CAJ | 학술논문

目的我国面临着艾滋病母婴垂直传播的挑战.发展经济、有效的新生儿HIV-1感染早期诊断方法至关重要.本研究拟结合降落PCR、巢式PCR、多重PCR技术,以期建立一种能够在同一个PCR反应体系中同时扩增Env、Gag、Pol三个基因区的单管降落巢式三重PCR方法用于我国新生儿滤纸干血斑样本(DBS)中的HIV-1前病毒DNA检测,并对所建立的方法进行初步评价.方法样本采自云南、新疆、广西、河南四省HIV-1阳性母亲所生婴儿.用18个DBS样本进行方法优化(感染婴儿及未感染婴儿样本分别9个).采用由重组质粒及8E5细胞制备的涵盖HIV-1各亚型的DNA稀释盘确定方法的亚型适用性及检测下限.采用134个DBS样本进行临床评价(含25个感染婴儿的64个DBS样本及30个未感染婴儿的70个DBS样本).结果所建方法对HIV-1 A、AE、AG、B、BC、C、D、F、H亚型均可适用,检测下限为3拷贝/PCR反应体系；其临床特异度100％,阳性检出率在3月龄时接近95％、6月龄时可达到100％.结论本研究建立的方法能够检测出我国新生儿DBS样本中各亚型的HIV-1前病毒DNA,且省时、省力、节约成本,在我国新生儿HIV-1感染的早期诊断领域具备应用潜力.
목적 아국면림착애자병모영수직전파적도전.발전경제、유효적신생인HIV-1감염조기진단방법지관중요.본연구의결합강락PCR、소식PCR、다중PCR기술,이기건립일충능구재동일개PCR반응체계중동시확증Env、Gag、Pol삼개기인구적단관강락소식삼중PCR방법용우아국신생인려지간혈반양본(DBS)중적HIV-1전병독DNA검측,병대소건립적방법진행초보평개.방법 양본채자운남、신강、엄서、하남사성HIV-1양성모친소생영인.용18개DBS양본진행방법우화(감염영인급미감염영인양본분별9개).채용유중조질립급8E5세포제비적함개HIV-1각아형적DNA희석반학정방법적아형괄용성급검측하한.채용134개DBS양본진행림상평개(함25개감염영인적64개DBS양본급30개미감염영인적70개DBS양본).결과 소건방법대HIV-1 A、AE、AG、B、BC、C、D、F、H아형균가괄용,검측하한위3고패/PCR반응체계；기림상특이도100％,양성검출솔재3월령시접근95％、6월령시가체도100％.결론 본연구건립적방법능구검측출아국신생인DBS양본중각아형적HIV-1전병독DNA,차성시、성력、절약성본,재아국신생인HIV-1감염적조기진단영역구비응용잠력.
Objective HIV-1 mother-to-child transmission remains a challenge in China.The development of an efficient and affordable assay for early infant HIV-1 diagnosis is crucial.In this study,an in-house one-tube PCR assay combining touchdown PCR,nested PCR and triplex PCR techniques for Chinese infant HIV-I proviral DNA detection in dried blood spot(DBS) samples was developed and optimized to amplify Env,Gag,and Pol gene fragments in one PCR tube.Primary evaluation of the assay was made.Methods DBS from infants born to HIV-1 positive mothers in Guangxi,Henan,Xinjiang and Yunnan provinces were sampled for study.9 infected samples and 9 uninfected samples were used for optimization of the assay.An HIV-1 genome dilution panel derived from recombinant plasmids and 8E5 cells was prepared for determining subtype applicability of the assay and low limit of detection.64 samples from 25 infected infants and 70 samples from 30 uninfected babies were used for clinical evaluation.Results The established assay is applicable to HIV-1 subtypes A、AE、AG、B、BC、C、D、F and H.Low limit of the detection is3copies/PCR reaction.Clinical specificity was 100％.Positive detection rate achieved nearly 95％ at 3-month age and 100％ at 6-month age.Conclusions The assay can detect HIV-1 proviral DNA of multiple subtypes in Chinese infants' DBS samples.It is time-saving,labor saving and economic,showing good potential for early infant HIV-l diagnosis in China.