High-sensitivity PCR detection of parvovirus B19 in plasma.
Parvovirus B19 (B19) is a human pathogen transmitted to susceptible individuals via respiratory secretions and contaminated blood or blood products. B19 levels in pooled plasma of less than 10(4) genome equivalents/ml may not be infectious, while those greater than 10(7)/ml are capable of transmitting infection. A World Health Organization (WHO) B19 DNA international standard has been recently introduced. The purpose of the present work was to develop a PCR-enzyme-linked immunosorbent assay (PCR-ELISA) calibrated against the WHO B19 DNA international standard which could easily and reliably detect B19 DNA levels in plasma above 10(4) IU/ml (6.5 x 10(3) genome equivalents/ml). A B19 PCR-ELISA system was developed which uses a dinitrophenylated oligonucleotide probe to detect immobilized biotinylated amplicons following single-round PCR amplification. The level of B19 DNA (in international units per milliliter) in individual and pooled plasma specimens was evaluated. Proteinase K treatment of plasma was found to be sufficient to quantitatively release B19 DNA. The B19 PCR-ELISA had a sensitivity of detection of 1.6 x 10(3) IU/ml B19 DNA and a dynamic range extending from 8 to 1,000 IU of B19 DNA (equivalent to 1.6 x 10(3) to 2 x 10(5) IU of B19 DNA/ml). Furthermore, the antibody profile of pooled plasma products was determined in terms of B19 immunoglobulin G (IgG) (in international units per milliliter). The B19 IgG level was found to be 64.7 +/- 17.5 IU/ml (mean +/- standard deviation). The B19 PCR-ELISA, which is calibrated against the B19 DNA international standard, may have an application for the rapid screening of plasma minipools for B19 DNA, thereby leading to an improvement in blood product safety.
Daly
P
P
Department of Biology, National University of Ireland, Maynooth, County Kildare, Ireland.
Corcoran
A
A
Mahon
B P
BP
Doyle
S
S
D019921:Biotinylation; D015342:DNA Probes; D004279:DNA, Viral / Q000097:blood*; D004140:Dinitrophenols; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000592:standards; D006801:Humans; D016732:Parvovirus B19, Human / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity; D014944:World Health Organization
D015342:DNA Probes; D004279:DNA, Viral; D004140:Dinitrophenols