用于我国新生儿HIV-1感染早期诊断的单管降落巢式三重PCR检测方法的建立  

Establishment of a one tube touchdown nested three-monoplex PCR assay for HIV-1 early diagnosis in newborns in China

作　　者：苏雪丽[1,2] 姚均 蒋岩 王临虹 韩剑锋 张麒 卢红艳[1] 贺雄[1] 

机构地区：[1]北京市疾病预防控制中心 [2]国家疾病预防控制中心

出　　处：《国际病毒学杂志》2012年第3期102-108,共7页International Journal of Virology

基　　金：“十五”国家科技攻关计划课题资助项目（课题名称：我国艾滋病母婴传播模式及综合预防策略的研究,课题编号：2004BA719A03）、克林顿基金项目.

摘　　要：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-1A、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。Objective HIV-1 mother-to-child transmission remains a challenge in China. The de- velopment 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 Chi- nese infant HIV-1 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 in- fants 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 po- tential for early infant HIV-1 diagnosis in China.

关 键 词：新生儿 HIV-1 诊断 单管 三重PCR 干血斑 

分 类 号：R722.1[医药卫生—儿科]