【摘要】 目的比较治疗前静脉血浆和全血干血斑(DBS)人类免疫缺陷病毒1型(HIV-1)Pol区基因型耐药检测结果的差异,并分析DBS样品用于HIV-1基因型耐药检测的可行性。方法采集44例HIV-1感染者治疗前静脉全血,分别制备全血DBS样品和血浆样品,提取RNA,检测病毒载量。采用逆转录PCR(RT-PCR)扩增Pol区基因序列,对PCR产物进行Sanger测序,采用Sequencher 5.4.5软件对序列进行清理和拼接,采用BioEdit 7.2软件对参考株序列和样品序列进行比对、系统进化树分析及基因型耐药分析。结果治疗前44例血浆样品检出率100.0%,DBS样品检出30例,检出率为68.2%;24例血浆样品平均病毒载量达到5.02×105IU/mL,中位数为1.76×105 IU/mL,DBS样品平均病毒载量为1.46×103 IU/mL,中位数为7.95×102IU/mL;44例患者HIV-1亚型主要为流行重组株(CRF)07-BC(24/44)、CRF01-AE(16/44);血浆和DBS样品序列相似值100%26例(26/30),相似值99%3例(3/30),相似值97%1例(1/30),其中有6例发生不同程度的耐药突变,DBS样品耐药结果与血浆符合率100%,但在其他位点的突变存在差异。结论治疗前全血DBS样品耐药结果及序列与血浆样品具有高度的一致性,全血DBS样品可用于耐药性检测、HIV-1亚型分析,但检出率比血浆低。更多还原

【Abstract】 Objective To compare the differences of genotypic resistance in human immunodeficiency virus type 1(HIV-1)Pol regions between plasma and whole blood dried blood spot(DBS),and to analyze the feasibility of using DBS for HIV-1 genotype resistance detection.Methods The whole venous blood of 44 HIV-1 infected patients from the hospital were collected,and whole blood DBS samples and plasma samples were prepared for RNA extraction,load detection.The Pol region gene sequences were amplified by reverse transcription PCR(RT-PCR),and the PCR products were sequenced by Sanger sequencing.Sequencher 5.4.5 software was for sequence analysis,and BioEdit 7.2 software was used to conduct comparison between the reference strain sequence and sample sequence,phylogenetic tree analysis and genotype resistance analysis.Results The detection rate of 44 plasma samples was 100.0%,and 30 DBS samples were successfully detected,the detection rate was 68.2%.The average viral load of the 24 plasma samples detected by the load test reached 5.02×105 IU/mL,median 1.76×105 IU/mL,and the average load of DBS samples was 1.46×103 IU/mL,median7.95×102 IU/mL,which is relatively low.The 44 patients with HIV-1 virus subtypes were mainly epidemic recombinant strains(CRF)07-BC(24/44)and CRF01-AE(16/44).Sequence similarity between plasma and DBS samples was 100%in 26 cases(26/30),99%in 3 cases(3/30),and 97%in 1 case(1/30),among which6 cases had drug-resistant mutations of different degrees,the coincidence rate of DBS resistance results and plasma was 100%,but there were differences in other mutation sites.Conclusion The drug resistance results of DBS samples are consistent with plasma samples,and the sequences are also highly consistent.DBS samples could be used for drug resistance test and HIV-1 subtype analysis,but the detection rate is lower than plasma.更多还原