篇名:
建立快速區別豬瘟野外毒及兔化豬瘟疫苗毒之RT-PCR檢測方法Rapid detection and differentiation of wild-type and attenuated lapinized vaccine strains of classical swine fever virus by reverse-transcription polymerase chain reaction
作者:
潘居祥;鍾明華;黃有良;黃天祥;趙磐華;賴秀穗*
中文摘要:
本試驗基於兔化豬瘟疫苗毒核酸序列中有一段T-rich插入之特性發展單一步驟RT-PCR檢測方法,使用單步驟RT-PCR或?式RT-PCR增幅,經傳統洋菜膠或毛細管電泳後,可同時檢測及區別臨床檢體中的豬瘟野外毒及兔化豬瘟疫苗毒。本方法至少可應用於LPC、HCLV及C-strain等三種兔化豬瘟疫苗毒,對於豬瘟野外毒檢測的敏感度RT-PCR及?式RT-PCR分別為6.3及0.63 TCID50/ml。前人報告指出,兔化豬瘟疫苗毒核酸序列3' 端未轉譯區有一段12至13個T-rich插入之特性,然而我們發現LPC/PRK及LPC/TS兩株疫苗毒T-rich插入片段長度分別多達42及36個核?酸,這些不同大小的T-rich插入片段增加RT-PCR產物的大小,可當做很好的基因標記藉以快速區別豬瘟野外毒及不同兔化豬瘟疫苗毒株。
英文摘要:
A simple one-step reverse transcription-polymerase chain reaction (RT-PCR) method was developed based on T-rich insertions in the viral genome for simultaneous detection and differentiation of wild-type and vaccine strains of classical swine fever virus (CSFV). The CSFV-specific primers were designed to contain the sequences of the T-rich insertion sites that exist uniquely in the 3' nontranslated regions (3' NTR) of the genome of lapinized CSFV vaccine strains. Using a one-step RT-PCR or a semi-nested RT-PCR followed by an agarose gel electrophoresis or a multi-capillary electrophoresis, the wild-type and lapinized vaccine strains of CSFV in clinical samples could be detected and accurately distinguished. These assays can be applied to at least three attenuated lapinized vaccine strains, LPC (lapinized Philippines Coronel), HCLV (hog cholera lapinized virus), and C (Chinese)-strain. The detection limit of the wild-type virus was 6.3 TCID50 (50% tissue culture infective dose)/ml for RT-PCR and 0.63 TCID50/ml for semi-nested RT-PCR. In previous studies, notable T-rich insertions of 12–13 nucleotides (nts) were found in the 3' NTR of the genome of lapinized vaccine strains of CSFV. However, this study discovered that two T-rich insertions, 42 and 36 nts in length, are present in the viral genome of lapinized vaccine strains LPC/PRK (primary rabbit kidney) and LPC/TS (Tam-Sui), respectively. These T-rich insertions of 12, 36, and 42 nts length increases the size of PCR fragments, which are favorable genetic markers for rapid detection and differentiation of wild-type and different lapinized vaccine strains of CSFV.
備註:
年份:西元 2004 年
期別:第 40 期
附件下載:
附件名稱:
011
行政院農業委員會家畜衛生試驗所版權所有
服務信箱:info@mail.nvri.gov.tw
Tel:0800-068112、02-26212111
FAX:02-2622-5345

免責聲明
 新北市淡水區中正路376號