摘要
目的通过对不同病毒载量的同一研究对象同时进行血浆HIV-1 RNA与干血斑HIV-1 DNA的基因型耐药检测并对结果进行比较,探讨HIV-1 DNA用于耐药检测的可行性和用途。方法2021年12月采集云南省、广西壮族自治区及新疆维吾尔自治区接受ART后1年以上的HIV/AIDS患者静脉血5 mL,EDTA抗凝,分离血浆和制备成干血斑样本,分别提取病毒DNA和RNA进行pol区扩增,比较两种方法的扩增效率;并对同时扩增成功的序列利用MEGA7构建系统进化树并分析序列一致性。利用斯坦福大学HIV耐药数据库进行耐药位点分析,比较耐药性结果。结果209例样本中,来自云南82份(39.2%),来自广西壮族自治区69份(33.0%),来自新疆维吾尔自治区58份(27.8%)。105例VL<20拷贝/mL,25例20拷贝/mL≤VL<200拷贝/mL,42例200拷贝/mL≤VL<1000拷贝/mL,37例VL≥1000拷贝/mL。不同病毒载量的样本分类中,使用血浆中HIV-RNA pol区扩增成功率分别为12.4%、28.0%、69.0%、89.2%;使用干血斑中HIV-DNA pol区扩增成功率分别为39.0%、52.0%、59.5%、73.0%;血浆结合干血斑各组的扩增成功率为41.0%、56.0%、76.2%、89.2%。两种耐药检测方法同时扩增成功66例,耐药结果完全一致为98.5%,序列一致性99.7%。其中有58例(58/66,87.9%)配对样本耐药位点完全一致,8例配对样本耐药位点不完全一致。耐药位点不完全一致的配对样本中仅1例导致耐药结果不同,其他主要由混合碱基导致但未对耐药结果产生影响。结论应用干血斑HIV-1 DNA进行基因型耐药检测可以弥补目前血浆HIV-1 RNA耐药检测不足,特别是对病毒载量<1000拷贝/mL的样本,能够提高耐药检测效率。同时二者检测结果基因序列与耐药位点的一致性很好。结合干血斑样本容易制备、保存与运输的优点,提高了在偏远欠发达地区进行耐药监测的可及性。
Objective To explore the feasibility and use of HIV-1 DNA in drug resistance detection,the genotypes of HIV-1 RNA in plasma and HIV-1 DNA in dried blood spots of the same subjects with different viral loads were detected simultaneously and the results were compared.Methods In December 2021,5 mL of venous blood of HIV/AIDS patients who received ART for more than one year in Yunnan Province,Guangxi Zhuang Autonomous Region and Xinjiang Uygur Autonomous Region was collected,EDTA anticoagulation,plasma isolation and dry blood spot samples were prepared.RNA and DNA were extracted for pol amplification,respectively.The amplification efficiency of the two methods was compared.MEGA7 was used to construct a phylogenetic tree and analyze sequence consistency.The Stanford HIV drug resistance database was used to analyze drug resistance sites and compare drug resistance results.Results Among the 209 samples,82(39.2%)were from Yunnan,69(33.0%)were from Guangxi Zhuang Autonomous Region and 58(27.8%)were from Xinjiang Uygur Autonomous Region.105 patients had VL<20 copies/mL,25 patients had 20 copies/mL≤VL<200 copies/mL,42 patients had 200 copies/mL≤VL<1000 copies/mL and 37 patients had VL≥1000 copies/mL.The success rates of drug resistance amplification of HIV-1 RNA extracted from plasma were 12.4%,28.0%,69.01%and 89.2%,respectively.The drug resistance success rates of HIV-1 DNA extracted from dried blood spots were 39.0%,52.0%,59.5%,and 73.0%,respectively.The amplification efficiency of plasma combined with dried blood spots was 41.0%,56.0%,76.2%,and 89.2%.The two drug resistance detection methods successfully amplified the 66 cases,with the resistance results as 98.5%consistent and the sequence consistency as 99.7%.Among them,58(58/66,87.9%)matched samples had entirely consistent drug resistance sites and 8 matched samples had incomplete drug resistance sites.Only 1 matched sample with inconsistent drug resistance sites resulted in different drug resistance results,while the others were mainly caused by mixed bases but did not affect the drug resistance results.Conclusions The application of HIV-1 DNA extracted from dry blood spots for genotypic resistance detection can make up for the insufficient current resistance of plasma HIV-1 RNA,especially for samples with viral load<1000 copies/mL,which can improve the efficiency of drug resistance detection.At the same time,the gene sequences and drug resistance sites of the two detection results were in good agreement.Combined with the advantages of easy preparation,preservation,and transportation of dried blood spots,the accessibility of drug resistance testing in remote and underdeveloped areas is improved.
作者
梅朋飞
朱禹静
周德
吕毅
金聪
米云婷
邓煜川
姚均
MEI Pengfei;ZHU Yujing;ZHOU De;LYU Yi;JIN Cong;MI Yunting;DENG Yuchuan;YAO Jun(National Center for AIDS/STD Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China;Suqian First People's Hospital,Suqian 223800,Jiangsu;Yunnan Maternal and Child Health Care Hospital,Kunming 650000,Yunnan;Beijing Center for Disease Prevention and Control,Beijing 100013)
出处
《中国艾滋病性病》
CAS
CSCD
北大核心
2023年第1期9-13,共5页
Chinese Journal of Aids & STD
基金
国家科技重大专项(2015ZX10001001002)。
