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中文摘要：
目的建立1种基于实时PCR的检测HIV-1准种的终点有限稀释PCR技术并评价其应用价值。方法采用基于第2轮实时和常规PCR的多重有限稀释巢式PCR、终点PCR及测序等技术对3例低病毒载量HIV-1感染者不同时间点PBMC中的HIV-1前病毒准种ew基因e2-v3-c3区域和gag基因p17区域进行扩增和序列测定；序列经确证后进行系统进化树分析并对氨基酸序列进行比对；用QUALITY程序计算HIV-1DNA拷贝数。结果3例患者所有随访时间点标本共进行1207个巢式实时PCR，从第二轮常规巢式PCR共获得65条c2-v3-c3准种序列、101条p17准种序列；HIV-1前病毒水平介于1．34～53．76拷贝／10。个PBMC之间。c2-v3-c3及p17核苷酸序列系统进化树分析表明，不同患者的序列分开、同一患者的序列特异聚集在一起。氨基酸序列比对分析表明，HIV-1感染早期患者体内HIV-1准种序列比较单一，随着感染时间的增加，HIV-1准种趋于复杂化。结论本研究建立的独特终点有限稀释PCR技术检测HIV-1准种灵敏度高、特异性好、高通量，可用于低水平病毒载量HIV-1感染者HIV-1准种研究。
英文摘要：
Objective To establish a novel end-point limiting-dilution PCR （EPLD-PCR） assay to detect human immunodeficiency virus type 1 （HIV-1） quasispecies and evaluate its application value. Methods The multiplex limiting-dilution nested PCR base on the second round TaqMan real time PCR （N-RT PCR） and regular PCR, end-point PCR and sequencing technology were used for detection of the sequences of HIV-1 quasispecies env gene c2-v3-c3 region and gag gene p17 region from the PBMCs of 3 HIV-1 infected patients with low level viral load. All sequences of c2-v3-c3 and p17 were confirmed by sequencing. Phylogenetic tree analysis for nucleotide sequences and alignment analysis for amino acid sequences were performed to evaluate the result of HIV-1 quasispecies assay. The HIV-1 DNA copy number in per million PBMCs was calculated by QUALITY program. Results Total 1 207 N-RT PCRs were carried out for all 3 patients＇ samples from different visiting time points. Sixty-five sequences of c2-v3-c3 and 101 sequences of p17 were obtained from regular nested second round PCRs; the quantification of HIV-1 DNA in per million PBMCs from 3 patients was between 1.34 copies to 53.76 copies. Phylogenetic tree analysis for all nucleotide sequences showed that the sequences in different patients were separated and they got together specially in the same patient. Amino acid sequences alignment analysis showed that HIV-1 quasispecies sequences were simplex at early stage of infection and became more complex alone with increase of infected time. Conclusions The end-point limiting-dilution real-time PCR technology described in this study is a novel, sensitive, specific and high throughput PCR assay to detect human immunodeficiency virus type 1 quasispecies. This PCR assay could be applied for detection of HIV-1 quasispeeies in HIV-1 infected populations with low level viral load.
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