犬瘟熱病毒之分子檢測及其親緣關係之探討

Detection of Canine Distemper Virus and Phylogenetic Analysis by Molecular Techniques

周應欽(Ying-Chin Chou)

指導教授 ： 廖明輝

國立屏東科技大學/農學院/獸醫學系所/碩士(2013年)

https://doi.org/10.6346/NPUST.2013.00083

摘要

犬瘟熱(canine distemper, CD)是犬隻中重要且盛行已久之疾病，所有年齡的犬隻皆具有感受性，尤其是3~6月齡的幼犬，感染後會引起全身性及中樞神經系統性之症狀，屬於高發生、高傳染及高死亡率之疾病。因其臨床屬多系統性(multi-systemic)之症狀，因此在檢測上通常需花費許多時間來等待結果，故在臨床診斷上通常較不易確診，須藉由分子生物學診斷技術來輔助並加以確診。本實驗主要是應用反轉錄聚合酶連鎖反應(Reverse Transcriptase Polymerase Chain Reaction, RT-PCR）及隔絕式恒溫聚合酶連鎖反應(Insulated Isothermal Polymerase Chain Reaction, iiPCR)來檢測臨床上疑似感染犬瘟熱病毒(canine distemper virus, CDV)的檢體，並比較其陽性率及其親緣關係。在RT-PCR檢測引子對的敏感性為100 pg/μL，特異性檢測中僅對CDV才可檢測出。在疑似病例檢測陽性率方面，結果發現RT-PCR的陽性率為42%（42/100），而iiPCR的陽性率為41%（41/100）。雖然應用iiPCR檢測確診時間較快，但反應後產物無法直接進行核酸序列分析來比較親緣關係，而RT-PCR能直接進行核酸序列分析來比較親緣關係。從陽性檢體中隨機抽取10株病毒株，進行核酸序列分析，其中7株病毒序列與各國分離株相似度為99.3 -94.9%，但發現有3株病毒株序列與其他7株病毒株序列在親緣關係上差異性較大，值得再進一步的探討分析。

關鍵字

犬瘟熱病毒 ； 隔絕式恒溫聚合酶連鎖反應 ； 反轉錄聚合酶連鎖反應

並列摘要

Canine distemper virus (CDV) is an important disease and has a high prevalence for a long time. All ages are susceptible to infections, particularly in 3-6 months of age puppy. CDV is a high incidence, high prevalence and lethal disease, it causes systemic infection and central nervous system symptoms in affected dogs. CDV caused multisystem disorders and difficult to diagnose in clinical. This problem can be solved by molecular technique for disease diagnosis. The aim of this study was survey prevalence of suspected CDV infection by use reverse transcriptase polymerase chain reaction (RT-PCR), and insulated isothermal polymerase chain reaction (iiPCR), and established the phylogenetic relationship of wild-type CDV between different countries. The sensitivity of RT-PCR is 100 pg/L, the specificity was also established. The positive rate of RT-PCR and iiPCR were 42%, 41%, respectively. Although iiPCR is a time-saving technique, the reaction product can not subject to sequencing. Comparing the nucleic acid sequences obtained by RT-PCR with published strains in the GenBank. Seven of ten viral strain from positive specimen showed 99.3-94.9% identity with other country, but the other three wild-type strains had low identity. Further investigation is required for understanding the phylogenetic difference.

並列關鍵字

canine distemper virus ； iiPCR ； RT-PCR

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