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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

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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.

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Acknowledgements

This work was supported by the National Science and Technology Development Agency (Discovery Based Development Grant), the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission, and the Faculty of Medicine, Khon Kaen University, Thailand.

Conflict of interest

This study was in compliance with the Thai law. No conflict of interest is declared.

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Authors and Affiliations

  1. Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand

    Oranuch Sanpool, Pewpan M. Intapan, Penchom Janwan & Wanchai Maleewong

  2. Research and Diagnostic Center for Emerging Infectious Diseases, Khon Kaen University, Khon Kaen, Thailand

    Oranuch Sanpool, Pewpan M. Intapan, Tongjit Thanchomnang, Penchom Janwan, Viraphong Lulitanond & Wanchai Maleewong

  3. Faculty of Medicine, Mahasarakham University, Mahasarakham, Thailand

    Tongjit Thanchomnang

  4. Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand

    Viraphong Lulitanond

  5. Department of Parasitology, Institute of Ecology and Biological Resources, 18-Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam

    Pham Ngoc Doanh & Hoang Van Hien

  6. Department of Parasitology, National Institute of Malariology, Parasitology and Entomology, Hanoi, Vietnam

    Do Trung Dung

  7. Research Division, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand

    Yukifumi Nawa

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  1. Oranuch Sanpool
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Corresponding author

Correspondence to Pewpan M. Intapan.

Appendix A. Supplementary data

Below is the link to the electronic supplementary material.

Online Supplementary Resource 1

The analytical sensitivity of real-time PCR (DOC 233 kb)

Online Supplementary Resource 2

Real-time PCR for detection of C. sinensis and O. viverrini eggs artificially mixed in uninfected human feces (DOC 141 kb)

Online Supplementary Resource 3

Ethidium bromide stained patterns of the PCR products (DOC 523 kb)

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Sanpool, O., Intapan, P.M., Thanchomnang, T. et al. 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. Parasitol Res 111, 89–96 (2012). https://doi.org/10.1007/s00436-011-2804-7

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  • DOI: https://doi.org/10.1007/s00436-011-2804-7

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