CAJ | 학술논문

针对J亚群禽白血病病毒（ALV-J）基因序列保守区域设计一对特异性引物和一条特异性探针，通过构建重组阳性标准质粒作为阳性标准品，建立了检测ALV-J核酸的荧光定量PCR方法。优化反应体系和条件后进行特异性、敏感性、重复性试验。结果显示，该检测方法特异性强，与其它禽源病毒如A亚群禽白血病病毒（ALV-A）、B亚群禽白血病病毒（ALV-B）、新城疫病毒（ NDV）、禽流感病毒（AIV）、鸡传染性贫血病毒（CIAV）和马立克病病毒（MDV）均不发生交叉反应；该方法可检测到3.2×102拷贝/μL的病毒核酸，与常规RT-PCR相比，敏感性高100倍；重复性试验的变异系数小于2%。研究结果表明，建立的Real-time RT-PCR 检测方法特异性强、灵敏度高、可重复性好，可用于ALV-J的定量检测。
침대J아군금백혈병병독（ALV-J）기인서렬보수구역설계일대특이성인물화일조특이성탐침，통과구건중조양성표준질립작위양성표준품，건립료검측ALV-J핵산적형광정량PCR방법。우화반응체계화조건후진행특이성、민감성、중복성시험。결과현시，해검측방법특이성강，여기타금원병독여A아군금백혈병병독（ALV-A）、B아군금백혈병병독（ALV-B）、신성역병독（ NDV）、금류감병독（AIV）、계전염성빈혈병독（CIAV）화마립극병병독（MDV）균불발생교차반응；해방법가검측도3.2×102고패/μL적병독핵산，여상규RT-PCR상비，민감성고100배；중복성시험적변이계수소우2%。연구결과표명，건립적Real-time RT-PCR 검측방법특이성강、령민도고、가중복성호，가용우ALV-J적정량검측。
A pair of specific primers and a specific probe was designed according to the ALV-J conserved gene sequence and a plasmid containing the target gene was constructed as a standard control. A real-time PCR assay was developed for detection of ALV-J. After optimization of the reaction conditions,the assay was tested for its specificity,sensitiv-ity and repeatability. Result showed that the assay was highly specific and there were no cross-reactions with other avian pathogens including ALV-A,ALV-B,NDV,AIV,CIAV and MDV. The assay had a detection limit of 3.2×102 cop-ies/μL viral RNA,100 times more sensitive than the conventional PCR,and its coefficient of variations was less than 2%in the reproducible test. The results indicated that this real-time RT-PCR assay was of high specificity,sensitivity and reproducibility,and could be used for the quantitative detection of ALV-J.