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高通量测序平台用于一起疑似经性传播HIV的溯源调查
作者:赵琦 时丽丽 蒋岩 温玉洁 潘品良 张桂云 邱茂锋

摘要:

目的建立分析HIV准种的Miseq高通量测序(简称Miseq测序)技术,并尝试用于一起疑似经性传播HIV的溯源调查。方法采集疑似有传播关系的2例HIV感染者(编号P1、P2)血液样本,并以同一城市与他们无直接传播关系的2例HIV感染者(编号P3、P4)为对照。提取血浆中RNA,逆转录后进行HIV基因亚型分析和Miseq测序。根据测序结果分析样本中HIV的准种分布,比较使用优势准种数目由少到多(5、20、100、500、全部)时所得的基因离散率及系统进化树。结果4份样本中HIV的基因亚型相同,均为HIV1重组亚型(CRF)01_AE。采用Miseq测序,每份样本获得23 788~37 397条有效核酸序列,代表1 229~1 412个独特准种。P2与P1间的平均基因离散率(35%~45%)远小于与对照样本间(103%~196%,P<001)。系统进化树结果显示,P1和P2的准种聚集在一起,而P3和P4的各自单独聚为一簇;当所用的优势准种数目为20或更多时,P1对P2存在并系关系,提示HIV的传播方向是从P1到P2。结论Miseq测序在HIV感染溯源调查中可以推断传播关系和传播方向,其操作较简便、检测成本较低,具有较高的实用价值。

关键词:HIV;分子流行病学;疾病传播;新一代测序技术;准种

Abstract:

ObjectiveAn approach for analysis of HIV quasispecies using Miseq highthroughput sequencing platform (hereinafter referred to as Miseq platform) was established and applied to contact tracing for a possible case of HIV sexual transmission MethodsFour plasma specimens were collected from 2 HIV infections (P1 and P2) suspected to be involved in the sexual transmission and 2 local HIV infections as controls (P3 and P4) The RNAs were extracted from the specimens and then reversetranscribed into cDNA After HIV subtyping, Miseq platform was performed to detect and sequence the HIV quasispecies (352 bp) in each specimen The frequency of quasispecies was counted and ranked Intrapersonal and interpersonal genetic distance and phylogenetic tree were calculated by using the top 5, 20, 100, 500, and all quasispecies, respectively ResultsThe subtypes of HIV from all 4 specimens were CRF01_AE 23 788 to 37 397 cleaned sequences representing 1 229 to 1 412 unique HIV quasispecies were obtained from these specimens by using Miseq platform The average genetic distance (35%45%) between quasispecies from specimens P2 and P1 was significantly lower than that (103%196%) between quasispecies from P2 and the controls (P3 or P4) Phylogenetic tree analysis indicated that sequences from specimens P1 and P2 clustered together while sequences from P3 and P4 exhibited completely independent clusters When the top 20 or more quasispecies from each specimen were analyzed, sequences from P1 showed a paraphyletic relationship with those from P2, which may indicated that the direction of HIV transmission was from P1 to P2 ConclusionWith the feature of convenient and economic operation, Miseq platform has high practical value in contact tracing of possible HIV transmission

Key words: HIV;Molecular epidemiology;Disease transmission;New generation sequencing technology;Quasispecies

发表日期:2014/6

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