CAJ | 학술논문

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.
목적 건립분석HIV준충적Miseq고통량측서(간칭Miseq측서)기술,병상시용우일기의사경성전파HIV적소원조사.방법 채집의사유전파관계적2례HIV감염자(편호P1、P2)혈액양본,병이동일성시여타문무직접전파관계적2례HIV감염자(편호P3、P4)위대조.제취혈장중RNA,역전록후진행HIV기인아형분석화Miseq측서.근거측서결과분석양본중HIV적준충분포,비교사용우세준충수목유소도다(5、20、100、500、전부)시소득적기인리산솔급계통진화수.결과 4빈양본중HIV적기인아형상동,균위HIV-1중조아형(CRF) 01 _AE.채용Miseq측서,매빈양본획득23 788～37 397조유효핵산서렬,대표1 229～1 412개독특준충.P2여P1간적평균기인리산솔(3.5％～4.5％)원소우여대조양본간(10.3％～ 19.6％,P＜0.01).계통진화수결과현시,P1화P2적준충취집재일기,이P3화P4적각자단독취위일족;당소용적우세준충수목위20혹경다시,P1대P2존재병계관계,제시HIV적전파방향시종P1도P2.결론 Miseq측서재HIV감염소원조사중가이추단전파관계화전파방향,기조작교간편、검측성본교저,구유교고적실용개치.
Objective An 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.Methods Four 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 reversetranscribed 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.Results The 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.Conclusion With the feature of convenient and economic operation,Miseq platform has high practical value in contact tracing of possible HIV transmission.