High resolution melting real-time PCR for rapid discrimination between Brugia malayi and Brugia pahangi.
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).


METHODS
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.


CONCLUSIONS
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.
Areekit
Supatra
S
Department of Biochemistry, Faculty of Medicine, Srinakharinwirot University, Bangkok, Thailand.
Kanjanavas
Pornpimon
P
Pakpitchareon
Arda
A
Khawsak
Paisarn
P
Khuchareontaworn
Sintawee
S
Sriyaphai
Thayat
T
Chansiri
Kosum
K
D000818:Animals; D017178:Brugia malayi / Q000235:genetics / Q000302:isolation & purification*; D017179:Brugia pahangi / Q000235:genetics / Q000302:isolation & purification*; D002415:Cats; D006360:Heat-Shock Proteins / Q000235:genetics; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D012091:Repetitive Sequences, Nucleic Acid; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation*; D017422:Sequence Analysis, DNA; D013997:Time Factors
D006360:Heat-Shock Proteins