Comparison of techniques for extracting viral RNA from isolation-negative serum for dengue diagnosis by the polymerase chain reaction.

Aiming at the improvement of the molecular diagnosis of dengue, three well-established methods of RNA extraction from serum of patients with clinical symptoms of dengue were compared. The methods were based on the QIAamp Viral RNA kit, the Chomczynski-Sacchi technique and TRIzol. One hundred samples were examined using the same protocol for reverse transcription-polymerase chain reaction (RT-PCR). Out of the 100 samples tested, none was positive by either the Chomczynski-Sacchi technique or TRIzol, and six were positive using the QIAamp viral RNA kit. Of the six positive samples, only one was collected before 5 days of the beginning of the disease, and it was also positive for viral isolation. These results were confirmed later by serology (MAC-ELISA) that showed that 19 samples were positive for IgM antibodies against dengue. These data indicate that PCR is a useful method for detection of dengue virus infections in IgM-positive samples, and the best method of RNA extraction from clinical samples, to be used for dengue diagnosis by PCR is the QIAamp Viral RNA kit.