Development of multiplex real-time TaqMan reverse-transcription PCR (RT-PCR) for rapid diagnosis of hand, foot and mouth disease.

• Main Author: Lee, Si Qi.
• Other Authors: School of Biological Sciences
• Format: Final Year Project
• Language: English
• Published: 2009
• Subjects: DRNTU::Science::Biological sciences::Genetics
• Online Access: http://hdl.handle.net/10356/16904
• Institution: Nanyang Technological University

Hand, foot and mouth disease (HFMD) is a viral illness that causes ulcerating on hands, foot and mouth. The major causing agents are Enterovirus-71 (EV71) and Coxsackievirus A16 (CA16). HFMD caused by EV71 were found to associate with severe neurological complications that result in deaths. With rising concerns of HFMD in these current years, it is essential to develop a rapid and specific diagnostic method to detect and distinguish the different enterovirus serotypes as the currently used tissue culture method (the “gold standard”) is time consuming and not as sensitive. In this study, a multiplex real-time TaqMan reverse transcriptase polymerase chain reaction (RT-PCR) was developed to demonstrate a rapid and sensitive method in detecting and differentiating EV71 and CA16, using CFX96 Real-Time PCR Detection System (Bio-Rad, USA). Primers and probes were specifically designed to target the VP1 region. The data demonstrated specific detection of EV71 and CA16 in singleplex reactions and duplex reactions. Quantitative analysis of EV71 and CA16 showed high sensitivity of up to the unit of femtogram. Furthermore, 10 clinical specimens obtained from 10 paediatrics patients who were suspected of HFMD were evaluated. Five specimens were tested positive for EV71 and among them, two were co-infected with CA16.