A simple and sensitive 'in-house' method for determining genotypic drug resistance in HIV-1. 2003

Anna Lindström, and Jan Albert
Division of Clinical Virology, Department of Immunology, Microbiology and Pathology (IMPI), Karolinska Institute, Huddinge University Hospital, S-141 86 Huddinge, Stockholm, Sweden.

Antiretroviral combination therapy is a major advance in the treatment of HIV infection, but development of antiretroviral resistance is still an important cause of treatment failure. Therefore, resistance testing was recommended recently for follow-up of HIV-1 infected individuals. The aim of this study was to develop a new genotypic resistance assay because simple and affordable assays with sufficient sensitivity for different genetic subtypes and low copy number samples are still lacking. Different methods and primers for RNA extraction from plasma, cDNA synthesis, nested PCR and sequencing on an ABI310 automated sequencer were evaluated and optimised. The PCR was designed to amplify a fragment covering the protease and the first half of the reverse transcriptase (RT), which harbour most known resistance mutations to licensed antiretroviral drugs. Resistance mutations were identified using resistance analysis tools available over the Internet. The optimised assay had a sensitivity of approximately 200 RNA copies per ml. The method was evaluated on plasma samples from treated patients infected with different subtypes of HIV-1 and appeared to have similar sensitivity for all subtypes. Samples that had failed previously in routine testing were analysed successfully with the new assay. The new assay is more sensitive and robust than the current routine method.

UI MeSH Term Description Entries
D008828 Microbiological Techniques Techniques used in microbiology. Microbiologic Technic,Microbiologic Technics,Microbiologic Technique,Microbiological Technics,Technic, Microbiologic,Technics, Microbiological,Technique, Microbiologic,Techniques, Microbiologic,Microbiologic Techniques,Microbiological Technic,Microbiological Technique,Technic, Microbiological,Technics, Microbiologic,Technique, Microbiological,Techniques, Microbiological
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D005838 Genotype The genetic constitution of the individual, comprising the ALLELES present at each GENETIC LOCUS. Genogroup,Genogroups,Genotypes
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000595 Amino Acid Sequence The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION. Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
D012367 RNA, Viral Ribonucleic acid that makes up the genetic material of viruses. Viral RNA
D012680 Sensitivity and Specificity Binary classification measures to assess test results. Sensitivity or recall rate is the proportion of true positives. Specificity is the probability of correctly determining the absence of a condition. (From Last, Dictionary of Epidemiology, 2d ed) Specificity,Sensitivity,Specificity and Sensitivity
D015497 HIV-1 The type species of LENTIVIRUS and the etiologic agent of AIDS. It is characterized by its cytopathic effect and affinity for the T4-lymphocyte. Human immunodeficiency virus 1,HIV-I,Human Immunodeficiency Virus Type 1,Immunodeficiency Virus Type 1, Human
D016133 Polymerase Chain Reaction In vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short oligonucleotide flanking sequences (primers). The essential steps include thermal denaturation of the double-stranded target molecules, annealing of the primers to their complementary sequences, and extension of the annealed primers by enzymatic synthesis with DNA polymerase. The reaction is efficient, specific, and extremely sensitive. Uses for the reaction include disease diagnosis, detection of difficult-to-isolate pathogens, mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships. Anchored PCR,Inverse PCR,Nested PCR,PCR,Anchored Polymerase Chain Reaction,Inverse Polymerase Chain Reaction,Nested Polymerase Chain Reaction,PCR, Anchored,PCR, Inverse,PCR, Nested,Polymerase Chain Reactions,Reaction, Polymerase Chain,Reactions, Polymerase Chain
D018076 DNA, Complementary Single-stranded complementary DNA synthesized from an RNA template by the action of RNA-dependent DNA polymerase. cDNA (i.e., complementary DNA, not circular DNA, not C-DNA) is used in a variety of molecular cloning experiments as well as serving as a specific hybridization probe. Complementary DNA,cDNA,cDNA Probes,Probes, cDNA

Related Publications

Anna Lindström, and Jan Albert
January 2003, Scandinavian journal of infectious diseases. Supplementum,
Anna Lindström, and Jan Albert
September 2007, Bioinformatics (Oxford, England),
Anna Lindström, and Jan Albert
January 2007, Anales de la Real Academia Nacional de Medicina,
Anna Lindström, and Jan Albert
September 2006, Antiviral research,
Anna Lindström, and Jan Albert
July 2010, Archives of virology,
Anna Lindström, and Jan Albert
March 2003, Annals of clinical microbiology and antimicrobials,
Copied contents to your clipboard!