Rapid and real-time detection of hepatitis A virus by reverse transcription loop-mediated isothermal amplification assay.
A one-step, single tube, real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detecting sequences of the untranslated region in the genome of hepatitis A virus (HAV). The RT-LAMP assay reported in this study was very simple and rapid; the HAV-specific amplification was obtained in 50 min under isothermal conditions at 62.5 degrees C by employing a set of seven primers. The RNAs of three cell-adapted HAV strains belonging to different subgenotypes (IA, IB and IIIB) were equally well amplified. The detection limits of the RT-LAMP assay for these HAV strains were 0.4-0.8 focus forming units (FFU)/reaction. The results of the calibration using the WHO international standard indicated that the RT-LAMP assay had similar sensitivity to the conventional RT-PCR method. A comparison of the results from the RT-LAMP and the LightCycler PCR assay using clinical samples in feces revealed that the findings were similar between the two methods. Although several genotypes remain to be tested, it is concluded that the new real-time RT-LAMP assay is very suitable for detection and quantitation of most prevalent genotypes of HAV in diagnostic laboratories.
Yoneyama
Tetsuo
T
Department of Virology II, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama, Tokyo 208-0011, Japan. tyoneyam@nih.go.jp
Kiyohara
Tomoko
T
Shimasaki
Noriko
N
Kobayashi
Gen
G
Ota
Yoshinori
Y
Notomi
Tsugunori
T
Totsuka
Atsuko
A
Wakita
Takaji
T
D001483:Base Sequence; D030041:Hepatitis A virus / Q000302:isolation & purification*; D006801:Humans; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity