Journal
EXPERIMENTAL PARASITOLOGY
Volume 132, Issue 2, Pages 237-242Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.exppara.2012.07.007
Keywords
Opisthorchis viverrini; Haplorchis taichui; Multiplex detection; HAT-RAPD
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Funding
- Thailand Research Fund through the Applied Technology in Biodiversity Research Unit, Institute for Science and Technology Research, Chiang Mai University
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Specific primers for the detection of Opisthorchis viverrini and Haplorchis taichui were investigated by using the HAT-RAPD PCR method. Fourteen arbitrary primers (Operon Technologies) were performed for the generation of polymorphic DNA profiles. The results showed that a 319 bp fragment generated from the OPA-04 primer was expected to be O. viverrini-specific while a 256 bp fragment generated from the OPP-11 primer was considered to be H. taichui-specific. Based on each sequence data, two pairs of specific primers were designed and sequences of each primer were as follows; H. taichui; Hapt_F5'-GGCCAACGCAATCGTCATCC-3'and Hapt_R1 5'-CTCTCGACCTCCTCTAGAAT-3' which yielded a 170 bp PCR product. For O. viverrini, OpV-1F: 5'-AATCGGGCTGCATATTGACCGAT-3' and OpV-1R: 5'-CGGTGTTGCTT ATTTTGCAGACAA-3' which generated a 319 bp PCR product. These specific primers were tested for efficacy and specific detection for all parasites DNA samples. The results showed that 170 and 319 bp specific PCR products were generated as equivalent to positive result in H. taichui and O. viverrini, respectively by having no cross-reaction with any parasites tested. PCR conditions are recommended at 68 C annealing temperature and with 0.5 mM magnesium chloride (Mg Cl-2). Additionally, specific primers developed in this study were effective to determine the presence of both parasites in fish and snail intermediate hosts, which the DNA of O. viverrini was artificially spiked since it is rarely found in northern Thailand. The H. taichui and O. viverrini-specific primers successfully developed in this study can be use for epidemiological monitoring, preventing management and control programs. (c) 2012 Elsevier Inc. All rights reserved.
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