Evaluation of the Applicability of a One-step Multiplex, Real-time, Taqman® Reverse Transcriptase PCR (RT-PCR) for the Rapid Detection and Serotyping of the Dengue Virus (DENV-1, 2, 3, 4) in the Philippine Setting

Abstract

Dengue Virus (DENV) is considered as one of the world’s most important viral pathogens. It can cause illnesses ranging from mild fevers to fatal febrile illnesses. Accurate and quick diagnostic methods are needed to be able to give the proper and immediate treatment to the infected patient. The current methods for the detection and serotyping of DENV are virus isolation, serological assays, and nucleic-acid amplification methods. The third is currently gaining ground in detecting different diseases caused by viral pathogens. In this study, the one-step, multiplex, TaqMan® real-time RT-PCR protocol developed by Kong et al. (2006) for the simultaneous detection and serotyping of DENV was evaluated for its applicability for Philippine clinical samples. Each 25 pl reaction contained 0.25 pM of each of the probes, 0.5 pM of each of the primers, and 0.1 U/pl of SuperScriptm/Tat?. The optimum temperature of the assay was determined to be 61.9°C, and the optimum [Mg *] is 5mM. The multiplex assay for DENV-2 and -3 was validated by comparing their Cq values to the Cq values of their corresponding monoplex assays, and their differences were no more than 2 cycle units. In all real-time PCR runs, amplified DENV-4 products were never detected. Conventional PCR and DNA sequencing confirmed the amplification of the right target sequence. Sequence alignment of the published DENV-4 probe and DENV-4 strains 814669 and H241, however, showed 4 and 3 base mismatches respectively, indicating that the probe may not have annealed to the target sequence. These findings suggest that the protocol developed by Kong et al. (2006) may not be applicable for the simultaneous detection and serotyping of DENV in the Philippines. New DENV-4 probes were designed using the NS5 gene sequences of DENV-4 strains H241, China GuangZhou/B5, Thailand 0087/77, Thailand 0348/91, and Thailand 0485/01. It is recommended that experiments be carried out to test the validity of the designed probes, optimize the DENV-4 monoplex reaction, and optimize the DENV-1-4 multiplex reaction protocol. Furthermore, studies regarding probe functionality should be undertaken to investigate the Kong et al. DSQ4 probe.