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.
De Paula
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Laboratory of Molecular Virology, Department of Clinical Medicine, School of Medicine of Ribeirão Preto, School of Medicine of Ribeirão Preto, University of São Paulo, Avenida dos Bandeirantes, 3900, 14049-900 Ribeirao Preto, San Paulo, CEP, Brazil.
Nunes
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Matos
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de Oliveira
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Lima
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da Fonseca
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D000914:Antibodies, Viral / Q000097:blood*; D020029:Base Pairing; D052160:Benzothiazoles; D002478:Cells, Cultured; D003715:Dengue / Q000175:diagnosis* / Q000276:immunology / Q000821:virology; D003716:Dengue Virus / Q000145:classification / Q000276:immunology / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007075:Immunoglobulin M / Q000276:immunology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000302:isolation & purification*; D011933:Reagent Kits, Diagnostic / Q000821:virology; D013451:Sulfonic Acids
D000914:Antibodies, Viral; D052160:Benzothiazoles; D007075:Immunoglobulin M; D012367:RNA, Viral; D011933:Reagent Kits, Diagnostic; D013451:Sulfonic Acids; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid