高通量测序技术用于丙型肝炎病毒感染溯源调查  

Tracing hepatitis C virus transmission by using high-throughput sequencing technique

作　　者：张静娜[1] 王译葵 蒋岩[1] 龙玉存[3] 王继宝[2] 冯凯迪 唐仁海[2] 张志敏[1] 段松[2] 赵琦[4] 张桂云[1] 邱茂锋[1] 

机构地区：[1]中国疾病预防控制中心性病艾滋病预防控制中心,北京102206 [2]云南省德宏州疾病预防控制中心性病艾滋病防制科 [3]云南省德宏州陇川县疾病预防控制中心美沙酮门诊 [4]山东大学第二医院信息中心

出　　处：《中华预防医学杂志》2016年第6期530-534,共5页Chinese Journal of Preventive Medicine

基　　金：国家丙型肝炎防治项目@@@@National Funding for Hepatitis C Virus Control and Prevention

摘　　要：目的：建立检测丙型肝炎病毒（HCV）准种群的Hiseq高通量测序（Hiseq测序）技术，并调查一起疑似通过共用针具传播HCV的案例。方法针对2015年1月15日在美沙酮门诊发现的1例HCV抗体阳转静脉注射吸毒者（编号P1），调查在其可能感染HCV的时期内（2014年3月24日之后）与其共用针具、HCV抗体阳性的4例静脉注射吸毒者（编号P2～P5）的流行病学信息，并以与P1在同一美沙酮门诊服药或者居住在同一乡镇、HCV抗体阳性的28例静脉注射吸毒者（编号C1～C28）作为研究对照。分别采集上述33名的全血样品5 ml，提取血浆中的RNA并逆转录后进行HCV基因亚型分析，对亚型与P1相同的样品进行Hiseq测序，分析样品中HCV的准种分布情况，计算样品内和样品间的基因离散率，绘制系统进化树。结果 P1样品的基因亚型为3b，与其亚型相同的样品有P2、C7、C12、C14、C15、C16、C19、C20、C28。其中9份成功进行了Hiseq测序，获得的有效序列数为249753～1086333条（平均869608条）。剔除重复序列后，获得3～172个（平均48个）独特准种群的序列。9份样品的样品内基因离散率中位数（P50）为0.4%～12.3%；P1与其余8份样品间基因离散率的P50（P25，P75）分别为19.0%（18.4%，19.8%）、10.4%（2.8%，18.3%）、19.6%（17.8%，21.4%）、24.9%（23.8%，26.1%）、19.8%（18.7%，20.7%）、20.1%（18.9%，21.2%）、20.6%（20.0%，21.1%）、23.6%（22.4%，24.8%），差异无统计学意义（T=9.40，P=0.100）；P1与C7个别准种群间的基因离散率为0。系统进化分析显示， P1与P2之间无传播关系，但与C7之间可能存在传播关系。结论 Hiseq测序技术可以在准种水平上有效地用于HCV感染溯源调查，具有高通量、操作较简便、成本相对低、实用价值较高的特点。Objective An approach for analysis of hepatitis C virus （HCV） quasispecies using Hiseq high-throughput sequencing （hereinafter referred to as Hiseq sequencing） technique was developed and then applied to investigate a possible case of HCV needle sharing transmission. Methods One case of HCV antibody seroconversion （P1） was found in a methadone clinic on January 15, 2015. Four HCV antibody positive injecting drug users （IDUs）, P2 to P5, suspected to be involved in needle sharing transmission with P1 during the period （after March 24, 2014） that P1 may be infected with HCV were investigated, and another 28 HCV antibody positive IDUs were selected as controls （C1 to C28）. These controls came from the same methadone clinic or lived in the same town with P1. The RNAs were extracted from the plasma specimens and then reverse-transcribed into cDNA. After HCV subtyping, Hiseq sequencing was performed to detect and sequence the HCV quasispecies （263 bp） in the specimens with the same subtype as P1. The frequency of quasispecies was counted and ranked. Intrapersonal and interpersonal genetic distance and phylogenetic tree were calculated. Results The HCV subtype of specimen P1 was 3b. All the other specimens with the same subtype were P2, C7, C12, C14, C15, C16, C19, C20 and C28. Hiseq sequencing was successfully performed in 9 out of these 10 specimens, and 249 753 to 1 086 333 （average 869 608） cleaned sequences representing 3 to 172 （average 48） unique HCV quasispecies were obtained. The medians （P50） of intrapersonal genetic diversities from the 9 specimens were 0.4% to 12.3%. The P50 （P25, P75） of genetic diversities between P1 and the other 8 specimens were 19.0% （18.4%, 19.8%）, 10.4%（2.8%, 18.3%）, 19.6% （17.8%, 21.4%）,24.9% （23.8%, 26.1%）, 19.8% （18.7%, 20.7%）, 20.1% （18.9%, 21.2%）, 20.6% （20.0%, 21.1%）, 23.6% （22.4%, 24.8%）. There were no significant difference between the genetic diversities of P1 and P2 and those of P1 and other 7 specime

关 键 词：肝炎 丙型 分子流行病学 高通量测序技术 

分 类 号：R512.63[医药卫生—内科学]