Rapid detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini eggs in human fecal samples using a duplex real-time fluorescence resonance energy transfer PCR and melting curve analysis

Abstract

We developed a single step duplex real-time fluorescence resonance energy transfer (FRET) PCR merged with melting curve analysis for the fast detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini eggs in human fecal samples. Two species of mitochondrial NADH dehydrogenase subunit 2 (nad2) DNA elements, the 165-bp nad2 product of C. sinensis and the 209-bp nad2 product of O. viverrini, were amplified by species-specific primers, and the fluorescence melting curve analyses were generated from hybrid of amplicons and two pairs of species-specific fluorophore-labeled probes. By their different fluorescence channels and melting temperatures, both C. sinensis and O. viverrini eggs in infected human fecal samples were detected and differentiated with high (100%) sensitivity and specificity. Detection limit was as little as a single C. sinensis egg and two O. viverrini eggs in 100 mg of fecal sample. The assay could distinguish the DNA of both parasites from the DNA of negative fecal samples and fecal samples with other parasitosis, as well as from the well-defined genomic DNA of human leukocytes and other parasites. It can reduce labor time of microscopic examination and is not prone to carry over contamination of agarose electrophoresis. Our duplex real-time FRET PCR method would be useful to determine the accurate range of endemic areas and/or to discover the co-endemic areas of two liver flukes, C. sinensis and O. viverrini, in Asia. This method also would be helpful for the differential diagnosis of the suspected cases of liver fluke infections among travelers who had visited the endemic countries of those parasites.

First report and molecular identification of Opisthorchis viverrini infection in human communities from Lower Myanmar

“…However, it is difficult to differentiate opisthorchiid eggs morphologically from those of other trematodes such as lecithodendriids and heterophyids, prompting the development of molecular approaches for this purpose [19–20]. A lack of awareness of potential O .…”

Development of a PCR assay and pyrosequencing for identification of important human fish-borne trematodes and its potential use for detection in fecal specimens

“…Many molecular techniques have been developed for the detection of a range of FBT, i.e., multiplex PCR and real-time PCR for identification of C. sinensis and O. viverrini [27,28], random amplified polymorphic DNA-PCR method for differential detection of O. viverrini and H. taichui [29], PCR targeting ITS regions for O. viverrini , C. sinensis , H. pumilio and H. taichui [30], PCR-restriction fragment length polymorphism (RFLP) for detection of O. viverrini , C. sinensis , and H. taichui [31] and for H. taichui , H. pumilio , H. yokogawai , Procerovum varium , S. falcatus , and Centrocestus formosanus [32]. Our study could identify life cycle stages of O. viverrini , eggs in infected feces, metacercariae from infected fishes, and cercarial stages from infected snails; C. sinensis eggs in infected feces as well as H. pumilio , H. taichui and S. falcatus metacercariae from infected fishes at the species level by a pyrosequencing approach.…”

“…This result is quite similar with previous reports for the egg detection limits of either O. viverrini or C. sinensis single egg by PCR in fecal samples [31,34] as well as can be detected with ranges of 5–10 EPG in fecal samples by a single plex Taqman probe-based real-time PCR assay for C. sinensis eggs [35,36], by a single plex FRET-probe-based real-time PCR assay for O. viverrini eggs [37] and by a duplex real-time FRET PCR assay for C. sinensis eggs [28]. Variations in the detection limits noted in various reports were possibly due to inhibitors remaining in the fecal samples.…”

The Liver Flukes: Clonorchis sinensis, Opisthorchis spp, and Metorchis spp.

“…Distinguishing liver fluke eggs from the intestinal heterophyid fluke eggs can also be difficult (Ditrich et al, 1992), and molecular methods applicable to egg identification have also been developed (Sato et al, 2009;Sanpool et al, 2012;Armignacco et al, 2008).…”