Rapid detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini using real-time PCR and high resolution melting analysis.

Xian-Quan Cai,Xing-Quan Zhu,Qiao-Yun Yue,Yan Deng,De-Yi Qiu,Rong Li,Hai-Qiong Yu,Guo-Hua Liu,Jian-Shan Bai

doi:10.1155/2014/893981

Abstract

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.

Highlights

Opisthorchis viverrini and Clonorchis sinensis are pathologically important members of the family Opisthorchiidae
Opisthorchiasis caused by O. viverrini is considered an important food-borne parasitic disease in Southeast Asia including Thailand, Lao PDR, Vietnam, and Cambodia; approximately 67.3 million people are at risk of the infection
A BLAST search of the chosen primer and probe sequences resulted in a hit of the target sequence in C. sinensis and O. viverrini, suggesting the specificity of the primers

Summary

Introduction

Opisthorchis viverrini and Clonorchis sinensis are pathologically important members of the family Opisthorchiidae Chronic infections with these liver flukes closely associate with the development of the bile duct cancer (cholangiocarcinoma) and the liver cancer (hepatocarcinoma) in humans [1]. Crude or recombinant antigens have been evaluated for immunodiagnosis; the specificity and sensitivity of these methods are still in great need of improvement [11, 12] Both conventional PCR and LAMP have high risk of contamination. The real-time PCR platform with HRM is a single-step closed tube, reduces turnaround time of the assay reported here to almost 1 h, eliminates the risk of contamination, and saves expense These features make it advantageous for use in laboratories. We developed a real-time PCR assay coupled with HRM analysis for rapid identification and differentiation of C. sinensis and O. viverrini

Materials and Methods

Results

Discussion

Conclusion