Detection of parvovirus B19 by dot-blot and polymerase chain reaction.
Our studies compare detection of parvovirus B19 DNA by dot-blot hybridization and by the polymerase chain reaction (PCR). Compared to detection by dot-blot hybridization with a 32P-oligonucleotide probe, the sensitivity of PCR product detection by ethidium bromide fluorescence is not compromised when the size of the amplification target and number of cycles of amplification are properly selected. Our PCR assay simultaneously amplifies two targets in the B19 genome and yields a sensitivity 10 times greater than 32P-labelled oligonucleotide--dot-blot hybridization (dot-blot). In 126 serum samples from cases of suspected parvovirus B19 infection, viral DNA was detected by PCR in 17% (21/126), whereas dot-blot detected B19 DNA in 0.8% (1/126). Parvovirus B19-specific antibodies were detected in 69% (87/126) of the suspected clinical cases by immunoblot. Nineteen of the antibody-positive specimens were DNA positive of which three were IgM-positive/IgG-negative, 11 IgM-positive/IgG-positive and five IgM-negative/IgG-positive. The only dot-blot DNA positive sample was also PCR-DNA positive but was B19 IgM-negative/IgG-negative. Eighty-six B19 antibody-negative, clinically uninfected controls were also negative for B19 DNA by PCR and by dot-blot. These studies confirm the increased sensitivity of gene amplification by PCR for detection of B19 DNA and demonstrate that two targets in B19 DNA can be amplified simultaneously with resultant increased ease of interpretation. Finally, detection of the two B19-specific products by ethidium bromide fluorescence is documented.
Sevall
J S
JS
Specialty Laboratories, Inc., Santa Monica, California 90404.
D000914:Antibodies, Viral / Q000032:analysis; D001483:Base Sequence; D015153:Blotting, Western; D015342:DNA Probes; D004279:DNA, Viral / Q000302:isolation & purification*; D005784:Gene Amplification; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010321:Parvoviridae / Q000276:immunology / Q000302:isolation & purification*; D010322:Parvoviridae Infections / Q000175:diagnosis* / Q000382:microbiology; D016133:Polymerase Chain Reaction; D011237:Predictive Value of Tests
D000914:Antibodies, Viral; D015342:DNA Probes; D004279:DNA, Viral