Tone Kazuya, Fujisaki Ryuichi, Yamazaki Takashi, Makimura Koichi
Graduate School of Medicine, Teikyo University, Tokyo 173-8605, Japan; Teikyo University Institute of Medical Mycology, 539 Otsuka, Hachioji, Tokyo 192-0395, Japan.
Department of Emergency Medicine, Faculty of Medicine, Teikyo University, Tokyo, Japan.
J Microbiol Methods. 2017 Jan;132:41-45. doi: 10.1016/j.mimet.2016.10.020. Epub 2016 Oct 27.
Loop-mediated isothermal amplification (LAMP) is widely used for differentiating causative agents in infectious diseases. Melting curve analysis (MCA) in conjunction with the LAMP method reduces both the labor required to conduct an assay and contamination of the products. However, two factors influence the melting temperature (Tm) of LAMP products: an inconsistent concentration of Mg ion due to the precipitation of MgPO, and the guanine-cytosine (GC) content of the starting dumbbell-like structure. In this study, we investigated the influence of inorganic pyrophosphatase (PPase), an enzyme that inhibits the production of MgPO, on the Tm of LAMP products, and examined the correlation between the above factors and the Tm value using MCA. A set of LAMP primers that amplify the ribosomal DNA of the large subunit of Aspergillus fumigatus, Penicillium expansum, Penicillium marneffei, and Histoplasma capsulatum was designed, and the LAMP reaction was performed using serial concentrations of these fungal genomic DNAs as templates in the presence and absence of PPase. We compared the Tm values obtained from the PPase-free group and the PPase-containing group, and the relationship between the GC content of the theoretical starting dumbbell-like structure and the Tm values of the LAMP product from each fungus was analyzed. The range of Tm values obtained for several fungi overlapped in the PPase-free group. In contrast, in the PPase-containing group, the variance in Tm values was smaller and there was no overlap in the Tm values obtained for all fungi tested: the LAMP product of each fungus had a specific Tm value, and the average Tm value increased as the GC% of the starting dumbbell-like structure increased. The use of PPase therefore reduced the variance in the Tm value and allowed the differentiation of these pathogenic fungi using the MCA method.
环介导等温扩增技术（LAMP）被广泛用于鉴别传染病中的病原体。结合LAMP方法的熔解曲线分析（MCA）减少了检测所需的工作量以及产物污染。然而，有两个因素会影响LAMP产物的熔解温度（Tm）：由于MgPO沉淀导致的Mg离子浓度不一致，以及起始哑铃状结构的鸟嘌呤-胞嘧啶（GC）含量。在本研究中，我们研究了抑制MgPO产生的无机焦磷酸酶（PPase）对LAMP产物Tm的影响，并使用MCA研究了上述因素与Tm值之间的相关性。设计了一组用于扩增烟曲霉、扩展青霉、马尔尼菲青霉和荚膜组织胞浆菌大亚基核糖体DNA的LAMP引物，并在有和没有PPase的情况下，以这些真菌基因组DNA的系列浓度作为模板进行LAMP反应。我们比较了无PPase组和含PPase组获得的Tm值，并分析了理论起始哑铃状结构的GC含量与每种真菌LAMP产物Tm值之间的关系。在无PPase组中，几种真菌获得的Tm值范围有重叠。相比之下，在含PPase组中，Tm值的差异较小，所有测试真菌获得的Tm值没有重叠：每种真菌的LAMP产物都有特定的Tm值，并且平均Tm值随着起始哑铃状结构的GC%增加而增加。因此，使用PPase减少了Tm值的差异，并允许使用MCA方法区分这些致病真菌。
