title: Development and customization of a color-coded microbeads-based assay for drug resistance in HIV-1 reverse transcriptase.
abstract: BACKGROUND
  Drug resistance (DR) of HIV-1 can be examined genotypically or phenotypically. Although sequencing is the gold standard of the genotypic resistance testing (GRT), high-throughput GRT targeted to the codons responsible for DR may be more appropriate for epidemiological studies and public health research.
  
  
  METHODS
  We used a Japanese database to design and synthesize sequence-specific oligonucleotide probes (SSOP) for the detection of wild-type sequences and 6 DR mutations in the clade B HIV-1 reverse transcriptase region. We coupled SSOP to microbeads of the Luminex 100 xMAP system and developed a GRT based on the polymerase chain reaction (PCR)-SSOP-Luminex method.
  
  
  RESULTS
  Sixteen oligoprobes for discriminating DR mutations from wild-type sequences at 6 loci were designed and synthesized, and their sensitivity and specificity were confirmed using isogenic plasmids. The PCR-SSOP-Luminex DR assay was then compared to direct sequencing using 74 plasma specimens from treatment-naïve patients or those on failing treatment. In the majority of specimens, the results of the PCR-SSOP-Luminex DR assay were concordant with sequencing results: 62/74 (83.8%) for M41, 43/74 (58.1%) for K65, 70/74 (94.6%) for K70, 55/73 (75.3%) for K103, 63/73 (86.3%) for M184 and 68/73 (93.2%) for T215. There were a number of specimens without any positive signals, especially for K65. The nucleotide position of A2723G, A2747G and C2750T were frequent polymorphisms for the wild-type amino acids K65, K66 and D67, respectively, and 14 specimens had the D67N mutation encoded by G2748A. We synthesized 14 additional oligoprobes for K65, and the sensitivity for K65 loci improved from 43/74 (58.1%) to 68/74 (91.9%).
  
  
  CONCLUSIONS
  We developed a rapid high-throughput assay for clade B HIV-1 DR mutations, which could be customized by synthesizing oligoprobes suitable for the circulating viruses. The assay could be a useful tool especially for public health research in both resource-rich and resource-limited settings.
authors.affiliation: Research Center for Asian Infectious Diseases, the Institute of Medical Science, the University of Tokyo, Tokyo, Japan; Japan-China Joint Laboratory of Molecular Immunology and Molecular Microbiology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, P. R. China.
authors.forename: Lijun
authors.initials: L
authors.lastname: Gu
authors.affiliation: Division of Infectious Diseases, Advanced Clinical Research Center, the Institute of Medical Science, the University of Tokyo, Tokyo, Japan.
authors.forename: Ai
authors.initials: A
authors.lastname: Kawana-Tachikawa
authors.affiliation: AIDS Research Center, National Institute of Infectious Diseases, Tokyo, Japan.
authors.forename: Teiichiro
authors.initials: T
authors.lastname: Shiino
authors.affiliation: Department of Infectious Disease Control, International Research Center for Infectious Diseases, the Institute of Medical Science, the University of Tokyo, Tokyo, Japan; Division of Infectious Diseases and Applied Immunology, Research Hospital, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
authors.forename: Hitomi
authors.initials: H
authors.lastname: Nakamura
authors.affiliation: Division of Infectious Diseases, Advanced Clinical Research Center, the Institute of Medical Science, the University of Tokyo, Tokyo, Japan; Division of Infectious Diseases and Applied Immunology, Research Hospital, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
authors.forename: Michiko
authors.initials: M
authors.lastname: Koga
authors.affiliation: Division of Infectious Diseases, Advanced Clinical Research Center, the Institute of Medical Science, the University of Tokyo, Tokyo, Japan; Division of Infectious Diseases and Applied Immunology, Research Hospital, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
authors.forename: Tadashi
authors.initials: T
authors.lastname: Kikuchi
authors.affiliation: Division of Infectious Diseases and Applied Immunology, Research Hospital, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
authors.forename: Eisuke
authors.initials: E
authors.lastname: Adachi
authors.affiliation: Division of Infectious Diseases and Applied Immunology, Research Hospital, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
authors.forename: Tomohiko
authors.initials: T
authors.lastname: Koibuchi
authors.affiliation: Research Center for Asian Infectious Diseases, the Institute of Medical Science, the University of Tokyo, Tokyo, Japan; Japan-China Joint Laboratory of Molecular Immunology and Molecular Microbiology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, P. R. China.
authors.forename: Takaomi
authors.initials: T
authors.lastname: Ishida
authors.affiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, P. R. China.
authors.forename: George F
authors.initials: GF
authors.lastname: Gao
authors.affiliation: Biotech Research and Development, Wakunaga Pharmaceutical Corporation, Hiroshima, Japan.
authors.forename: Masaki
authors.initials: M
authors.lastname: Matsushita
authors.affiliation: AIDS Research Center, National Institute of Infectious Diseases, Tokyo, Japan.
authors.forename: Wataru
authors.initials: W
authors.lastname: Sugiura
authors.affiliation: Research Center for Asian Infectious Diseases, the Institute of Medical Science, the University of Tokyo, Tokyo, Japan; Division of Infectious Diseases, Advanced Clinical Research Center, the Institute of Medical Science, the University of Tokyo, Tokyo, Japan; Department of Infectious Disease Control, International Research Center for Infectious Diseases, the Institute of Medical Science, the University of Tokyo, Tokyo, Japan; Division of Infectious Diseases and Applied Immunology, Research Hospital, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
authors.forename: Aikichi
authors.initials: A
authors.lastname: Iwamoto
authors.affiliation: Division of Infectious Diseases, Advanced Clinical Research Center, the Institute of Medical Science, the University of Tokyo, Tokyo, Japan; Department of Infectious Disease Control, International Research Center for Infectious Diseases, the Institute of Medical Science, the University of Tokyo, Tokyo, Japan.
authors.forename: Noriaki
authors.initials: N
authors.lastname: Hosoya
mesh_terms: D001483:Base Sequence; D004252:DNA Mutational Analysis; D024882:Drug Resistance, Viral / Q000235:genetics*; D015658:HIV Infections / Q000188:drug therapy / Q000821:virology*; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors / Q000235:genetics*; D015497:HIV-1 / Q000201:enzymology / Q000235:genetics*; D006801:Humans; D008863:Microspheres; D058889:Molecular Typing; D016133:Polymerase Chain Reaction; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology* / Q000627:therapeutic use; D012680:Sensitivity and Specificity
chemical_list: D018894:Reverse Transcriptase Inhibitors; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase