動物族群遺傳資訊之建立及以恆溫核酸擴增偵測平台應用於動物病毒性病原之快速診斷

The establishment of animal population genetic information and the application of rapid nucleic acid detection platform for diagnosis of animal viral pathogens

凃柏安(Po-An Tu)

指導教授 ： 王佩華

國立臺灣大學/生物資源暨農學院/動物科學技術學研究所/博士(2019年)

https://doi.org/10.6342/NTU201902496

摘要

家畜禽遺傳保育之目的為維持有效族群大小、保存遺傳多樣性、遺傳資源及擴展本地原生種家畜禽之用途。早期用於鑑別動物品種及遺傳特徵之方法，主要是透過外觀形態上之表型鑑別，但無法有效應用於鑑別遺傳改進或是動物遺傳資源保存之目的。因此，以分子遺傳技術剖析個別動物體之遺傳特性並建立遺傳資訊庫，可用於家畜禽品種鑑別之用。本研究第一部分為建立臺灣黃牛及臺灣黑山羊族群分子遺傳特徵，並分析其與其他國內外不同牛羊品種之類緣關係。不同牛羊品種之遺傳多樣性及其地緣分布之差異，提供過去前人研究對於臺灣本地種牛羊族群之起源及遺傳關係之證據，並提供不同族群個體曾經被引進以控制特定族群近親效應之證據。這些基礎資訊可用於建立一個可靠的家畜禽遺傳保育計畫，更可延伸用於了解臺灣本地牛羊品種之起源及育種歷史。
重組聚合酶擴增反應（recombinase polymerase amplification, RPA）為一個高敏感性且高專一性的恆溫擴增技術，反應溫度於37至42˚C，且僅需要簡單的樣品處理便可進行擴增反應，可在20分鐘內偵測10個copies的DNA或RNA標的。本研究第二部分成功應用此技術放大及偵測不同常見動物疾病之標的，包含山羊關節炎腦炎（caprine arthritis-encephalitis virus, CAEV）、牛白血病（bovine leukemia virus, BLV）及家禽白血病J型（avian leukosis virus subgroup J, ALV-J）；搭配側層流分析法（lateral flow dipstick, LFD）進行偵測，並與傳統現行主流疾病診斷方法進行比較。
家畜禽為國內重要且可更新之動物遺傳資源，容易受到自然環境及人為開發之影響。因此了解目前家畜禽之遺傳類緣關係對於育種管理極為重要。此外，農民也應對於常見之動物傳染性疾病之預防、診斷、控制及治療有簡易之瞭解，並與獸醫密切合作擬定及實施牧場之健康管理計畫；本研究所開發之RPA-LFD快速檢測技術可用於牧場簡易及快速診斷特定動物傳染性疾病，並提供良好之敏感性及特異性，適合現場快速診斷使用。此外，本平台更可進一步用於發展成為其他病原體及遺傳標幟之鑑別偵測，且僅需輕便易攜帶之基礎試驗設備。結合此二部分，可提升家畜禽飼養之農民及產業之生產力及建立更健康之種畜禽族群。

關鍵字

臺灣黃牛 ； 臺灣黑山羊 ； 遺傳類緣分析 ； 恆溫核酸擴增 ； 快速疾病診斷

並列摘要

The Genetic Preservation of Domestic Livestock was set to maintain an effective population size, preserve genetic diversity, genetic resources, and extend their utilization. In early times, characterizations of domestic livestock breeds were through observation of phenotypic heterogeneity, and could not justify accurately for the purpose of genetic improvement or to sustain the genetic resources preservation in Taiwan. The application of molecular genetics profiling provides advanced hereditary information for each individual, and thus enable us to better define the differences among livestock populations. The aim of this study was to characterize the current genetic structure of these Taiwan yellow cattle and Taiwan black goat populations, as well as to assess their phylogenetic relationship. The spatial distribution of the different breeds of cattle and goat populations and the high contribution of these populations to overall gene diversity supported the hypothesis that the present herd was derived from different genetic lineages that were incorporated to control the level of inbreeding. This information will be essential for establishing a reliable breeding and conservation plan and are also crucial for understanding the origin and breeding history of domestic cattle and goat breeds in Taiwan.
Recombinase polymerase amplification (RPA) is a highly sensitive and selective isothermal amplification technique, operating at 37-42˚C, with minimal sample preparation and capable of amplifying as low as 10 DNA or RNA target copies in about 20 min. We have used the technique to amplify diverse targets, including caprine arthritis-encephalitis virus (CAEV), bovine leukemia virus (BLV), and avian leukosis virus subgroup J (ALV-J) from a wide variety of target animals. Furthermore, different detection strategies integrated with RPA has been successfully performed using lateral flow strips detection. This study focuses on the comparison of different detection methodologies and targets related to RPA-LFD with other disease diagnostic technologies.
To sum up, livestock and poultry breeds are an important part of the biological genetic resources. They are renewable or changeable, and are influenced by the natural ecological environments and by social development. Understanding the origin and genetic phylogenetic relationship of domestic cattle and goat breeds are important for breeding management. Meanwhile, farmers and stockmen should know how to prevent, control and treat animal diseases through farm health planning and close working with vets. The developed RPA-LFD assays are simple and sensitive enough for point-of-care diagnostics. It also serves as a model platform that could be further adapted to detect other pathogens or genetic markers of interest using basic laboratory equipment. Animal husbandry would be better improved with more productive and healthy livestock and poultry in Taiwan.

並列關鍵字

Taiwan yellow cattle ； Taiwan black goat ； phylogenetic analysis ； isothermal nucleic acid amplification ； rapid disease diagnosis

參考文獻

Aida, Y., H. Murakami, M. Takahashi, and S.-N. Takeshima. 2013. Mechanisms of pathogenesis induced by bovine leukemia virus as a model for human T-cell leukemia virus. Front. Microbiol. 4: 328-339.

Al-Qudah, K., A. M. Al-Majali, and Z. B. Ismail. 2006. Epidemiological studies on caprine arthritis-encephalitis virus infection in Jordan. Small Rumin. Res. 66: 181-186.

Ali Al Ahmad, M. Z., F. Fieni, J. L. Pellerin, F. Guiguen, Y. Cherel, G. Chatagnon, A. B. Bouzar, and Y. Chebloune. 2008. Detection of viral genomes of caprine arthritis-encephalitis virus (CAEV) in semen and in genital tract tissues of male goat. Theriogenology 69: 473-480.

An, L., W. Tang, T. A. Ranalli, H. -J. Kim, J. Wytiaz, and H. Kong. 2005. Characterization of a thermostable UvrD helicase and its participation in helicase-dependent amplification. J. Biol. Chem. 280: 28952-28958.

Andras, S. C., J. B. Power, E. C. Cocking, and M. R. Davey. 2001. Strategies for signal amplification in nucleic acid detection. Mol. Biotechnol. 19: 29-44.