CAJ | 학술논문

目的利用环媒恒温扩增技术(loop-mediated isothermal amplification,LAMP),建立检测原型泡沫病毒(prototype foamy virus,PFV)的方法.方法根据PFV的整合酶核心区基因序列设计了3对LAMP引物,利用有链取代活性的Bst DNA聚合酶在63℃对靶序列进行扩增.优化检测条件,建立PFV的检测方法.结果以含目的片段的质粒为模板建立了PFV的检测方法,运用此方法可特异地检测出PFV,而人免疫缺陷病毒、牛免疫缺陷病毒、牛泡沫病毒检测均为阴性,检测灵敏度为50拷贝,较聚合酶链反应高一个数量级.系统可以在15 min内完成扩增,同时可以在细胞基因组干扰下检出病毒,对混有病毒基因的人外周血单个核细胞(PBMC)总DNA的检测呈现特异阳性反应.结论建立基于PFV整合酶基因的LAMP检测方法,在PFV感染检测方面有应用前景.
목적 이용배매항온확증기술(loop-mediated isothermal amplification,LAMP),건립검측원형포말병독(prototype foamy virus,PFV)적방법.방법 근거PFV적정합매핵심구기인서렬설계료3대LAMP인물,이용유련취대활성적Bst DNA취합매재63℃대파서렬진행확증.우화검측조건,건립PFV적검측방법.결과 이함목적편단적질립위모판건립료PFV적검측방법,운용차방법가특이지검측출PFV,이인면역결함병독、우면역결함병독、우포말병독검측균위음성,검측령민도위50고패,교취합매련반응고일개수량급.계통가이재15 min내완성확증,동시가이재세포기인조간우하검출병독,대혼유병독기인적인외주혈단개핵세포(PBMC)총DNA적검측정현특이양성반응.결론 건립기우PFV정합매기인적LAMP검측방법,재PFV감염검측방면유응용전경.
Objective To develop prototype foamy virus (PFV) detection method by loop-mediated isothermal amplification. Methods Three pairs of primers targeting core region of PFV integrase were designed in this study and Bst DNA polymerase was used to amplify target sequence at 63℃. The system was established with all the conditions optimized. Results The method was established with the plasmid containing target sequence as the template. This method could specifically detect PFV infectious clone, no crossreaction was observed with human immunodeficieney virus infectious clone, bovine immunodefieiency virus infectious clone and bovine foamy virus infectious clone as templates. The detection capability of this system was 50 copy, one order more sensitive than PCR. The amplification could be finished in 15 min and human genomic DNA did not adversely affect the amplification efficiency. Conclusion The PFV detection method by loop-mediated isothermal amplification was established and it had potential usefulness in PFV detection.