結合恆溫環形核酸增幅法與螢光標示引子區別野外豬瘟病毒株與兔化豬瘟疫苗株

Differentiating Wild Type and LPC Strains of Classical Swine Fever Virus by Using the Combination of RT-LAMP and Fluorophore Labeling Primer Methods

鄧明中(Ming-Chung Deng) ； 林育如(Yu-Ju Lin) ； 宣詩玲(Shih-Ling Hsuan) ； 李敏旭(Tien-Shine Huang) ； 黃天祥(Ming-Shiuh Lee) ； 黃金城(Chih-Cheng Huang) ； 簡茂盛(Maw-Sheng Chien)

《臺灣獸醫學雜誌》 34卷2期 (2008/06) Pp. 106-115

https://doi.org/10.7009/TVJ.200806.0106

摘要

台灣使用兔化豬瘟疫苗進行豬瘟防疫，但由於兔化豬瘟疫苗株與野外毒株無法有效地以血清學或病毒分離的方式進行鑑別，因此建立快速而敏感的區別診斷方法對於豬瘟防疫是非常重要的一環。恆溫環形核酸增幅法由於所需設備簡便且操作容易，目前已開發完成快速偵測豬瘟病毒核酸的方法，但此法若需進一步應用於區別診斷，則必須增設引子及進行雙重反應等，對於臨床應用容易徒增勞力與引起交叉污染。我們結合特異性螢光標示引子－LUX引子，配合快速敏感的RT-LAMP反應，藉由定量PCR反應儀偵測特異性螢光訊號，可同時於單一反應管中達到快速區別診斷豬瘟野外毒與疫苗株的目的。實驗結果顯示此項新技術之偵測極限範圍分別為10^0.6 TCID50（野外株）與10^1.0 TCID50（疫苗株），遠較一般常用於區別診斷之鐵克曼探針法來的方便、快速且敏感，此成功開發之新方法對於日後應用於豬瘟疫情防疫將有很大的幫助。

關鍵字

豬瘟病毒 ； 鐵克曼探針 ； 反轉錄恆溫環式核酸增幅法

並列摘要

In Taiwan, the immunization of pigs with lapinized hog cholera vaccine (LPCV) has been generally used for the control of classical swine fever (CSF) disease. It has rather difficulty to differentiate LPC strain and field strain using conventional methods of virus isolation or serologic examination. Therefore, it is desperately needed to develop a rapid, accurate and efficient method for screening CSF virus. The loop-mediated isothermal amplification (LAMP) assay with less equipment requirement and easy to be operated has been developed for rapid identification of CSF virus nucleic acids. However, for differential diagnosis of viruses, LAMP method may need the time-consuming designation of specific primers and cause cross-contamination by duplicated reactions. We have developed a method by combining RT-LAMP and Lux (Light Upon eXtension) fluorogenic primer technology to rapidly differentiate LPC from field strains in a single tube using RT-PCR analyzer for detection of fluorescent signal from the LUX primer. The results show that detection limitation of the novel method to detect virus are 1006 TCID50 for field strain and 10 TCID50 for LPC strain, respectively. The method developed is rather convenient, rapid and sensitive than the current TaqMan probe method for differentiating various viruses and which will be greatly helpful for disease control and prevention.

並列關鍵字

classical swine fever virus ； taqMan probe ； reverse transcriptase-loop-mediated isothermal amplification