Application of Nested-qPCR-High Resolution Melting (HRM) Technology on Strongyloides stercoralis Isolates from Iran.

Abstract

Strongyloides stercoralis is a parasite with special characteristics presenting it as a unique nematode. Iran is an endemic area for S. stercoralis. In this study, nested-qPCR-high resolution melting (HRM) technology was applied on some human isolates of S. stercoralis from this country by focusing on evolutionary genetics analysis. Twelve human isolates of S. stercoralis were collected from four endemic provinces of Iran. Genomic DNA was extracted from a single filariform larva for every isolate. Using specific primers targeting partial regions in cox1 gene, nested-qPCR-HRM was performed and melting-curve profiles were analyzed alongside the evaluation of genetic proximity and phylogenetic analysis using MEGA7 and DnaSP5 software. The melting temperature (Tm) values of the isolates were 77.9°C-78.3°C. All isolates from Guilan, Mazandaran, and Khouzestan Provinces shared Tm values of 78.2°C to 78.3°C, while the isolates from Hormozgan Province showed Tm values of 77.9°C, 78.0°C, and 78.1°C. The phylogenetic tree illustrated that thesequences of the current study included nine haplotypes. Tajima's D index analyses showed that cox1 gene in S. stercoralis isolates was negative (Tajima's D = - 0.27). The isolates were divided into five temperature groups. Although HRM assay compared to PCR sequencing identified more limited genetic changes, it revealed that the mean of Tm of the isolates from Hormozgan Province was lower than those of other provinces and represented specific haplotypes for this geographical region on the phylogenetic tree.