rapid detection and identification of human hookworm infections through high resolution melting (hrm) analysis快速检测和识别人体钩虫感染通过高分辨率融化(人力资源管理)分析.pdfVIP

Rapid Detection and Identification of Human Hookworm Infections through High Resolution Melting (HRM) Analysis 1 1 2 Romano Ngui , Yvonne A. L. Lim , Kek Heng Chua * 1 Department of Parasitology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia, 2 Department of Molecular Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia Abstract Background: Hookworm infections are still endemic in low and middle income tropical countries with greater impact on the socioeconomic and public health of the bottom billion of the world’s poorest people. In this study, a real-time polymerase chain reaction (PCR) coupled with high resolution melting-curve (HRM) analysis was evaluated for an accurate, rapid and sensitive tool for species identification focusing on the five human hookworm species. Methods: Real-time PCR coupled with HRM analysis targeting the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA as the genetic marker was used to identify and distinguish hookworm species in human samples. Unique and distinct characteristics of HRM patterns were produced for each of the five hookworm species. The melting curves were characterized by peaks of 79.24 60.05uC and 83.00 60.04uC for Necator americanus, 79.12 60.10uC for Ancylostoma duodenale, 79.40 60.10uC for Ancylostoma ceylanicum, 79.63 60.05uC for Ancylostoma caninum and 79.70 60.14uC for Ancylostoma braziliense. An evaluation of the method’s sensitivity and specificity revealed that this assay was able to detect as low as 0.01 ng/ml hookworm DNA and amplification was only recorded for hookworm positive samples. Conclusion: The HRM assay developed in this study is a rapid and straightforward method for the diagnosis, identificati