Korean J Parasitol.  2013 Dec;51(6):689-694. 10.3347/kjp.2013.51.6.689.

Molecular Differentiation of Opisthorchis viverrini and Clonorchis sinensis Eggs by Multiplex Real-Time PCR with High Resolution Melting Analysis

Affiliations
  • 1Research and Diagnostic Center for Emerging Infectious Diseases, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand. wanch_ma@kku.ac.th
  • 2Department of Biochemistry, Faculty of Medical Science, Naresuan University, Phitsanulok 65000, Thailand.
  • 3Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand.
  • 4Faculty of Medicine, Mahasarakham University, Mahasarakham 44000, Thailand.
  • 5Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand.
  • 6Department of Parasitology, Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology, Hanoi, Vietnam.

Abstract

Opisthorchis viverrini and Clonorchis sinensis are parasites known to be carcinogenic and causative agents of cholangiocarcinoma in Asia. The standard method for diagnosis for those parasite infections is stool examination to detect parasite eggs. However, the method has low sensitivity, and eggs of O. viverrini and C. sinensis are difficult to distinguish from each other and from those of some other trematodes. Here, we report a multiplex real-time PCR coupled with high resolution melting (HRM) analysis for the differentiation of O. viverrini and C. sinensis eggs in fecal samples. Using 2 pairs of species-specific primers, DNA sequences from a portion of the mitochondrial NADH dehydrogenase subunit 2 (nad 2) gene, were amplified to generate 209 and 165 bp products for O. viverrini and C. sinensis, respectively. The distinct characteristics of HRM patterns were analyzed, and the melting temperatures peaked at 82.4+/-0.09degrees C and 85.9+/-0.08degrees C for O. viverrini and C. sinensis, respectively. This technique was able to detect as few as 1 egg of O. viverrini and 2 eggs of C. sinensis in a 150 mg fecal sample, which is equivalent to 7 and 14 eggs per gram of feces, respectively. The method is species-specific, rapid, simple, and does not require fluorescent probes or post-PCR processing for discrimination of eggs of the 2 species. It offers a new tool for differentiation and detection of Asian liver fluke infections in stool specimens.

Keyword

Opisthorchis viverrini; Clonorchis sinensis; high resolution melting analysis; multiplex real-time PCR; differentiation; detection
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