Development and evaluation of a SYBR green-based real-time multiplex RT-PCR assay for simultaneous detection and serotyping of Dengue and Chikungunya viruses
Autor(es): Chen Huixin, Parimelalagan Mariya, Lai Yee Ling, Lee Kim Sung, Koay Evelyn Siew-Chuan, Hapuarachchi Hapuarachchige C, Ng Lee Ching, Ho Phui San, Chu Justin Jang Hann
Resumo: Chikungunya virus (CHIKV) - dengue virus (DENV) have emerged as the two most important arbovirus diseases of global health significance. Similar clinical manifestations, transmission vectors, geographical distribution, - seasonal correlation often result in misdiagnosis of chikungunya infections as dengue cases - vice versa. In this study, we developed a rapid - accurate laboratory confirmative method to simultaneously detect, quantify, - differentiate DENV serotypes 1, 2, 3, - 4 - CHIKV. This SYBR Green I-based one-step multiplex real-time RT-PCR assay is highly sensitive - specific for CHIKV - DENV. Melting temperature analysis of PCR amplicons was used to serotype DENV - to differentiate from CHIKV. The detection limit of the assay was 20, 10, 50, 5, - 10 RNA copies/reaction for DENV-1, DENV-2, DENV-3, DENV-4, - CHIKV, respectively. Our assay did not cross-react with a panel of viruses that included other flaviviruses, alphaviruses, influenza viruses, human enteroviruses, - human coronaviruses. The feasibility of using this assay for clinical diagnosis was evaluated in DENV- - CHIKV-positive patient sera. Accordingly, the assay sensitivity for DENV-1, DENV-2, DENV-3, DENV-4, - CHIKV was 89.66%, 96.67%, 96.67%, 94.12%, - 95.74%, respectively, with 100% specificity. These findings confirmed the potential of our assay to be used as a rapid test for simultaneous detection - serotyping of DENV - CHIKV in clinical samples.
Imprenta: The Journal of Molecular Diagnostics, v. 17, n. 6, p. 722-728, 2015
Identificador do objeto digital: 10.1016/j.jmoldx.2015.06.008
Descritores: Chikungunya virus - Arbovirus ; Chikungunya virus - Cytopathology ; Chikungunya virus - RNA ; Chikungunya virus - Viral infections ; Chikungunya virus - PCR detection ; Chikungunya virus - Real Time PCR ; Chikungunya virus - RT-PCR ; Chikungunya Virus - Virus ; Chikungunya virus - Molecular screening ; Chikungunya virus - Transmission ; Chikungunya virus - Dengue
Data de publicação: 2015
