Yoneyama Tetsuo, Kiyohara Tomoko, Shimasaki Noriko, Kobayashi Gen, Ota Yoshinori, Notomi Tsugunori, Totsuka Atsuko, Wakita Takaji
Department of Virology II, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama, Tokyo 208-0011, Japan.
J Virol Methods. 2007 Nov;145(2):162-8. doi: 10.1016/j.jviromet.2007.05.023. Epub 2007 Jun 29.
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.
开发了一种一步法、单管实时逆转录环介导等温扩增（RT-LAMP）检测方法，用于检测甲型肝炎病毒（HAV）基因组中未翻译区域的序列。本研究报道的RT-LAMP检测方法非常简单快速；通过使用一组七条引物，在62.5℃等温条件下50分钟内即可获得HAV特异性扩增产物。属于不同亚基因型（IA、IB和IIIB）的三种细胞适应型HAV毒株的RNA扩增效果同样良好。该RT-LAMP检测方法对这些HAV毒株的检测限为0.4-0.8个焦点形成单位（FFU）/反应。使用世界卫生组织国际标准进行校准的结果表明，RT-LAMP检测方法与传统RT-PCR方法具有相似的灵敏度。对粪便临床样本进行RT-LAMP和LightCycler PCR检测结果的比较显示，两种方法的结果相似。尽管仍有几种基因型有待检测，但得出结论，新的实时RT-LAMP检测方法非常适合诊断实验室检测和定量HAV的大多数流行基因型。
