寡核酸微陣列在鳥類性別鑑定及新城病與禽流感病毒篩檢之應用

Abstract

國內每年救傷收容或新進檢疫的野生鳥禽（如各縣市野鳥協會，南投集集特有生物中心，各公私立動物園等）數量龐大。 然而由於多數無法由外觀判定性別，使得後續的繁殖、保育及管理等工作窒礙難行。此外野生鳥禽攜帶人畜共同傳染病病原的機會相當高，且常為不發病的潛伏帶原者，故如何篩檢帶原疾病並確定病原的種類， 以採取對應措施而及時杜絕疾病的傳播，是疫病防治上亟待解決的問題。另外由於鳥類天生的易緊迫性，如何減少因採樣緊迫所帶來的損傷，也是本研究考量的重點之一。DNA微陣列(DNA microarray)，又稱為DNA晶片，依探針的長短可分為cDNA微陣列及寡核酸微陣列兩種，為近十年來發展的新興技術。藉由樣本核酸分子與探針之間專一性結合後所顯示的訊號，可在極小的晶片上迅速得到大量的基因分子訊息，其對遺傳基因的分析或是病原的偵測診斷，提供了一個快速方便的檢測途徑。此研究的目的是希望利用DNA微陣列可以同步檢測多重核酸分子的特性，更精準快速地鑑定各類鳥種的性別，以突破現行各種鑑定法的瓶頸。本研究同時針對兩項重要的人鳥共同傳染病-新城病(Newcastle disease, ND) 與家禽流行性感冒(Avian influenza, AI)，作同步檢測與病毒分型，以檢視鳥禽帶原狀況並及時防堵病原的入侵。研究方法如下：首先對各類鳥禽的性聯基因作定序，擷取基因庫中各型家禽流行性感冒與新城病病毒的核酸序列，再經由進一步的比對分析，分別設計高度專一性的引子與探針，接著各自開發單一的PCR檢測法，並建立陽性對照組；再逐一修正合併成多引子聚合酵素連鎖反應(Multiplex PCR)的模式，以增進多重核酸分子的檢測效能。將設計好的各個探針點漬在晶片不同的位置上，完成DNA微陣列的製作；再把待測樣本的核酸標的分子以多引子聚合酵素連鎖反應方式大量增殖後，與晶片上的探針作雜交反應。配合陽性對照反應的結果，進行雜交反應訊號的比對與解讀，由此判斷受檢個體的性別，或是作病毒的篩檢與分型。在我們的研究結果中發現羽毛的羽軸，特別是靠基部的前半部是一個很好的體基因DNA (genomic DNA)的來源，此意謂著羽毛檢體並不需要用拔毛的方式獲得，以剪取的方式取得羽軸前半部即可，此法可大大降低受檢鳥體所受的傷害與緊迫。在鳥類性別檢測方面，我們併用兩個CHD1基因的插入子(intron)作多引子聚合酵素連鎖反應，透過寡核酸微陣列上所設計的9個探針的同步作用，已可檢測至少80種涵蓋26科(family)以上不同的鳥類。在病毒檢測方面，我們將ND病毒的融合蛋白質(fusion protein)基因， AI病毒的基質蛋白質(matrix protein)基因以及AI病毒的血球凝集素(haemagglutinin) 基因，作多引子聚合酵素連鎖反應的同步增殖，再利用寡核酸微陣列上所設計的12個探針之聯合作用，已可成功同步檢測ND病毒與AI病毒並作病毒的分型。寡核酸微陣列檢測系統在鳥類性別與人畜共同傳染病的開發與應用，將提供另一快速檢測的途徑，有助於野生鳥類的保育繁殖與疾病防疫體制的建立。

There are numbers of wild birds rescued or quarantined in Taiwan each year. Most of them are endangered species. Their rarity poses great responsibility of us on breeding. Information of an individual bird’s sex, thus, is critical to the breeding strategies and conservation programs. Wild birds are important carriers for many pathogens, e.g. Newcastle disease virus, avian influenza virus and others. Early detection of these pathogens from carrier birds is important for the disease prevention from spreading to susceptible herds. DNA microarray, also called DNA chip, is a newly developed technique this decade. It is divided into cDNA microarray and oligonucleotide microarray based on the size of probes. Lots of molecular information is obtained at the same time through hybridization between target DNA and probes on the microarray. It provides a rapid approach to the gene analysis and pathogen detection. The purpose of this study is to develop an oligonucleotide microarray system which can precisely determine the gender of diversified avian species. Simultaneous detection and differentiation of the most two important zoonotic viruses, Newcastle disease virus and avian influenza virus, is also the main objective of this research. Most birds are vulnerable to stress. How to decrease injuries while handling, therefore, is a major consideration. Sex-related gene markers of various avian species were sequenced. The sequencing nucleotides, including the sequences retrieved from the GenBank, were aligned and analyzed, and specific primers and probes were designed. Each respective PCR method was developed, and then gradually integrated into multiplex PCR. The probes were spotted to specific positions on the microarray polymer substrate. Hybridization between target DNA and probes was performed on the array surface. The information of the gender and the carried pathogens were then obtained according to the hybridization pattern on the microarrays. We found that a cut feather including the scapus, instead of a plucked feather, is a good source of genomic DNA for bird sexing. It means this cutting method could substantially decrease injury to the examined birds. The nine designed probes on microarrays combined with two-CHD1 intron approach have successfully identified the sex of 80 avian species covering 26 families. Moreover, the 12 designed probes on microarrays targeting fusion protein gene of NDV, matrix protein gene of AIV and haemagglutinin gene of AIV also have successfully detected and differentiated different types of viruses simultaneously. Oligonucleotide microarrays, therefore, may provide potential for rapid sexing and zoonosis detection. It would be conducive to the conservation and zoonosis prevention of wild birds.