High Resolution Melting Real-Time PCR for Rapid Discrimination between Brugia malayi and Brugia Pahangi
Supatra Areekit MSc*, Pornpimon Kanjanavas PhD*, Arda Pakpitchareon MSc*, Paisarn Khawsak MSc*, Sintawee Khuchareontaworn MSc*, Thayat Sriyaphai MSc*, Kosum Chansiri PhD*
Affiliation : * Department of Biochemistry, Faculty of Medicine, Srinakharinwirot University, Bangkok, Thailand
Objective : To identify two closely related Brugia malayi and B. pahangi in cat reservoirs by using high
resolution melting real-time PCR (HRM real-time PCR)
Material and Method: HRM analysis on the Corbett Rotor-Gene 6000 instrument was used to test 5 Brugia
specimens by using five sets of specific primers for HhaI repetitive region (HR), small heat shock protein
(SHP), small subunit ribosomal DNA (18S rDNA), internal transcribed spacer region (ITS), and trans-spliced
leading Exon I gene (SLX1).
Results : HRM analysis of ITS and SLX clearly generated 2 profiles of B. malayi and B. pahangi while those of
HR, 18S rDNA, and SHP could classify B. pahangi.
Conclusion : HRM is a simple and rapid method for identification of two closely related B. malayi and B.
pahangi in which it can detect both parasites within 30 min after real-time PCR detection. This assay is probe-
free HRM and reduces a risk of PCR carryover. It does not require multiplex methods and DNA sequencing;
therefore, HRM provides a new approach for genetic screening and rapid detection of closely related species
in a clinical laboratory.
Keywords : Brugia, HhaI repetitive region, Small heat shock protein, Small subunit ribosomal DNA, Internal transcribed spacer, Tran-spliced leading Exon I gene, High- melting resolution, PCR, High resolution melting
