title: Improvement in allele-specific PCR assay with the use of polymorphism-specific primers for the analysis of minor variant drug resistance in HIV-1 subtype C.
abstract: In recent years, highly sensitive assays have been developed that detect HIV-1 drug resistance mutations when present at less than 1% of the viral population. These assays are powerful tools when attempting to determine the clinical implications of these low level resistant virions after the administration of single-dose nevirapine. This report demonstrates that non-drug resistant polymorphisms in the primer-binding site for the allele-specific PCR (ASPCR) assay impact primer binding resulting in significant discrepancies in the assay's performance. Specifically, the use of a "universal" set of ASPCR primers caused an overestimation of the K103N (ntAAC) mutation at position 103 of reverse transcriptase when primer binding site polymorphisms resided close to the 3' end of the allele-specific primer. Drug resistance was predicted at values ranging from 0.69% to 7.69% for a sample containing only 1% resistance mutations and 3.35-31.84% for a sample containing 5% mutations. Conversely, the use of polymorphism-specific primers detected 1.15-1.36% and 5.20-5.71% resistance for the same 1% and 5% samples. The results demonstrate the need to account for sequence polymorphisms when designing and implementing this highly specific assay.
authors.affiliation: Division of Infectious Diseases, Beth Israel Deaconess Medical Center, Lowry Building, Suite GB, 110 Francis Street, Boston, MA 02215, USA. crowley1@bidmc.harvard.edu <crowley1@bidmc.harvard.edu>
authors.forename: Christopher F
authors.initials: CF
authors.lastname: Rowley
authors.forename: Christian L
authors.initials: CL
authors.lastname: Boutwell
authors.forename: Shahin
authors.initials: S
authors.lastname: Lockman
authors.forename: M
authors.initials: M
authors.lastname: Essex
mesh_terms: D000483:Alleles; D019380:Anti-HIV Agents / Q000494:pharmacology* / Q000627:therapeutic use; D001665:Binding Sites; D001902:Botswana; D017931:DNA Primers; D024882:Drug Resistance, Viral / Q000235:genetics*; D005260:Female; D015658:HIV Infections / Q000188:drug therapy / Q000821:virology*; D054303:HIV Reverse Transcriptase / Q000235:genetics; D015497:HIV-1 / Q000187:drug effects / Q000235:genetics*; D006801:Humans; D019829:Nevirapine / Q000494:pharmacology / Q000627:therapeutic use; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology / Q000627:therapeutic use
chemical_list: D019380:Anti-HIV Agents; D017931:DNA Primers; D018894:Reverse Transcriptase Inhibitors; D019829:Nevirapine; D054303:HIV Reverse Transcriptase