Methods and parameters of melting curve analysis for identification of Leishmania species: A scoping review

Leishmaniasis is a set of diseases with a worldwide distribution that affects mainly economically underprivileged populations in developing countries. It has a major impact on public health, with a global cost of billions of dollars per year. The treatment and control of leishmaniasis vary according to the Leishmania species involved, which require reliable methods for species identification. Since most of the currently used methods have limitations, there is a need for assays that allow rapid, precise identification of the offending species. Real-time polymerase chain reactions in conjunction with dissociation curve analysis have been used to detect differences in the DNA composition of selected genes of Leishmania spp. Kinetoplast DNA is the main molecular target used because of its high copy number per parasite, but other targets have also been studied. As part of an effort to establish melting temperature standards for each target gene, we have reviewed the pertinent literature available in public databases, including PubMed, Web of Science, SciELO and LILACS, using the keywords 'Leishmania', 'leishmaniasis', 'real-time PCR', 'melting temperature', and 'melting curve', alone or in combination. After applying eligibility criteria, 27 articles were selected for analysis. A considerable variation in the methodologies analyzed was found regarding molecular targets, standardization of the methods, reproducibility and specificity. Because of this, statistical analysis was not performed. In most cases, the methods were able to differentiate the parasite at the subgenus level or few species regardless of the target chosen.

Detection of Low-abundance Point Mutations by Competitive Strand Assisted Endonuclease Ⅳ Signal Amplification System / 华中科技大学学报（医学）（英德文版）

Genetic mutations are important molecular biomarkers for cancer diagnosis and surveillance.Therefore,the development of methods for mutation detection characterized with straightforward,highly specific and sensitive to low-level mutations within various sequence contexts is extremely needed.Although some of the currently available methods have shown very encouraging results,their discrimination efficiency is still very low.Herein,we demonstrate a fluorescent probe coupled with blocker and property of melting temperature discrimination,which is able to identify the presence of known or unknown single-base variations at abundances down to 0.1％ within 20 min.The discrimination factors between the perfect-match target and single-base mismatched target are determined to be 10.15-38.48.The method is sequence independent,which assures a wide range of application.The new method would be an ideal choice for high-throughput in vitro diagnosis and precise clinical treatment.

Modified low cost SNP genotyping technique using cycle threshold (Ct) & melting temperature (Tm) values in allele specific real-time PCR.

Background & objectives: Genotyping has now become one of the major diagnostic means for almost all diseases. Among the advanced techniques that are used to study single nucleotide polymorphisms (SNPs), only a few are applicable for routine disease diagnosis. Their applicability mainly depends on three factors: cost, time, and accuracy. The primary objective of this study was to propose allele-specific real-time PCR as a rapid, low cost and simple genotyping method for routine diagnostics. Methods: Two SNPs, rs3014866 and rs2149356 were analysed using allele-specific real-time PCR. The polymerase chain reaction was carried out using RealQ PCR master mix containing SYBR Green DNA I dye followed by melt curve analysis. The results were validated by agarose gel electrophoresis and DNA sequencing. Results: The allelic discrimination and zygosity of the two SNPs were assessed by combined cycle threshold (Ct) and melting temperature (Tm) values. Variations in Ct and Tm values among the two alleles were observed in both rs3014866 (Ct: C allele - 24±1, T allele - 27±1; Tm: C allele - 82.5±0.3, T allele - 86.3±0.2) and rs2149356 (Ct: C allele - 24±1, A allele - 26±1; Tm: C allele - 79.4±0.2, A allele - 80.4±0.3). Based on the variations, homozygous and heterozygous alleles were detected. Agarose gel electrophoresis and DNA sequencing also confirmed the allelic variation and zygosity observed in real-time PCR. Interpretation & conclusions: In diagnostic settings where a large number of samples are analysed daily, allele-specific real-time PCR assay may serve as a simple, low cost and efficient method of genotyping

Interaction of aromatic ionen oligomers with DNA.

A novel polycationic ionen was synthesized and fractionated on carboxymethyl- Sephadex using a salt gradient in 7M urea. A series of oligomers of discrete length were characterised by ultraviolet spectra. The ultraviolet spectra of oligomers revealed a new band centred at 232.5 nm which was probably due to exciton splitting. Thermal denaturation studies indicated both stabilization of the helix conformation and a higher degree of cooperativity in the melting of DNA (oligomers)n complex as compared to native calf thymus DNA. Ionen oligomers exhibited large extrinsic Cotton effect at 232.5 nm which could be attributed to exciton interaction.