Korean J Parasitol.
2013 Dec;51(6):645-650. 10.3347/kjp.2013.51.6.645.
Rapid Detection and Identification of Wuchereria bancrofti, Brugia malayi, B. pahangi, and Dirofilaria immitis in Mosquito Vectors and Blood Samples by High Resolution Melting Real-Time PCR
- Affiliations
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- 1Research and Diagnostic Center for Emerging Infectious Diseases, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand. wanch_ma@kku.ac.th
- 2Faculty of Medicine, Mahasarakham University, Mahasarakham 44000, Thailand.
- 3Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand.
- 4Division of Cell Biology, Department of Preclinical Sciences, Faculty of Medicine, Thammasat University, Rangsit Campus, Pathum Thani 12121, Thailand.
- 5Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand.
- 6Parasitology Unit, Department of Pathology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand.
- 7Department of Biochemistry, Faculty of Medical Science, Naresuan University, Phitsanulok 65000, Thailand.
- 8Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand.
- KMID: 1712216
- DOI: http://doi.org/10.3347/kjp.2013.51.6.645
Abstract
- A simple, rapid, and high-throughput method for detection and identification of Wuchereria bancrofti, Brugia malayi, Brugia pahangi, and Dirofilaria immitis in mosquito vectors and blood samples was developed using a real-time PCR combined with high-resolution melting (HRM) analysis. Amplicons of the 4 filarial species were generated from 5S rRNA and spliced leader sequences by the real-time PCR and their melting temperatures were determined by the HRM method. Melting of amplicons from W. bancrofti, B. malayi, D. immitis, and B. pahangi peaked at 81.5+/-0.2degrees C, 79.0+/-0.3degrees C, 76.8+/-0.1degrees C, and 79.9+/-0.1degrees C, respectively. This assay is relatively cheap since it does not require synthesis of hybridization probes. Its sensitivity and specificity were 100%. It is a rapid and technically simple approach, and an important tool for population surveys as well as molecular xenomonitoring of parasites in vectors.
Keyword
MeSH Terms
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Animals
Blood/*parasitology
Brugia/classification/genetics/*isolation & purification
Cats
Culicidae/*parasitology
Dirofilaria immitis/classification/genetics/*isolation & purification
Dogs
Humans
Male
Parasitology/*methods
RNA, Helminth/genetics
RNA, Ribosomal, 5S/genetics
Real-Time Polymerase Chain Reaction/*methods
Sensitivity and Specificity
Transition Temperature
Wuchereria bancrofti/classification/genetics/*isolation & purification
RNA, Helminth
RNA, Ribosomal, 5S
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