應用反轉錄環圈式恆溫核酸增幅法快速偵測藍舌病病毒

藍舌病是由藍舌病病毒(Bluetongue virus, BTV)所引起， BTV主要藉由節肢動物叮咬傳播並感染野生及馴養之反芻獸，其中以綿羊和白尾鹿最具感受性，但引起之臨床症狀因病毒株、動物個體及品種的影響而有差異。除綿羊外，本病於其他反芻動物多為不顯性感染，易造成病原的隱藏及疾病的擴散，遂檢測潛在病原為重要議題。本研究應用反轉錄環圈式恆溫核酸增幅法(Reverse Transcription Loop-mediated Isothermal Amplification, RT-LAMP)進行BTV核酸擴增。本實驗應用四條特殊設計之引子標定BTV第五個片段基因上的六個位置，使用耐高溫之反轉錄酶(AMV reverse transcription)及具有取代股能力之Bst DNA polymerase，於65℃一小時內擴增目標序列。特異度方面，RT-LAMP於其他測試四個常見之病原的核酸並無交叉反應。本實驗自中部某牛場採集田間健康牛隻之血液樣本以競爭性酵素免疫分析法、反轉錄聚合酶連鎖反應 (RT-PCR) 與RT-LAMP進行檢測，其血清抗體陽性率為80% (12/15) 、 RT-PCR以及RT-LAMP陽性率分別為0% (0/15)和20% (3/15)，顯示RT-LAMP有較高之敏感度。本研究建立一簡單、準確、具高敏感度及高經濟效益之RT-LAMP法進行偵測BTV，期許未來能增進檢測潛在藍舌病感染之野外樣本陽性率以及提供於資源受限之情況下快速篩檢BTV之工具。

關鍵字

藍舌病；反轉錄環圈式恆溫核酸增幅法；反轉錄聚合酶連鎖反應

並列摘要

Bluetongue, an arthropod-borne viral disease, is caused by bluetongue virus (BTV), belonging to the Orbivirus genus of the family Reoviridae. Most species of ruminants can be infected with BTV, and most infected ruminants usually present mild or no clinical signs. These ‘reservoir hosts’ may potentially further the viral transmission and expansion of the disease resulting in severe economic loss; thus, detection of subclinical infection is an important issue. To detect the BTV, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed. Four specially designed primers were employed to target six regions of the segment 5 gene. The whole process was completed in one hour at one temperature (65℃), and the products can be specifically digested with MboII enzyme. There was no cross-reaction with other tested common ruminant infectious agents. By screening fifteen field blood samples from clinically healthy dairy cattle in central Taiwan, the diagnostic sensitivity of RT-LAMP assay was compared with reverse transcription polymerase chain reaction (RT-PCR). The sera samples from the same animals were also screened by competitive enzyme-linked immunoassay. The seroprevalence rate was 80% (12/15) and the positive rates of RT-PCR and RT-LAMP were 0% (0/15) and 20% (3/15), respectively. The results indicated the high sensitivity of the RT-LAMP assay. In this study, we establish a novel RT-LAMP assay to provide a simple, highly efficient and sensitive method to detect BTV specifically and is suitable for the screening of field samples, especially in resource-shortage conditions.

並列關鍵字

bluetongue virus (BTV) ； reverse transcription loop-mediated isothermal amplification (RT-LAMP) ； reverse transcription polymerase chain reaction (RT-PCR)

參考文獻

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應用反轉錄環圈式恆溫核酸增幅法快速偵測藍舌病病毒

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