利用即時聚合酶連鎖反應定量BK病毒及JC病毒

Using Real-time PCR Assay to Quantify BKV and JCV

陳瀅旭(Ying-Hsu Chen)

指導教授 ： 李君男

國立臺灣大學/醫學院/醫學檢驗暨生物技術學研究所/碩士(2009年)

https://doi.org/10.6342/NTU.2009.10239

摘要

JC病毒及BK病毒為多瘤病毒科（Polyomaviridae）中會感染人類的病毒。JC病毒及BK病毒初次感染多發生於幼年時期，之後多會潛伏於人體。正常人受感染後通常不會產生臨床症狀，然而於免疫能力低下的病人，會產生嚴重的臨床症狀。因此本研究希望發展快速且敏1感度高的檢測方法，可以在免疫能力低下之病人偵測JC病毒及BK病毒於體內復發的情形。
早期偵測人類多瘤病毒以細胞培養、直接以電子顯微鏡觀察或免疫螢光染色，爾後發展出聚合酶連鎖反應（PCR）之方法提高了檢驗速度，但仍須有許多後續的工作以協助判定為何種病毒。近年來即時聚合酶連鎖反應（real-time PCR）已廣泛用於臨床檢驗上，因此本研究希望利用即時聚合酶連鎖反應的方法同時偵測並定量JC病毒及BK病毒。首先針對JC病毒及BK病毒之VP2基因設計具有特異性之引子及探針，並測試其特異性、敏感度、再現性。結果發現偵測JC及BK病毒之探針具有高度特異性，兩者之間並不會有交互反應之情形。在敏感度方面，BK病毒之偵測極限（detection limit）為一個反應中有10個DNA分子；JC病毒之偵測極限為一個反應中有3個DNA分子。而即時聚合酶反應之再現性，不論是組間（inter-plate）或組內（intra-plate）測試其CV值均小於5%，顯示具有高度再現性。因此可利用此方法定量JC及BK病毒以了解病毒於病人體內復發的情形。此外，本研究希望於反應中加入內對照品（internal control）以了解試驗的有效性，並降低偽陰性的檢出率，經測試後發現針對內對照品所設計之引子及探針具有高度特異性，並不會與JC病毒或BK病毒間產生交互反應。但內對照品和JC病毒或BK病毒同時存在下，彼此間會產生干擾，並降低偵測JC病毒或BK病毒之敏感度。經即時聚合酶連鎖反應分析79位骨髓移植病患之血漿檢體及尿液檢體，結果發現分別有51（64%）、54（68%）個病人具有BK病毒，且發現兩者之間具有相關性。接著分析在此79位骨髓移植病患和出血性膀胱炎之相關性，結果發現年齡、性別、診斷結果、接受骨髓移植來源及有無發生GVHD和出血性膀胱炎無顯著相關。綜合以上結果，本研究已建立以即時聚合酶連鎖反應定量JC病毒及BK病毒之方法，未來希望改善內對照品使干擾降低，以達降低成本之目標。

關鍵字

BK病毒 ； JC病毒 ； 即時聚合酶連鎖反應 ； 內對照品

並列摘要

JC virus (JCV) and BK virus (BKV) are two species of Polyomaviridae that can infect humans. Primary infections usually occur during childhood and remain latent in the body. Infections from both viruses are generally asymptomatic in immunocompetent hosts. However, reactivation of these viruses may occur in immunosuppressed individuals and could cause severe diseases. This study intended to develop a rapid and highly sensitive method of diagnosis that could detect reactivation of JCV and BKV in immunosuppressed patients.
In early stages, time-consuming and less sensitive methods, such as cell culture, electron microscopy, and immunofluorescence were used for the detection of human polyomavirus. Recently, polymerase chain reaction (PCR) and real-time PCR are widely used for detection of viral DNA in clinical diagnosis. The goal of this study is application of real-time PCR in detection and quantification of JCV and BKV. At first, we as established to monitor the efficiency of the assay and to avoid false negative results. The results showed that that the primers and probes of internal control did not interact with JCV and BKV. However, there were some interferences between the internal control and target genes. We analyzed plasma and urine samples of 79 bone marrow transplant patients by real-time PCR, 51 (64%) and 54 (68%) were detected as BKV positive, respectively. The results correlated well between plasma and urine samples. Then we analyzed the factors associated with hemorrhagic cystitis (HC) in bone marrow transplant patients. There were no relationship between HC and age, sex, diagnosis, or source of bone marrow transplant.

並列關鍵字

BK virus ； JC virus ； real-time PCR ； internal control

參考文獻

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