caa a22 4500 445339683 CHVBK 20180317142808.0 cr unu---uuuuu 170323e20111201xx s 000 0 eng 10.1007/s00436-011-2419-z doi (NATIONALLICENCE)springer-10.1007/s00436-011-2419-z Rapid detection of Opisthorchis viverrini and Strongyloides stercoralis in human fecal samples using a duplex real-time PCR and melting curve analysis [Elektronische Daten] [Penchom Janwan, Pewpan Intapan, Tongjit Thanchomnang, Viraphong Lulitanond, Witthaya Anamnart, Wanchai Maleewong] Human opisthorchiasis caused by the liver fluke Opisthorchis viverrini is an endemic disease in Southeast Asian countries including the Lao People's Democratic Republic, Cambodia, Vietnam, and Thailand. Infection with the soil-transmitted roundworm Strongyloides stercoralis is an important problem worldwide. In some areas, both parasitic infections are reported as co-infections. A duplex real-time fluorescence resonance energy transfer (FRET) PCR merged with melting curve analysis was developed for the rapid detection of O. viverrini and S. stercoralis in human fecal samples. Duplex real-time FRET PCR is based on fluorescence melting curve analysis of a hybrid of amplicons generated from two genera of DNA elements: the 162bp pOV-A6 DNA sequence specific to O. viverrini and the 244bp 18S rRNA sequence specific to S. stercoralis, and two pairs of specific fluorophore-labeled probes. Both O. viverrini and S. stercoralis can be differentially detected in infected human fecal samples by this process through their different fluorescence channels and melting temperatures. Detection limit of the method was as little as two O. viverrini eggs and four S. stercoralis larvae in 100mg of fecal sample. The assay could distinguish the DNA of both parasites from the DNA of negative fecal samples and fecal samples with other parasite materials, as well as from the DNA of human leukocytes and other control parasites. The technique showed 100% sensitivity and specificity. The introduced duplex real-time FRET PCR can reduce labor time and reagent costs and is not prone to carry over contamination. The method is important for simultaneous detection especially in areas where both parasites overlap incidence and is useful as the screening tool in the returning travelers and immigrants to industrialized countries where number of samples in the diagnostic units will become increasing. Springer-Verlag, 2011 Janwan Penchom Department of Parasitology, Faculty of Medicine, Khon Kaen University, 40002, Khon Kaen, Thailand aut Intapan Pewpan Department of Parasitology, Faculty of Medicine, Khon Kaen University, 40002, Khon Kaen, Thailand aut Thanchomnang Tongjit Faculty of Medicine, Mahasarakham University, 44000, Mahasarakham, Thailand aut Lulitanond Viraphong Department of Microbiology, Faculty of Medicine, Khon Kaen University, 40002, Khon Kaen, Thailand aut Anamnart Witthaya Department of Medical Technology, School of Allied Health Sciences and Public Health, Walailak University, 80160, Thasala, Nakhon Si Thammarat, Thailand aut Maleewong Wanchai Department of Parasitology, Faculty of Medicine, Khon Kaen University, 40002, Khon Kaen, Thailand aut Parasitology Research Springer-Verlag 109/6(2011-12-01), 1593-1601 0932-0113 109:6<1593 2011 109 436 https://doi.org/10.1007/s00436-011-2419-z text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s00436-011-2419-z text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Janwan Penchom Department of Parasitology, Faculty of Medicine, Khon Kaen University, 40002, Khon Kaen, Thailand aut NATIONALLICENCE 700 1- Intapan Pewpan Department of Parasitology, Faculty of Medicine, Khon Kaen University, 40002, Khon Kaen, Thailand aut NATIONALLICENCE 700 1- Thanchomnang Tongjit Faculty of Medicine, Mahasarakham University, 44000, Mahasarakham, Thailand aut NATIONALLICENCE 700 1- Lulitanond Viraphong Department of Microbiology, Faculty of Medicine, Khon Kaen University, 40002, Khon Kaen, Thailand aut NATIONALLICENCE 700 1- Anamnart Witthaya Department of Medical Technology, School of Allied Health Sciences and Public Health, Walailak University, 80160, Thasala, Nakhon Si Thammarat, Thailand aut NATIONALLICENCE 700 1- Maleewong Wanchai Department of Parasitology, Faculty of Medicine, Khon Kaen University, 40002, Khon Kaen, Thailand aut NATIONALLICENCE 773 0- Parasitology Research Springer-Verlag 109/6(2011-12-01), 1593-1601 0932-0113 109:6<1593 2011 109 436 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer