title: Single genome amplification of proviral HIV-1 DNA from dried blood spot specimens collected during early infant screening programs in Lusaka, Zambia.
abstract: The ability to evaluate individual HIV-1 virions from the quasispecies of vertically infected infants was evaluated in a field setting at the Centre for Infectious Disease Research in Zambia. Infant heel-prick blood specimens were spotted onto dried blood spot (DBS) filter paper cards at government health clinics. Nucleic acid was extracted and used as a template for HIV-1 proviral DNA detection by a commercial Amplicor HIV-1 PCR test (Roche, version 1.5). On samples that tested positive by commercial diagnostic assay, amplification of DNA was performed using an in-house assay of the 5' and 3' region of the HIV-1 genome. Additionally, fragments covering 1200 nucleotides within pol (full length protease and partial reverse transcriptase) and 1400 nucleotides within env (variable 1-variable 5 region) were further analyzed by single genome amplification (SGA). In summary, we have demonstrated an in-house assay for amplifying the 5' and 3' proviral HIV-1 DNA as well as pol and env proviral DNA fragments from DBS cards collected and analyzed entirely in Zambia. In conclusion, this study shows the feasibility of utilizing DBS cards to amplify the whole proviral HIV-1 genome as well as perform SGA on key HIV-1 genes.
authors.affiliation: Centre for Infectious Disease Research Zambia, 5032 Great North Road, P.O. Box 34681, Lusaka, Zambia; University of Alabama at Birmingham, Division of Infectious Diseases, 1900 University Boulevard, THT 215E, Birmingham, AL 35294, United States. Electronic address: ls2026@uab.edu.
authors.forename: Lillian
authors.initials: L
authors.lastname: Seu
authors.affiliation: University of Alabama at Birmingham, Division of Infectious Diseases, 1900 University Boulevard, THT 215E, Birmingham, AL 35294, United States. Electronic address: Innocent.Mwape@cidrz.org.
authors.forename: Innocent
authors.initials: I
authors.lastname: Mwape
authors.affiliation: Centre for Infectious Disease Research Zambia, 5032 Great North Road, P.O. Box 34681, Lusaka, Zambia; University of Alabama at Birmingham, Division of Infectious Diseases, 1900 University Boulevard, THT 215E, Birmingham, AL 35294, United States. Electronic address: guffey@uab.edu.
authors.forename: M Bradford
authors.initials: MB
authors.lastname: Guffey
mesh_terms: D001769:Blood / Q000821:virology; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D003890:Desiccation; D005260:Female; D015684:Gene Products, pol / Q000235:genetics; D016679:Genome, Viral; D015658:HIV Infections / Q000175:diagnosis* / Q000635:transmission / Q000821:virology*; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D018445:Infectious Disease Transmission, Vertical; D008403:Mass Screening / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011247:Pregnancy; D011533:Proviruses / Q000235:genetics / Q000302:isolation & purification*; D013048:Specimen Handling / Q000379:methods; D015024:Zambia; D054299:env Gene Products, Human Immunodeficiency Virus / Q000235:genetics
chemical_list: D004279:DNA, Viral; D015684:Gene Products, pol; D054299:env Gene Products, Human Immunodeficiency Virus
keywords: Dried blood spots (DBS); HIV DNA PCR; Single genome amplification; Transmitted/founder viruses; Zambia