assay were assessed.
- Validate the RT-LAMP in clinical samples and artificially seeded oysters.

A reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay was developed for detection of rotavirus. Four primers were designed to recognize six distinct regions based on a highly conserved sequence in the VP7 region of the rotavirus genome. The optimal conditions for the LAMP assay were determined to 62 °C for 60 min with 8.0 mM MgSO4, 0.2 M betaine, 0.8 mM dNTPs, 0.2 μM each of outer primer, 1.6 μM each of inner primer. After amplification the products were detected either by observing a ladder pattern following gel electrophoresis, observation of turbidity, or a color change with the addition of SYBR Green I to the reaction tube. A ladder pattern of bands after gel electrophoresis was observed for only rotavirus isolates and showed that the rotavirus RT-LAMP assay was highly specific without any cross-reactivity with norovirus and astrovirus. The RT-LAMP assay was evaluated further using 79 fecal samples and 32 artificially seeded oyster samples. The detection limit of the RT-LAMP assay was 0.5 pg of rotavirus RNA. The sensitivity and simplicity of the test can served as a rapid and economic method for routine monitoring and risk assessment from clinical samples or marine products in both field conditions and laboratory settings.