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

Intapan, Pewpan M.; Thanchomnang, Tongjit; Lulitanond, Viraphong; Pongsaskulchoti, Phunthira; Maleewong, Wanchai

doi:10.4269/ajtmh.2009.09-0275

Cited by 17 publications

(10 citation statements)

References 22 publications

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“…The detection limit of our method was as little as one C. sinensis egg and two O. viverrini eggs in 100 mg of fecal sample, equivalent to 10 and 20 EPG, respectively. This result is similar with the egg detection limits in fecal samples of previous reports of 5-10 EPG by singleplex Taqman probe-based real-time PCR assay for C. sinensis eggs (Cai et al 2012;Rahman et al 2011) or of 10 EPG by singleplex FRET-probe-based real-time PCR assay for O. viverrini eggs (Intapan et al 2009a). Variations in the detection limits noted in various reports were possibly due to the analytical sensitivity examined the parasite DNA spiked in distilled water or fecal samples.…”

Section: Discussionsupporting

confidence: 89%

“…The real-time FRET PCR was sensitive enough to detect four copies of each artificial positive template controls. The detection limit for C. sinensis and O. viverrini genomic DNA was both 0.2 pg, which is quite similar to the detection ranges of 0.1-1 pg of C. sinensis genomic DNA (Cai et al 2012;Rahman et al 2011) and far smaller than that of O. viverrini genomic DNA in a real-time PCR method, which was 30 pg (Intapan et al 2009a). The detection limit of our method was as little as one C. sinensis egg and two O. viverrini eggs in 100 mg of fecal sample, equivalent to 10 and 20 EPG, respectively.…”

Section: Discussionsupporting

confidence: 70%

“…Some of the examples are multiplex PCR approaches (Le et al 2006), PCR and sequencing of nuclear ribosomal and mitochondrial DNA (Park 2007) and nuclear marker sequences (PM-int9) (Shekhovtsov et al 2009), PCR-RFLP analysis of the 18S-ITS1-5.8S nuclear ribosomal DNA region (Kang et al 2008), and PCR targeting ribosomal DNA ITS regions (Sato et al 2009). A rapid, high-throughput, specific, and sensitive realtime PCR has been applied for the detection of C. sinensis (Kim et al 2009;Cai et al 2012;Rahman et al 2011) and O. viverrini (Intapan et al 2008a, 2008b, 2009aSuksumek et al 2008;Sri-Aroon et al 2011;Janwan et al 2011) DNA. However, those methods have been applied for detecting both parasites in separate assays and are useful in endemic areas of either C. sinensis or O. viverrini alone but may be problematic where C. sinensis and O. viverrini are co-endemic.…”

Section: Introductionmentioning

confidence: 99%

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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

et al. 2012

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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.

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Section: Discussionsupporting

confidence: 89%

Section: Discussionsupporting

confidence: 70%

Section: Introductionmentioning

confidence: 99%

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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

et al. 2012

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“…Recently, a real-time-based PCR method proved to be sensitive and specific for the detection of either O. viverrini (Intapan et al 2009) or S. stercoralis (ten Hove et al 2009Verweij et al 2009;Kramme et al 2011;Basuni et al 2011;Taniuchi et al 2011) DNA in human stool samples. However, both detections were performed in separate assays.…”

Section: Introductionmentioning

confidence: 99%

Rapid detection of Opisthorchis viverrini and Strongyloides stercoralis in human fecal samples using a duplex real-time PCR and melting curve analysis

et al. 2011

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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 162 bp pOV-A6 DNA sequence specific to O. viverrini and the 244 bp 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 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 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.

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“…Currently, a rapid, specifi c, and sensitive real-time FRET (fl uorescence resonance energy transfer) PCR study has been developed for detection of O . viverrini in human stool samples, and the method is considered as a powerful tool for diagnosis of human opisthorchiasis [ 63 ].…”

Section: Diagnosismentioning

confidence: 99%

The Diagnosis and Classification of Parasitic Diseases of the Liver

Mandal

2013

Liver Immunology

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

Cited by 17 publications

References 22 publications

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

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

Rapid detection of Opisthorchis viverrini and Strongyloides stercoralis in human fecal samples using a duplex real-time PCR and melting curve analysis

The Diagnosis and Classification of Parasitic Diseases of the Liver

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