title: Allele-specific real-time PCR testing for minor HIV-1 drug resistance mutations: assay preparation and application to reveal dynamic of mutations in vivo.
abstract: BACKGROUND
  It is very important for the clinical management to test for minor HIV-1 resistance mutations accurately and sensitively. The conventional genotypic assays of HIV drug resistance detection based on sequencing can only discriminate the mutations which present in more than 20% - 30%. The aim of this study was to evaluate allele-specific real-time PCR (ASPCR) to detect the resistance-related mutations located at positions 103, 184 and 215.
  
  
  METHODS
  We developed the allele-specific PCR assay, using the most common drug resistance mutations in Chinese AIDS patients, K103N, M184V/I, T215F/Y as a model system. The standards were constructed by cloning the wild-type and mutant DNA fragments into the T-vector. We designed specific primers to discriminate mutant templates in the real-time PCR using SYBR green as a fluorescence reporter. And then we evaluated the ASPCR assay and tested 140 clinical samples using this method.
  
  
  RESULTS
  The sensitivities of ASPCR assay were 0.04% for K103N, 0.30% for M184I, 0.40% for M184V, 0.03% for T215F and 0.02% for T215Y. The intra-assay and inter-assay coefficients of variation were less than 0.42. One hundred and forty plasma samples were tested by ASPCR and dynamic resistance curves of ten patients were obtained.
  
  
  CONCLUSIONS
  Drug resistance emerged half a year after the start of antiretroviral therapy. The mutation of T215Y emerged 1 to 1.5 years after starting treatment and then increased rapidly. The ASPCR assay we developed was a sensitive, accurate and rapid method to detect the minor HIV-1 variants and it can provide earlier and more drug-resistance information for HIV research and AIDS antiretroviral therapy.
authors.affiliation: State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, China.
authors.forename: Dong-Xing
authors.initials: DX
authors.lastname: Guo
authors.forename: Han-Ping
authors.initials: HP
authors.lastname: Li
authors.forename: Lin
authors.initials: L
authors.lastname: Li
authors.forename: Dao-Min
authors.initials: DM
authors.lastname: Zhuang
authors.forename: Li-Yan
authors.initials: LY
authors.lastname: Jiao
authors.forename: Zheng
authors.initials: Z
authors.lastname: Wang
authors.forename: Zuo-Yi
authors.initials: ZY
authors.lastname: Bao
authors.forename: Si-Yang
authors.initials: SY
authors.lastname: Liu
authors.forename: Yong-Jian
authors.initials: YJ
authors.lastname: Liu
authors.forename: Jing-Yun
authors.initials: JY
authors.lastname: Li
mesh_terms: D000483:Alleles; D024882:Drug Resistance, Viral; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics*; D006801:Humans; D009154:Mutation; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity