Detection of Avian Reovirus Infection Using a Reverse-Transcription Loop-mediated Isothermal Amplification Assay

利用反轉錄廻路媒介恆溫增幅法診斷家禽里奧病毒感染

沈品均(Pin-Chun Shen) ； 楊佳玲(Jia-Ling Yang) ； 李龍湖(Long-Huw Lee)

《臺灣獸醫學雜誌》 39卷2期 (2013/06) Pp. 119-127

摘要

針對家禽里奧病毒（ARV）S1133株S1基因片段設計6個引子開發反轉錄廻路媒介恒溫增幅法（RT-LAMP），用於檢測ARV RNA。以供試三株ARV為樣品進行RT-LAMP測試，從每株病毒樣品中均可測得增幅產物，但增幅產物無法於其他4種重要家禽病毒，5種重要病原菌，和無ARV感染的對照組細胞及雞隻肌腱檢體中檢測到。本試驗利用純化的ARV基因體雙股RNA（dsRNA）測定結果做比較。結果顯示RT-LAMP檢測ARV RNA的極限值為0.01 pg。敏感度為反轉錄聚合鏈反應（RT-PCR）的10倍。除可自ARV S1133感染雞隻腳踝關節肌腱檢體中檢測出ARV RNA外，試驗結果顯示RT-LAMP也可從田間飼養肉雞腳踝關節肌腱檢體中（16/23）檢測出ARV RNA，且和RT-PCR檢測結果一致。因此，RT-LAMP具有快速、高特異性及高敏感度等特性，適合應用於診斷ARV感染。

關鍵字

針對家禽里奧病毒 ； 反轉錄廻路媒介恒溫增幅法 ； 反轉錄聚合鏈反應 ； 病毒性關節炎

並列摘要

The method of reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed for the detection of avian reovirus (ARV) infections, using primers specific to ARV S1. The amplification products were detected in three strains of ARV; whereas, none was obtained from the irrelevant controls. The sensitivity of RT-LAMP for ARV dsRNA was determined and compared with reverse transcription-polymerase chain reaction (RT-PCR) in parallel. The detection limit of RT-LAMP was 0.01 pg of dsRNA which was 10-fold more sensitive than that of RT-PCR. In addition to detecting ARV RNA in tendon tissues collected from S1133-infected chickens, RT-LAMP could also detect ARV RNA in 16 out of 23 tendon-tissue samples prepared from commercial broilers. The RT-LAMP detection results concorded 100% (16/16) with that detected by RT-PCR. This study demonstrates a speed, specificity and sensitivity RT-LAMP that is relevant for the diagnosis of ARV infection.

並列關鍵字

avian reovirus ； RT-LAMP ； RT-PCR ； viral arthritis