桿狀病毒的微量偵測與病毒株分析

Trace Detection and Strain Analysis of Baculovirus

桿狀病毒是感染節肢動物的專一性病毒，且被認為對脊椎動物及植物不具致病性或毒性。但隨著應用日益廣泛，它們暴露在環境中的機會及濃度也將隨之增加，偵測桿狀病毒的微量極限及病毒株的鑑定分析技術都是重要的課題。本論文針對桿狀病毒多角體蛋白基因，利用聚合酶連鎖反應來探討豆莢螟核多角體病毒可被偵測的微量極限。結果顯示0.15pg病毒基因體為偵測的最小量，大約有1,216個病毒基因體。此外，我們以限制酶片段長度多態性進行異種核多角病毒的分析，證實此技術可分辨出加州苜蓿夜蛾核多角體病毒與豆莢螟核多角體病毒的差異。另外，我們也利用斜紋夜蛾核多角體病毒為材料，以BamHI、PstI、EcoRI、BglII四種限制酶分析台灣四個不同地理區域，分別為台灣大學(TW)、大甲(DC)、霧峰(WF)、草屯(TT)所分離之病毒株基因體的差異性。結果顯示這四個病毒株的限制酶圖譜相似但不完全相同，BglII限制酶同時可區分出這四個病毒株的差異。本研究利用簡易的聚合酶連鎖反應技術及限制酶片段長度多態性技術，探討病毒偵測的微量極限、基因體差異性及篩選高感染力的病毒株，可實際應用於昆蟲桿狀病毒的安全性及在生物農藥的開發上。

Baculovirus are arthropod-specific viruses and considered not to be pathogenic or toxic agents in vertebrates and plants. The trace detection and strain identification of baculovirus have been regarded an important issue because the exposure and concentrations of baculovirus in the environment have increased following its wide application. In this paper, the polymerase chain reaction (PCR) toward the polihedrin gene of baculovirus was conducted to detect the minimum viral genome of MaviMNPV. It was revealed that at least a 0.15 pg equivalent 1,216 viral genome was detected. We identified heterogeneity of baculovirus by using restriction fragment length polymorphism (RFLP) to explore the difference between AcMNPV and MaviMNPV. We also analyzed the genomic heterogeneity of SpltMNPV which were isolated from National Taiwan University (TW), Dajia (DC), Wufeng (WF) and Caotun (TT) respectively, by restriction map analysis of BamHI, PstI, EcoRI and BglII. The results showed similar but not identical profiles of restriction map for these four viral strains. The BglII restriction enzyme map exhibited a difference among the four viral strains. Utilizing PCR and RFLP techniques for the purpose of trace detection, genomic heterogeneity, and screening for highly pathogenic strains, have provided practical approaches toward the security of baculovirus and biopesticide development.