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A duplex PCR for the simultaneous detection of Fasciola hepatica and Clonorchis sinensis

Yang, Yimin, Li, Minwei, Pan, Chen, Yang, Yi, Chen, Xueqiu, Yao, Chaoqun, Du, Aifang
Veterinary parasitology 2018 v.259 pp. 1-5
Ascaris, Clonorchis sinensis, DNA, Fasciola hepatica, Fasciolopsis, Haemonchus contortus, NAD (coenzyme), NADH dehydrogenase, detection limit, diagnostic techniques, eggs, epidemiological studies, farms, feces, genes, internal transcribed spacers, polymerase chain reaction, sheep, China, Japan, South East Asia, South Korea
Both Fasciola hepatica and Clonorchis sinensis are endemic in China, South Korea, Japan and other Southeast Asian countries. Reliable and sensitive diagnostic methods are needed for detecting their infections in humans and animals. Differential simplex and duplex polymerase chain reaction (PCR) methods were developed. The PCRs targeted the second internal transcribed spacer (its2) (408 bp) of F. hepatica, and NADH dehydrogenase subunit 2 gene (nad2) (527 bp) of C. sinensis. Both simplex PCRs detected as little as 2 pg genomic DNA in one microliter in a 25 μL PCR reaction system. The duplex PCR had similar detection limit as well, and detected as low as one egg in 200 mg feces. These methods were analytical specific with no amplification being observed from the gemonic DNA of Fasciolopsis buski, Haemonchus contortus, Ascaris ovis or Eimeri ahsata. Of 158 sheep fecal samples collected from various farms, four and one samples were PCR-positive for F. hepatica and C. sinensis, respectively. The duplex PCR method described here is time-saving and convenient, and may prove to be an invaluable tool for molecular detection and epidemiological investigation of F. hepatica and C. sinensis in endemic area.