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

Kou, Xiaoxia, Fan, Hongying, Wu, Qingping, Xue, Liang, Zhang, Jumei
Food control 2014 v.41 pp. 151-157
RNA, Rotavirus, betaine, color, cross reaction, detection limit, feces, gel electrophoresis, genome, magnesium sulfate, monitoring, oysters, reverse transcription loop-mediated isothermal amplification, risk assessment, turbidity
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.