CAJ | 학술논문

　　目的建立双重荧光PCR检测HIV前病毒DNA的方法，并应用于婴幼儿HIV感染的早期诊断。方法采用TaqMan技术，组建针对人类核糖核酸酶P（ RNase P）和HIV的长末端重复序列（ LTR）基因的双重荧光PCR体系；采用TA克隆技术构建pTG19-T重组质粒作为模板进行该方法灵敏度的评价；采用11例已知健康人的血样和98例已知HIV感染者血样进行该方法的特异性验证；收集2011年1月至2012年9月浙江省各地妇幼保健医疗机构上送的96份婴幼儿样本，用新方法进行HIV的早期诊断，并将检测结果与罗氏HIV DNA定性检测试剂盒作比较。结果双重荧光PCR新方法能特异性检测HIV前病毒DNA，特异性为100％，检测灵敏度为100拷贝/反应；新方法和罗氏HIV DNA定性检测试剂盒检测96份婴幼儿样本，结果完全一致（符合率为100％）。结论建立的双重荧光PCR方法经济便捷、特异性好、灵敏度高、结果准确、易于推广，有望用于婴幼儿HIV早期诊断，并为HIV前病毒DNA的检测提供了一个通用技术平台。
　　목적건립쌍중형광PCR검측HIV전병독DNA적방법，병응용우영유인HIV감염적조기진단。방법채용TaqMan기술，조건침대인류핵당핵산매P（ RNase P）화HIV적장말단중복서렬（ LTR）기인적쌍중형광PCR체계；채용TA극륭기술구건pTG19-T중조질립작위모판진행해방법령민도적평개；채용11례이지건강인적혈양화98례이지HIV감염자혈양진행해방법적특이성험증；수집2011년1월지2012년9월절강성각지부유보건의료궤구상송적96빈영유인양본，용신방법진행HIV적조기진단，병장검측결과여라씨HIV DNA정성검측시제합작비교。결과쌍중형광PCR신방법능특이성검측HIV전병독DNA，특이성위100％，검측령민도위100고패/반응；신방법화라씨HIV DNA정성검측시제합검측96빈영유인양본，결과완전일치（부합솔위100％）。결론건립적쌍중형광PCR방법경제편첩、특이성호、령민도고、결과준학、역우추엄，유망용우영유인HIV조기진단，병위HIV전병독DNA적검측제공료일개통용기술평태。
Objective To establish a duplex fluorescence PCR for detection of HIV proviral DNA and to evaluate its application for early diagnosis of HIV infection in infants .Methods A duplex fluores-cence PCR system was set up based on TaqMan technology for detection of human ribonuclease P ( RNase P) gene and long terminal repeat ( LTR) region of HIV.A recombinant plasmid containing the targeted gene fragment , pTG19-T, was constructed by TA cloning technique and used as the template for evaluation of sen -sitivity of the assay .Blood samples from 11 healthy individuals and 98 HIV-infected patients were collected and detected to validate the assay specificity .The assay of duplex fluorescence PCR was then carried out to detect 96 infant blood samples collected from several maternal and child health hospitals in Zhejiang province from January 2011 to September 2012 for early diagnosis of HIV infection .The results were compared with those by using the Roche HIV DNA qualitative detection kit .Results The established duplex fluorescence PCR could specifically detect HIV proviral DNA with a specificity of 100%and a detection sensitivity of 100 cps per reaction .The coincidence rate between the established assay and the Roche HIV DNA qualitative de -tection kit was 100%in the detection of 96 blood samples .Conclusion The duplex fluorescence PCR as-say showed advantages of cost-effectiveness , convenience , good specificity and accuracy with high sensitivi-ty.It could be used for early diagnosis of HIV infection in infants and also as a general technical platform for the detection of HIV proviral DNA .