PDF(Pubmed)
Abstract：
HIV genotyping is used to assess HIV susceptibility to antiretroviral drugs. The Applied Biosystems HIV-1 Genotyping Kit with Integrase (AB kit, Thermo Fisher Scientific) detects resistance-associated mutations (RAMs) in HIV protease (PR), reverse transcriptase (RT), and integrase (IN). We compared results from the AB kit with results obtained previously with the ViroSeq HIV-1 Genotyping System. DNA amplicons from the AB kit were also analyzed using next-generation sequencing (NGS). HIV RNA was extracted using the MagNA Pure 24 instrument (Roche Diagnostics; 96 plasma samples, HIV subtype B, viral load range: 530-737,741 copies/mL). FASTA files were generated from AB kit data using Exatype (Hyrax Biosciences). DNA amplicons from the AB kit were also analyzed by NGS using the Nextera XT kit (Illumina). Drug resistance was predicted using the Stanford HIV Drug Resistance Database. The mean genetic distance for sequences from ViroSeq and the AB kit was 0.02% for PR/RT and 0.04% for IN; 103 major RAMs were detected by both methods. Four additional major RAMs were detected by the AB kit only. These four major RAMs were also detected by NGS (detected in 18.1%-38.2% of NGS reads). NGS detected 27 major RAMs that were not detected with either of the Sanger sequencing-based kits. All major RAMs detected with ViroSeq were detected with the AB kit; additional RAMs were detected with the AB kit only. DNA amplicons from the AB kit can be used for NGS for more sensitive detection of RAMs.
摘要：
HIV基因分型用于评估HIV对抗逆转录病毒药物的易感性。带整合酶的应用生物系统HIV-1基因分型试剂盒(AB试剂盒，ThermoFisherScientific）检测HIV蛋白酶（PR）中的抗性相关突变（RAM），逆转录酶（RT），和整合酶(IN)。我们将AB试剂盒的结果与先前使用ViroSeqHIV-1基因分型系统获得的结果进行了比较。还使用下一代测序(NGS)分析来自AB试剂盒的DNA扩增子。使用MagNAPure24仪器（RocheDiagnostics；96个血浆样本，HIV亚型B,病毒载量范围：530-737,741拷贝/mL）。使用Exatype(HyraxBiosciences)从AB试剂盒数据产生FASTA文件。还使用NexteraXT试剂盒(Illumina)通过NGS分析来自AB试剂盒的DNA扩增子。使用斯坦福HIV耐药性数据库预测耐药性。来自ViroSeq和AB试剂盒的序列的平均遗传距离对于PR/RT为0.02％，对于IN为0.04％；两种方法均检测到103个主要RAM。AB试剂盒仅检测到另外四个主要RAM。这四个主要RAM也被NGS检测到(在18.1％-38.2％的NGS读数中检测到)。NGS检测到27种主要RAM，这两种基于Sanger测序的试剂盒均未检测到。用ViroSeq检测的所有主要RAM均用AB试剂盒检测;另外的RAM仅用AB试剂盒检测。来自AB试剂盒的DNA扩增子可用于NGS以更灵敏地检测RAM。
