Colorimetric detection of norovirus genotype GII by reverse transcription loop-mediated isothermal amplification

• Published in: Bingdu xuebao Vol. 28; no. 2; p. 165
• Main Authors: Luo, Jian-Ming, Wu, Xi-Yang, Xu, Zi-Qian, Luo, Le, Nie, Kai, Yang, Meng-Jie, Zeng, Ya-Lan, Duan, Zhao-Jun, Ma, Xue-Jun
• Format: Journal Article
• Language: Chinese
• Published: China 01.03.2012
• Subjects: Caliciviridae Infections - diagnosis; Caliciviridae Infections - virology; Colorimetry - methods; Feces - virology; Genotype; Humans; Norovirus - genetics; Norovirus - isolation & purification; Nucleic Acid Amplification Techniques - methods
• ISSN: 1000-8721

A simple, rapid and sensitive colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) method was established to detect norovirus genotype GII. The method employed a set of six specially designed primers that recognized eight distinct sequences of RNA-dependant RNA polymerase and capsid protein gene for amplification of nucleic acid under isothermal conditions at 65 degrees C for 60 minutes. The amplification process of RT-LAMP was monitored by the addition of HNB (Hydroxy naphthol blue) dye prior to amplification. A positive reaction was indicated by a color change from violet to sky blue and confirmed by agarose electrophoresis. The specificity of the RT-LAMP was validated by detecting several different diarrhea viruses including norovirus genotype GII. The sensitivity was determined by serial dilutions of RNA molecules from in vitro transcription of norovirus genotype GII in parallel with conventional RT-PCR detection. The assay was further evaluated with 93 clinical specimens of