台灣九孔鮑疱疹病毒DNA polymerase基因之分析及診斷方法的建立

The analysis of DNA polymerase gene of abalone herpesvirus Taiwan isolate and its application on the diagnosis

陳敏修(Min-Hsiu Chen)

指導教授 ： 張本恆

國立臺灣大學/獸醫專業學院/獸醫學研究所/博士(2014年)

https://doi.org/10.6342/NTU.2014.03048

摘要

台灣分離之九孔鮑 (Haliotis diversicolor supertexta)疱疹病毒 (AbHV) DNA經定序後，其中一段5781 bp基因序列，與澳洲的鮑魚疱疹病毒 (strain Victoria/AUS/2007, AbHV-1)的DNA聚合酶基因DNA序列有99% (5767/5779)的相同度 (identity)，胺基酸序列有99% (1923/1926)的高度相同度，與感染牡蠣等雙殼貝類的牡蠣疱疹病毒 (ostreid herpesvirus 1, OsHV-1) DNA聚合酶胺基酸序列則30% (563/1856) 的相同度。在本研究中，依據AbHV DNA聚合酶基因序列，開發了一個以PCR反應檢測九孔鮑疱疹病毒感染的方法；以引子對40f與146r進行PCR反應，預期產物大小為606bp。PCR檢測九孔鮑疱疹病毒具特異性，與其他疱疹病毒包括牡蠣疱疹病毒 (OsHV-1)、錦鯉疱疹病毒 (koi herpesvirus, KHV)、鰻魚疱疹病毒 (eel herpesvirus)、雞傳染性喉氣管炎病毒 (avian infectious laryngotracheitis virus, ILTV)進行PCR反應，結果都呈陰性反應。結合PCR與組織病理學檢查，能提供可靠的九孔鮑疱疹病毒感染的診斷。在本研究中，依據病毒DNA聚合酶基因序列，亦開發了一個以恆溫環形核酸增幅法 (loop-mediated isothermal amplification, LAMP)檢測九孔鮑疱疹病毒；最佳反應條件為溫度63℃下，反應60分鐘。LAMP反應結果可用電泳分析或添加螢光劑觀察。LAMP法檢測九孔鮑疱疹病毒具特異性，與其他疱疹病毒包括牡蠣疱疹病毒 (OsHV-1)、錦鯉疱疹病毒 (KHV)、鰻魚疱疹病毒 (eel herpesvirus)、雞傳染性喉氣管炎病毒 (ILTV)進行LAMP反應，結果都呈陰性反應。本試驗所開發的LAMP檢驗法敏感性為PCR的100倍，敏感性較SYBR Green PCR少10倍。顯示LAMP檢驗法是一個簡單、迅速、有高度特異性與敏感性及可靠的偵測九孔鮑疱疹病毒技術。

關鍵字

疱疹病毒 ； 九孔鮑 ； DNA聚合酶 ； 聚合酶鏈反應 (PCR) ； 恆溫環形核酸增幅法 (LAMP) ； SYBR green即時聚合酶鏈反應

並列摘要

A 5781-base pair (bp) fragment of genomic DNA was obtained from Taiwanese abalone herpesvirus and showed 99% (5767/5779) identity in the nucleotide sequence and 99% (1923/1926) in the amino acid sequence with the DNA polymerase gene of the abalone herpesvirus strain Victoria/AUS/2007. Identity of the amino acid sequence was 30% (563/1856) with the DNA polymerase of ostreid herpesvirus 1. In this study, a PCR-based procedure for detecting herpesvirus infection of abalone, Haliotis diversicolor supertexta, in Taiwan was developed. The method employed primer sets targeting the viral DNA polymerase gene, and was able to amplify DNA fragments of the expected size from infected samples. Primer sets of 40f and 146r were designed for amplification of an expected PCR product of 606 bp. Combining the new PCR protocol with histopathology, this assay can serve as a reliable diagnostic method for herpesvirus infections in abalone. A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of abalone herpesvirus DNA. Two pairs of primers were designed, based on the sequence of the DNA polymerase gene of abalone herpesvirus. The reaction temperature and time were optimized at 63℃ and 60 min, respectively. LAMP amplicons were analyzed by 2% agarose gel electrophoresis or by visual inspection of a colour change emitted by fluorescent dye. The method developed was specific for the detection of abalone herpesvirus, without cross-reactions with other tested herpesviruses including ostreid herpesvirus 1 (OsHV-1), European eel herpesvirus, koi herpesvirus (KHV) and an avian herpesvirus. The LAMP assay was 100 folds more sensitive than a conventional PCR and 10 folds less sensitive than a SYBR Green PCR. These results indicate that the developed LAMP assay is a simple, rapid, sensitive, specific and reliable technique for the detection of abalone herpesvirus.

並列關鍵字

Herpes virus ； Abalone ； Haliotis diversicolor supertexta ； DNA polymerase ； Polymerase chain reaction ； Loop-mediated isothermal amplification ； SYBR green PCR

參考文獻

巫文隆，2003。台灣貝類目錄 I，腹足綱-原始腹足目。行政院農業委員會。

黃之暘、李國誥、潘昭蓉、劉秉忠，2009。養殖鮑常見疾病，健康管理與防疫對策。海大漁推，39，1-37。

趙佳鴻，2012。即時聚合酶連鎖反應(Real-time PCR Real-time PCR)在植物病毒病害檢測上之應用。台中區農業改良場一○○年專題討論專集專，131-135。

蕭君倪，2010。建立偵測常見豬隻呼吸道病毒性疾病即時聚合酶鍊鎖反應技術。臺灣大學獸醫學研究所碩士論文。

林于勤、李淑英、陳豪勇、林鼎翔，2004。新型恆溫式圈環形核酸增幅法簡介與應用Loop-mediated Isothermal Amplification (LAMP)。疫情報導，20(6)，323-332。