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標題: 鴨環狀病毒DNA疫苗及恆溫式圈環形核酸增幅法檢測技術之研發
Development of DNA vaccine and methods for detecting duck circovirus by loop mediated isothermal amplification
作者: 黃朝祺
Huang, Chau-Chi
關鍵字: duck circovirus;鴨環狀病毒;loop mediated isothermal amplification (LAMP);恆溫式圈環形核酸增幅法
出版社: 微生物暨公共衛生學研究所
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鴨環狀病毒為新興的水禽疾病,受到鴨環狀病毒感染的鴨隻會有生長遲緩和免疫抑制現象,導致二次感染機會之增加。鴨環狀病毒的感染會對養殖業造成經濟損失,但目前仍沒有可使用之疫苗以控制疫情。另一方面,由於尚無方法可以使用細胞培養方式進行鴨環狀病毒的增殖,因此聚合酶連鎖反應仍為檢測此病毒的主要診斷方法。本研究的目的有兩個,首先為發展對抗鴨環狀病毒的DNA疫苗,另一為發展利用恆溫式圈環形核酸增幅法 (loop mediated isothermal amplification,LAMP)以檢測鴨環狀病毒。在DNA疫苗的製備方面,本研究將鴨環狀病毒的外殼蛋白 (Capsid protein)之基因選殖至真核表現系統之載體pcDNA3.1/TOPO中,構築為重組質體,再將此重組質體以肌肉注射方式來免疫鴨隻,使用的DNA質體劑量分別為0.5 mg和5 mg並使用磷酸鋁膠作為佐劑。攻毒試驗結果顯示此DNA疫苗並無法提供顯著的保護效力。LAMP檢測技術之開發,則是使用經由Primer Explorer V3系統所設計的6個特異性的引子來進行LAMP反應。利用LAMP僅需60分鐘以內的反應時間即可檢測到鴨環狀病毒,其敏感度較傳統PCR高達100倍。再者,針對鴨環狀病毒所設計的LAMP引子具有高特異性,對其他亦會感染鴨隻的病原不會有交互反應的情況發生。在田間試驗檢測方面,由LAMP和PCR檢測鴨環狀病毒的結果顯示,鴨環狀病毒普遍存在於田間鴨隻中,且LAMP檢測之敏感度優於PCR。因此,本實驗中發展LAMP技術可作為快速檢測鴨環狀病毒的方法。

Duck circovirus (DuCV) is an emerging disease in waterfowl. Infection with DuCV is associated with growth retardation and lymphoid depletion that leads immune suppression and increased susceptibility to second infections. Although diseases caused by DuCV might lead to economic loss, no vaccine is yet available for control of DuCV. Moreover, in the absence of method for growing DuCV in cell culture, polymerase chain reaction (PCR) is the major method for detection of this virus. The goal of this study is twofold; the first is to develop a DNA vaccine against DuCV and the second is to develop a method for detecting DuCV by loop mediated isothermal amplification (LAMP). For development of the DNA vaccine, a recombinant plasmid was constructed by cloning of the gene encoding the capsid protein of DuCV into the vector pcDNA3.1/TOPO. The ducks were immunized with this recombinant plasmid by intramuscular injection. The dosages used were 5 mg and 0.5 mg of plasmid DNA per duck and the adjuvant used was aluminum phosphate. The results of challenge tests showed that the DNA vaccine did not provide significant protection. The LAMP assay is based on a set of six specific primers designed using Primer Explorer V3 software. The time required for detecting DuCV by LAMP was found to be less than 60 minutes. The sensitivity of the LAMP assay was 100-fold more sensitive than conventional PCR assay. Moreover, the LAMP assay detected only DuCV but not other common pathogens found in ducks. Detection DuCV in field samples using LAMP and PCR assays showed that DuCV is highly prevalent in the field. In conclusion, the method of LAMP assay developed in this study could serve as a rapid and sensitive method for detection of DuCV.
其他識別: U0005-0407201114453600
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