超深度焦磷酸测序技术用于HIV-1 RT基因区原发性耐药突变的研究
The use of ultra deep sequencing technique in the screening program on HIV-1 drug resistance mutation among ART-na?ve patients in Hunan province
目的:利用超深度焦磷酸测序技术(UDS)研究湖南省HIV感染者中原发性耐药流行趋势。方法90份未接受抗病毒治疗的HIV感染者同时采用UDS测序和Sanger测序进行HIV基因型耐药检测,测序结果采用斯坦福大学HIV耐药突变数据库进行分析比对,分析湖南省HIV-1反转录(RT)基因区原发性耐药突变情况。结果 UDS检测成功获取90份测序结果,84.4%为AE亚型(76/90),发现38例(42.2%,38/90)针对RT基因区的耐药突变位点,其中14例样本仅发生核苷类反转录酶(NRTI)耐药突变(15.6%),15例仅发生非核苷类反转录酶(NNRTI)耐药突变(16.7%),9例样本同时发生了NRTI和NNRTI类耐药突变。在16个位置上共发现54次耐药突变。17例(18.9%,17/90)样本有针对RT基因区的中度及以上的耐药突变。传统Sanger法仅检出7例;其中针对NRTI类药物耐药的6例;针对NNRTI类药物耐药的5例。结论 Sanger法测序能够鉴定出显著的耐药性突变(≥20%),但不能及时发现尚未达到显著突变量的突变位点或稀有型抗药型突变(<20%)。而UDS则能更灵敏检测到低频突变毒株,这些耐药突变同样可在药物选择压力下快速的增长引起病毒学治疗失败,导致临床治疗失败。因此选择更高灵敏度和精确度的技术检测低水平耐药突变位点,对于HIV抗病毒临床治疗有着十分重大的意义。
更多Objective To determine the prevalence rates of nucleotide reverse-transcriptase inhibitor (NRTI) and nonnucleoside reverse transcriptase inhibitor (NNRTI) TDRs among HIV-1 ART-na?ve patients in Hunan province using the ultra deep sequencing(UDS) technique. Methods ART-na?ve subjects diagnosed in Hunan between 2010 and 2011 were evaluated by both UDS technique and Sanger sequencing techniques,to the 1%variant level. Mutations were scored using the Stanford HIVdb algorithm to infer the status on drug resistance. Results UDS method was performed on 90 ART-na?ve subjects that seeking service of care,in Hunan. In total,42.2%(38/90)of the subjects showed major NRTI or nonnucleoside reverse transcriptase inhibitor NNRTI TDRs by UDS technique,at a HIV variant frequency level of≥1%,15.6%(14/90)showed NRTI TDR,16.7%(15/90) showed a major NNRTI TDR and 10%(9/90)were both resistant to NRTI and NNRTI when variants were analyzed by Stanford HIVdb. Conclusion ART-na?ve subjects from Hunan province, which had been predominately infected by subtype AE,would frequently possess HIV variants with NRTI/NNRTI TDRs that would affect the use of first line ART in the region,identified by the UDS technique. Further studies were needed to describe the prevalence of TDRs and to gather related information.
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