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DETECTION AND DIFFERENTIATION OF FILARIAL PARASITES BY UNIVERSAL PRIMERS AND POLYMERASE CHAIN REACTION–RESTRICTION FRAGMENT LENGTH POLYMORPHISM ANALYSIS

Filarial nematode parasites are a serious cause of morbidity in humans and animals. Identification of filarial infection using traditional morphologic criteria can be difficult and lead to misdiagnosis. We report on a polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP)–based method to detect and differentiate a broad range of filarial species in a single PCR. The first internal transcribed spacer 1 (ITS1) along with the flanking 18S and 5.8S ribosomal DNA (rDNA) were isolated and cloned from Wuchereria bancrofti, Brugia malayi, and Brugia pahangi. Sequence analysis identified conserved sites in the 18S and 5.8S rDNA sequence that could be used as universal priming sites to generate ITS1-distinctive PCR products that were useful for distinguishing filariae at the genus level. The addition of a digestion of the ITS1 PCR product with the restriction endonuclease Ase I generated a fragment profile that allowed differentiation down to the species level for W. bancrofti, B. malayi, B. pahangi, Dirofilaria immitis, and D. repens. The PCR-RFLP of ITS1 rDNA will be useful in diagnosing and differentiating filarial parasites in human, animal reservoir hosts, and mosquito vectors in disease-endemic areas.

Received April 29, 2005. Accepted for publication May 27, 2005.

Acknowledgments: We thank Dr. Saravudh Suvannadabba, Dr. Suwich Thammapalo, Kobkarn Kanjanopas, Sumas Loymak, Surapong Chumpong, and officers at Filariasis Division and regional officers for their support during specimen collections. We also appreciate Dr. Suwannee Nithiuthai and Sudchit Chungpivat for their kind advice, and the staff of the Parasitology Unit, Department of Pathology, Faculty of Veterinary Sciences, and the staff of the Department of Parasitology, Faculty of Medicine, Chulalongkorn University, for their technical help. Lastly, we thank Dr. Mettanando Bhikkhu (Wat Nakprok, Bangkok) for reviewing the manuscript.

Financial support: This study was supported by the Molecular Biology Funds, Research Affairs, Faculty of Medicine, and the grants of Research Affairs for graduate students in public universities, Graduate School, Chulalongkorn University. Alisa Junpee was supported by the National Center for Genetic Engineering and Biotechnology. Yong Poovorawan is a Senior Research Scholar of the Thailand Research Fund.

^* Address correspondence to Surang Nuchprayoon, Department of Parasitology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand. E-mail: fmedstt{at}md2.md.chula.ac.th

Authors’ addresses: Surang Nuchprayoon and Alisa Junpee, Lymphatic Filariasis Research Unit, Department of Parasitology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand, Telephone: 66-2256-4387, Fax: 66-2252-4963, E-mails: fmedstt{at}md2.md.chula.ac.th and au_junpee{at}yahoo.com. Yong Poovorawan, Department of Pediatrics, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand, E-mail: yong.p{at}md.chuls.ac.th. Alan L. Scott, The W. Harry Feinstone Department of Molecular Microbiology and Immunology, The Johns Hopkins University, Bloomberg School of Hygiene and Public Health, Baltimore, MD 21287, E-mail: ascot{at}jhsph.edu.