Rapid Detection and Differentiation of Clonorchis sinensis and Opisthorchis viverrini Using Real-Time PCR and High Resolution Melting AnalysisReport as inadecuate




Rapid Detection and Differentiation of Clonorchis sinensis and Opisthorchis viverrini Using Real-Time PCR and High Resolution Melting Analysis - Download this document for free, or read online. Document in PDF available to download.

The Scientific World Journal - Volume 2014 2014, Article ID 893981, 6 pages -

Research Article

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

Technical Center, Guangdong Entry-Exit Inspection and Quarantine Bureau, Guangzhou, Guangdong Province 510630, China

Guangzhou Airport Entry-Exit Inspection and Quarantine Bureau, Guangzhou, Guangdong Province 510470, China

Received 4 March 2014; Revised 3 August 2014; Accepted 3 August 2014; Published 19 October 2014

Academic Editor: Rafael Toledo

Copyright © 2014 Xian-Quan Cai et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.





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

Source: https://www.hindawi.com/



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