Enhancing melting curve analysis for the discrimination of loop-mediated isothermal amplification products from four pathogenic molds: Use of inorganic pyrophosphatase and its effect in reducing the variance in melting temperature values.

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 Mg2+ ion due to the precipitation of Mg2P2O7, 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 Mg2P2O7, 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.