Application of QR-PCR for dengue clinical samples

• Main Authors: Wang, K. J., 王冠茹
• Other Authors: Chen, S. H.
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/76835936326247965821

碩士 === 國立成功大學 === 微生物暨免疫學研究所 === 89 === Dengue viruses, RNA virus classified to Flaviviridae, have four antigenically distinct serotypes. Patients with dengue virus infection show a wide range of clinical severities, from mild, self-limited dengue fever (DF) to severe and potentially life-threatening dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). The mechanisms involved in the pathogenesis remain elusive. In this study, we developed quantitative reverse transcriptase-polymerase chain reaction (QR-PCR) to determine virus load in blood samples and the kinetic of virus replication in the dengue patients with different disease outcomes. The detection limit of the assay specific for the serotype 3 dengue virus is approximately 3,000 to 18,000 copies genome per milliliter serum sample. Thirty-five serum samples collected from 7 DHF/ DSS and 12 DF patients during 1998 Tainan outbreak were analyzed. Virus genome could be detected mostly in samples before fever day 8. The virus genome we detected in patient serum is mostly likely from infectious virus, because virus could be recovered from 33% (2 out of 6) signal positive samples. Among eight patients have samples before fever day 8, 2 out of 2 DHF/DSS patients had virus in serum compared with 4 out of 6 DF patients. The QR-PCR was modified to investigate whether virus exists or even replicates in peripheral blood mononuclear cells. The detection limit for the assay remains the same as that used for serum samples. Among 30 samples we analyzed, actin RNA was readily detected in host samples, but type 3 virus genome was detected only in one sample. In our study, virus replication was not higher or persisted longer in DHF/DSS patients. Quantifying virus load in clinical samples using QR-PCR could provide new insight into the pathogenesis of dengue virus infection.