出版年:

2001

研究生:

王冠茹

研究生(英文姓名):

Wang, K. J.

論文名稱:

運用定量反轉錄-聚合酵素連鎖反應偵測登革病人檢體

英文論文名稱:

Application of QR-PCR for dengue clinical samples

指導教授:

陳舜華 老師

指導教授(英文姓名):

Chen, S. H.

學位類別:

碩士

校院名稱:

國立成功大學　

系所名稱:

微生物暨免疫學研究所

學號:

S46884040

學年度:

89

語文別:

中文

論文頁數:

0

關鍵詞:

登革出血熱或登革休克症 ; 登革病毒 ; 反轉錄-聚合酵素連鎖反應

英文關鍵詞:

DHF/DSS ; Dengue virus ; RT-PCR

被引用次數:

0

[ 摘要 ]

登革病毒是屬於黃病毒科RNA病毒，它可區分為四種血清型。感染登革病毒的病人表現出不同程度的臨床症狀，從輕微可自然痊癒的登革熱，以至於發展成嚴重性並可能威脅生命的登革出血熱或登革休克症(DHF/DSS)
，我們對登革病毒的致病機轉仍然有許多不了解之處。在本篇論文中，我們發展出定量反轉錄酵素-聚合酵素連鎖反應 (QR-PCR) 以決定重症與輕症的登革病人血中之病毒量，以探討病毒在人類感染登革的致病機轉中
所扮演的角色。血清型第三型登革病毒分析的敏感度是每一毫升血清檢體約可偵測約3000至18000 個病毒基因組。我們分析了1998年在台南地區爆發的登革病人血清檢體，共三十五個，包括七位登革出血熱或登革
休克症及十二位登革熱病人。絕大部分檢體中的病毒基因組在發燒後前八天可被偵測。我們從病人血清中所偵測到的病毒基因組，應是來自具有感染性的病毒，因為我們可以分離出33%具有感染性的病毒檢體(六位可偵測到病
毒的檢體中有二位具有感染性的病毒)。八位病人有發燒後前八天的檢體，二位DHF/DSS病人及六位DF病人中有四位皆可自血清中偵測到病毒存在。利用我們改良過的QR-PCR探討病毒是否存在或甚至在周邊單核血
球細胞中複製。分析周邊單核血球細胞的分析其敏感度和血清檢體相同。在我們所分析的三十個檢體中，可以偵測到絕大部分檢體中的actin RNA，然而只有在一個檢體中測得登革第三型病毒存在。在我的研究中，發現

[ 英文摘要 ]

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 shocksyndrome (DHF/DSS). The mechanisms involved in the pathogenesis remain elusive. In this study, wedeveloped 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 couldbe detected mostly in samples before fever day 8. The virus genome we detected in patient serum ismostly 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 patientshad 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. Inour 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