DETECTION OF HUMAN PARVOVIRUS B19 DNA BY NESTED POLYMERASE CHAIN-REACTION

We describe the development of a nested polymerase chain reaction (PCR ) technique used to detect human parvovirus B19 DNA. It was performed in a two-step reaction, first with a pair of outer primers, then with a pair of inner (ie, nested) primers. Primer sequences were selected i n the VP1 gene, corresponding to the capsid protein, of human parvovir us B19. To establish the nested PCR assay, the plasmid pGEM-1 contiain ing almost the entire coding sequence of a human parvovirus B19 isolat e was used. The nested PCR could detect up to 1.8x10(-3) ag of B19 DNA , equivalent to 10(-4) genomes, by electrophoresis when the reaction m ixture contained human placental DNA, cytomegalovirus DNA, and sterile distilled water as templates. We used this assay to evaluate four cas es of maternal B19 infection that were diagnosed by determination of t he presence of anti-B19 immunoglobin-M in maternal serum. The advantag es of our nested PCR for detecting B19 DNA are plating simplicity. Our results suggest that this method may have general applicability in th e evaluation of nonimmune hydrops fetalis and in the documentation of the natural history of fetal infection with B19 when applied to specim ens of amniotic fluid or fetal blood.