Rapid detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini using real-time PCR and high resolution melting analysis.
Clonorchis sinensis and Opisthorchis viverrini are both important fish-borne pathogens, causing serious public health problem in Asia. The present study developed an assay integrating real-time PCR and high resolution melting (HRM) analysis for the specific detection and rapid identification of C. sinensis and O. viverrini. Primers targeting COX1 gene were highly specific for these liver flukes, as evidenced by the negative amplification of closely related trematodes. Assays using genomic DNA extracted from the two flukes yielded specific amplification and their identity was confirmed by sequencing, having the accuracy of 100% in reference to conventional methods. The assay was proved to be highly sensitive with a detection limit below 1 pg of purified genomic DNA, 5 EPG, or 1 metacercaria of C. sinensis. Moreover, C. sinensis and O. viverrini were able to be differentiated by their HRM profiles. The method can reduce labor of microscopic examination and the contamination of agarose electrophoresis. Moreover, it can differentiate these two flukes which are difficult to be distinguished using other methods. The established method provides an alternative tool for rapid, simple, and duplex detection of C. sinensis and O. viverrini.
State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu Province 730046, China ; Technical Center, Zhongshan Entry-Exit Inspection and Quarantine Bureau, Zhongshan, Guangdong Province 528403, China.
Xian-Quan
XQ
Cai
Technical Center, Guangdong Entry-Exit Inspection and Quarantine Bureau, Guangzhou, Guangdong Province 510630, China.
Hai-Qiong
HQ
Yu
Technical Center, Zhongshan Entry-Exit Inspection and Quarantine Bureau, Zhongshan, Guangdong Province 528403, China.
Rong
R
Li
Technical Center, Zhongshan Entry-Exit Inspection and Quarantine Bureau, Zhongshan, Guangdong Province 528403, China.
Qiao-Yun
QY
Yue
State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu Province 730046, China.
Guo-Hua
GH
Liu
Guangzhou Airport Entry-Exit Inspection and Quarantine Bureau, Guangzhou, Guangdong Province 510470, China.
Jian-Shan
JS
Bai
Guangzhou Airport Entry-Exit Inspection and Quarantine Bureau, Guangzhou, Guangdong Province 510470, China.
Yan
Y
Deng
Technical Center, Zhongshan Entry-Exit Inspection and Quarantine Bureau, Zhongshan, Guangdong Province 528403, China.
De-Yi
DY
Qiu
State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu Province 730046, China.
Xing-Quan
XQ
Zhu
D000818:Animals; D001208:Asia; D003003:Clonorchiasis / Q000469:parasitology* / Q000635:transmission; D003004:Clonorchis sinensis / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity; D051545:Cyclooxygenase 1 / Q000235:genetics / Q000302:isolation & purification*; D005399:Fishes / Q000469:parasitology; D006801:Humans; D009889:Opisthorchiasis / Q000469:parasitology* / Q000635:transmission; D009891:Opisthorchis / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods
D051545:Cyclooxygenase 1