A specific PCR assay for the diagnosis of Clonorchis sinensis infection in humans, cats and fishes

Clonorchiasis caused by Clonorchis sinensis is a fish-borne parasitic disease which is endemic in a number of countries. Using the sequences of the internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA (rDNA) of C. sinensis as genetic markers, a pair of C. sinensis-specific primers was designed and used to establish a specific PCR assay for the diagnosis of C. sinensis infection in humans, cats and fish. This approach allowed the specific identification of C. sinensis after optimizing amplification conditions, with no amplicons being amplified from related heterogeneous DNA samples, and sequencing of amplicons confirmed the identity of the sequences amplified. The detection limit of this assay was 1.03 pg of adult C. sinensis, 1.1 metacercariae per gram of fish filet, and a single egg in human and cat feces. The PCR assay should provide a useful tool for the diagnosis and molecular epidemiological investigation of clonorchiasis in humans and animals.

Analysis of PCR products amplified from Clonorchis sinensis metacercariae in fish samples using the specific PCR assay by agarose gel electrophoresis. Lanes 1–12 represent infected fish samples, lanes 13–15 represent non-infected fish samples, lane 16 represents the “positive control”, and lane 17 represents no-DNA control. M represents a DNA size marker (ordinate values in bp).Figure optionsDownload as PowerPoint slideHighlights
► A PCR assay using ITS rDNA as genetic marker was established for the detection of C. sinensis DNA.
► This PCR assay was quite sensitive and specific.
► It provides a molecular tool for the diagnosis of C. sinensis infection in humans, cats and fish.