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Rapid Detection of Brugia malayi in Mosquito Vectors Using a Real-time Fluorescence Resonance Energy Transfer PCR and Melting Curve Analysis

We developed real-time fluorescence resonance energy transfer (FRET) polymerase chain reaction (PCR) combined with melting curve analysis for detection of Brugia malayi DNA in blood-fed mosquitoes. Real-time FRET PCR is based on a fluorescence melting curve analysis of hybrid formed between amplicons generated from a family of repeated DNA element, 153-bp HhaI repeated sequence, specific to genus Brugia and specific fluorophore-labeled probes. The B. malayi–infected mosquitoes were differentiated from Wuchereria bancrofti–infected and uninfected mosquitoes and from genomic DNA of Dirofilaria immitis– and Plasmodium falciparum–infected human red blood cells and human leukocytes by their melting temperature. Sensitivity and specificity were both 100%. Melting curve analysis produces a rapid, accurate, and sensitive alternative for specific detection of B. malayi in mosquitoes, allows high throughput, and can be performed on small samples. This method has the potential for endemic area mapping or monitoring effect of brugian filariasis mass treatment programs.

Received October 10, 2007. Accepted for publication December 8, 2007.

Acknowledgment: The authors thank Dr Mark Roselieb for assistance in the manuscript preparation.

Financial support: This study was supported by the Diagnostic and Research Center for Emerging Infectious Diseases and by Khon Kaen University grants.

^* Address correspondence to Wanchai Maleewong, Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand. E-mail: wanch_ma{at}kku.ac.th

Authors’ addresses: Tongjit Thanchomnang, Pewpan M. Intapan, Viraphong Lulitanond, Anonglak Manjai, Thidarat K. Prasongdee, and Wanchai Maleewong, Departments of Parasitology and Microbiology, Faculty of Medicine and Diagnostic Center for Emerging Infectious Diseases, Khon Kaen University, Khon Kaen 40002, Thailand. Wej Choochote, Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand.

Reprint requests: Wanchai Maleewong, Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand, Telephone: 66-43-348387, Fax: 66-43-202475, E-mail: wanch_ma{at}kku.ac.th.