A cost effective base-matching assay with low backgrounds.

Base-matching or so-called mini-sequencing is a powerful technique for genotyping and mutation identification. However, its application is often hampered by high background and high cost. We have decreased the background by approximately 5-fold by incorporating an end-blocking step and using only 1/10 of the usual nucleotide concentrations.

Identifying human immunodeficiency virus infection at birth: application of polymerase chain reaction to Guthrie cards.

Guthrie cards containing dried blood spots from 67 children now known to be infected with human immunodeficiency virus (HIV) and 63 children now classified as having had seroreversion were retrieved from the newborn infant archives from 1986 through 1991 to determine whether the polymerase chain reaction (PCR) could predict the infection at birth. The PCR assays operating at a sensitivity capable of detecting 2 to 10 copies of HIV proviral DNA per microgram were able to detect HIV proviral DNA in 52% (35/67) of the infected neonatal blood specimens. Longer storage times did not decrease PCR positivity rates, an advantage over assays for HIV antibody. Children whose clinical progression has been aggressive had high rates of PCR positivity in neonatal specimens, 50% (7/14) in those with low CD4 cell counts during the first year of life, 71% (10/14) in those with Pneumocystis pneumonia or disseminated cytomegalovirus infection by age 1 year, 62% (18/29) in those with onset of acquired immunodeficiency syndrome by 18 months, and 66% (14/21) in those who died of the disease by 36 months of age. No evidence of HIV proviral DNA was found in any of the 63 specimens from children with seroreversion. We conclude that PCR, using routinely available dried blood spots from neonates, has applications in early diagnosis and in epidemiologic projections going beyond current seroprevalence studies.