Detection of Opisthorchis viverrini in infected bithynid snails by real-time fluorescence resonance energy transfer PCR-based method and melting curve analysis

Abstract

A real-time fluorescence resonance energy transfer (FRET) PCR combined with melting curve analysis was developed for the detection of Opisthorchis viverrini in experimentally infected bithynid snails, its first intermediate hosts. The test is based on the fluorescence melting curve analysis of a hybrid between an amplicon from the pOV-A6-specific probe sequence, a 162-bp repeated sequence specific to O. viverrini and specific fluorophore-labeled probes. The real-time FRET PCR could detect as little as a single cercaria artificially introduced in a pool of 30 non-infected snails. The O. viverrini-infected snails were discriminated from non-infected snails and from genomic DNA of other parasite DNAs by their melting temperatures. Sensitivity and specificity of this method were both 100%. Melting curve analysis is a sensitive alternative for the specific detection of O. viverrini-infected snails; it is rapid, allows a high throughput, and can be done on small samples. The assay not only has a high potential for epidemiological surveys of O. viverrini-infected bithynid snails, but also for the detection of cercariae infestations of natural waterways when monitoring transmission sites.

Prevalence of gastrointestinal helminth parasites of zoonotic significance in dogs and cats in lower Northern Thailand

“…No false-positive amplification was observed when testing with DNA of min intestinal flukes, such as Centrocestus spp. and Haplorchis taichui or other trematode parasites, such as Fasciola gigantica and Paragonimus heterotremus [13, 36]. However, inhibitors present in fecal samples can inhibit PCR reaction as shown in many epidemiological studies including our study where PCR analysis failed to yield positive results in some microscopic positive samples [29, 31].…”

Rapid Molecular Detection of Opisthorchis viverrini in Human Fecal Samples by Real-Time Polymerase Chain Reaction

“…17 The real-time FRET PCR was performed in glass capillaries using the specific primer pair OV-F (5′CAG TGA GTG TCT ATT GGC TAA 3-′) and OV-R (5′-GTA CTA CTC ATA AGG TTG CGT-3′) (Proligo, Singapore) and a pair of adjacent oligoprobes. One probe was labeled at the 5′ end with the LightCycler Red 640 fluorophore (5′-Red 640-AGA AGG GCG AAA CCG GTC GTG G-Phosphate-3′) (OVLC640 probe) and the other was labeled at the 3′ end with 530 fluorescein (5′-GGG ACT GCG CCT ACC TGA TAG CCC-Flou 530-3′) (OVFL530 probe) (Tib Molbiol, Berlin, Germany).…”

“…22 Regarding the specificity of the probes and primers, no fluorescence signal appeared when purified DNA from Centrocestus spp., Haplorchis taichui , Fasciola gigantica , Echinostoma malayanum , Paragonimus heterotremus , Haplorchoides spp., Stellantchasmus spp., or animal schistosomes was tested. 17 The reaction mixture (20 μL…”

“…A positive DNA control plasmid was created by cloning a PCR product of the pOV-A6 specific DNA probe sequence into the pCR4-TOPO vector (Invitrogen, Carlsbad, CA). 17 The real-time FRET PCR results were calculated from duplicate experiments.…”

“…[17][18][19] The effectual real-time PCR has increasingly superseded the cPCR because of its greatly improved molecular detection and its capability to distinguish microorganisms belonging to the same genus. It is not only accurate, rapid, and can express the quantity of specific DNA in samples, 20 but it also discriminates species or strains of several pathogenic agents by melting curve analysis.…”