A novel polymerase chain reaction method for detection of human immunodeficiency virus in dried blood spots on filter paper.
A method for detection of proviral human immunodeficiency virus DNA in dried blood spots on filter paper by direct polymerase chain reaction (PCR) has been developed. To develop the method, a standard system was used which was prepared from cells each containing a single integrated provirus and titrated with normal donor blood. This rapid procedure provides virtually quantitative yields of nuclear DNA and exploits most of the standard methodology described for blood specimens. A nested PCR using SK38-SK39 gag as the internal primer pair was also designed; this PCR detected a single copy of provirus per filter at near theoretical frequency with SK19 probe. The utility of the procedure was demonstrated with clinical specimens. Blood spot filters from human immunodeficiency virus-infected and uninfected individuals were readily and unequivocally discriminated. The method is designed for ultimate use with large (1.5-ml) sample preparation tubes that are compatible as PCR tubes with thermal cyclers. This will permit convenient, direct single-tube PCR of dried blood specimens on filters. It should be adaptable to analysis of dried blood spots for a variety of infectious or genetic diseases.
Yourno
J
J
New York State Department of Health, Wadsworth Center for Laboratory and Research, Empire State Plaza, Albany, New York 12201-0509.
Conroy
J
J
D001483:Base Sequence; D001800:Blood Specimen Collection; D002648:Child; D002675:Child, Preschool; D004279:DNA, Viral / Q000097:blood / Q000235:genetics; D005069:Evaluation Studies as Topic; D015750:Genes, gag; D006678:HIV / Q000235:genetics* / Q000302:isolation & purification*; D015658:HIV Infections / Q000097:blood / Q000175:diagnosis* / Q000382:microbiology; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008969:Molecular Sequence Data; D010209:Paper; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012015:Reference Standards
D004279:DNA, Viral