CAJ | 학술논문

　　目的建立一种快速、敏感度高的实时荧光环介导等温定量检测HIV-1 DNA的方法。方法根据HIV病毒基因序列选择6个保守区域设计4条环介导等温扩增（ LAMP）引物，建立环介导等温扩增体系，同时在体系中加入SYBR GreenⅠ荧光染料，并评价该方法的特异性和敏感度。结果成功合成实时荧光环介导等温定量检测HIV-1 DNA反应体系，检测200例HIV感染者外周淋巴细胞HIV-1 DNA，其中有195例阳性，并检测50例健康对照结果均阴性。其中有HIV-1 DNA定量结果显示最低含量为51拷贝/ml，最高含量为8．21×106拷贝/ml，平均值含量为5．78×105拷贝/ml，其中病毒含量比较集中在105数量级范围，共162例占83．08％。采用10倍稀释法发现该方法最低检出限为50拷贝/ml。对乙型肝炎病毒、疱疹病毒、丙型肝炎病毒进行交叉实验结果均为阴性。结论HIV感染者外周血中几乎都存在HIV-DNA，实时荧光环介导等温扩增是一种快速、敏感度高、特异性强的方法，适合各级医院的临床检测。
　　목적건립일충쾌속、민감도고적실시형광배개도등온정량검측HIV-1 DNA적방법。방법근거HIV병독기인서렬선택6개보수구역설계4조배개도등온확증（ LAMP）인물，건립배개도등온확증체계，동시재체계중가입SYBR GreenⅠ형광염료，병평개해방법적특이성화민감도。결과성공합성실시형광배개도등온정량검측HIV-1 DNA반응체계，검측200례HIV감염자외주림파세포HIV-1 DNA，기중유195례양성，병검측50례건강대조결과균음성。기중유HIV-1 DNA정량결과현시최저함량위51고패/ml，최고함량위8．21×106고패/ml，평균치함량위5．78×105고패/ml，기중병독함량비교집중재105수량급범위，공162례점83．08％。채용10배희석법발현해방법최저검출한위50고패/ml。대을형간염병독、포진병독、병형간염병독진행교차실험결과균위음성。결론HIV감염자외주혈중궤호도존재HIV-DNA，실시형광배개도등온확증시일충쾌속、민감도고、특이성강적방법，괄합각급의원적림상검측。
Objective To establish a rapid and high sensitive assay of real-time fluorescence loop-mediated isothermal amplification assay for clinical detection of HIV-1 DNA.Methods Four loop primers were constructed for loop-mediated isothermal amplification ( LAMP) assay based on six conserved regions selected from HIV gene sequence .SYBR Green Ⅰdye was added into the established LAMP assay and its specificity and sensitivity were evaluated .Results The real-time fluorescence LAMP assay for the detection of HIV-1 DNA was successfully established .It was used for the detection of HIV-1 DNA among 200 patients with HIV infection, of which 195 cases were positive.Moreover, 50 healthy subjects were found HIV-1 DNA negative tested by the real-time fluorescence LAMP assay .Quantitative testing for HIV-1 DNA showed that the lowest and the highest detectable amount were 51 copies/ml and 8.21×106 copies/ml respectively, and the average amount was 5.78×105 copies/ml.HIV viral loads ranging from 1×105 to 10×105 were detected in 162 of 200 patients (83.08%).The ten times dilution method showed that the lowest detection limit of the assay was 50 copies/ml.The crossover experiment indicated that the specificity of the assay was 100%as none of HBV-DNA, HSV-DNA and HCV-RNA was determined by the assay .Conclusion The present study shows that 97.5%of the patients with HIV infection are confirmed HIV-1 DNA positive by the real-time fluorescence LAMP assay , suggesting that the real-time fluorescence LAMP assay is a rapid and sensi-tive assay with high specificity and could be applied for clinical detection of HIV-1 DNA.