Internally Controlled, Generic Real-Time PCR for Quantification and Multiplex Real-Time PCR with Serotype-Specific Probes for Serotyping of Dengue Virus Infections

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Dengue has become a global public health problem and a sensitive diagnostic test for early phase detection can be life saving. An internally controlled, generic real-time PCR was developed and validated by testing serial dilutions of a DENV positive control RNA in the presence of a fixed amount of IC with results showing a good linearity (R(2) = 0.9967) and a LOD of at least 1.95 × 10(4) copies/mL. Application of the generic PCR on 136 patient samples revealed a sensitivity of 95.8% and specificity of 100%. A newly developed multiplex real-time PCR with serotype-specific probes allowed the serotyping of DENV for 80 out of 92 (87%) generic real-time PCR positive patients. Combined these real-time PCRs offer a convenient diagnostic tool for the sensitive and specific quantification of DENV in clinical specimens with the possibility for serotyping.