Kuhara Tomoe, Yoshikawa Tetsushi, Ihira Masaru, Watanabe Daisuke, Tamada Yasuhiko, Katano Harutaka, Asano Yoshizo, Matsumoto Yoshinari
Department of Dermatology, Aichi Medical University School of Medicine, Aichi, Nagakute, Japan.
J Virol Methods. 2007 Sep;144(1-2):79-85. doi: 10.1016/j.jviromet.2007.03.021. Epub 2007 May 23.
The reliability of a loop-mediated isothermal amplification (LAMP) method for the detection of human herpesvirus 8 (HHV-8) DNA was evaluated. Although LAMP products were produced with the DNA sample extracted from BCP-1 cells, LAMP products were not produced with the DNAs from seven other human herpesviruses. The detection limit of the HHV-8 LAMP method was 100 copies of target sequence/tube. To determine whether the HHV-8 LAMP method could be used to quantify viral DNA, threshold times, which are defined as the time (in s) it takes to reach the threshold turbidity level (0.1), were measured for the amplification of serial dilutions of a DNA plasmid containing the target sequence. The standard curve possessed a correlation coefficient of 0.9428 with a slope of -84.079 and y-intercept value of 1936.2. Additionally, an attempt was made to detect viral DNA in 17 specimens collected from Kaposi's sarcomas and two cell lines obtained from primary effusion lymphomas. HHV-8 DNA was detected in 14 of the 17 Kaposi's sarcoma tissue samples and both of the primary effusion lymphoma cell lines. Viral DNA was not detected in HHV-8 LAMP-negative samples using the real-time PCR method.
评估了一种环介导等温扩增（LAMP）方法检测人类疱疹病毒8型（HHV-8）DNA的可靠性。虽然从BCP-1细胞提取的DNA样本产生了LAMP产物，但其他七种人类疱疹病毒的DNA未产生LAMP产物。HHV-8 LAMP方法的检测限为100个靶序列拷贝/管。为了确定HHV-8 LAMP方法是否可用于定量病毒DNA，对含有靶序列的DNA质粒系列稀释液的扩增测量了阈值时间，阈值时间定义为达到阈值浊度水平（0.1）所需的时间（以秒为单位）。标准曲线的相关系数为0.9428，斜率为-84.079，y轴截距值为1936.2。此外，还尝试检测从卡波西肉瘤收集的17个标本和从原发性渗出性淋巴瘤获得的两个细胞系中的病毒DNA。在17个卡波西肉瘤组织样本中的14个以及两个原发性渗出性淋巴瘤细胞系中均检测到HHV-8 DNA。使用实时PCR方法在HHV-8 LAMP阴性样本中未检测到病毒DNA。
