Molecular diagnosis of dengue
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Several protocols for genomic amplification using reverse transcription followed by polymerase chain reaction (RT-PCR), important in the identification of the infecting serotype, have been used in the rapid diagnosis of Dengue Virus (DENV) infections. The qualitative protocol described by Lanciotti et al. (J Clin Microbiol 30: 545–551, 1992) suggested by WHO detects the four DENV serotypes simultaneously in one procedure “semi-nested,” generating amplified products with specific sizes in base pairs for each serotype and it has been the most used in the past two decades. However, advances in molecular diagnosis have enabled the development of RT-PCR in real time (qRT-PCR) based on the use of dyes and probes (SYBR green and TaqMan), which is performed in a single step and is capable of providing quantitative data. In addition to quantification, the advantages of qRT-PCR over conventional RT-PCR include speed, greater sensitivity and specificity, and low rate of false positives. Several protocols for the diagnosis and/or quantification of DENV have already been described. Non-PCR-based methods such as reverse transcription loop-mediated isothermal amplification have shown high sensitivities and specificities. RT-PCR and qRT-PCR techniques can be performed using serum, plasma, infected cells, mosquitoes, fresh, and paraffin-embedded tissues. However, despite fast and accurate, they are limited to samples collected during the acute phase of infection (up to 7 days after the onset of symptoms) and require specialized equipment and trained staff.
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