High-sensitivity PCR detection of parvovirus B19 in plasma

Abstract

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.

FIG. 1.

PCR-ELISA quantitation using the WHO B19 DNA international standard. Half-log dilutions were extracted by proteinase K (solid line) or spin column (dashed line) methodologies, subjected to PCR using primers F1 and R1, and analyzed by ELISA. Absorbance at 450 and 630 nm is plotted versus the log amount of B19 DNA. The PCR-ELISA cutoff (0.159) is indicated by the horizontal black line and was calculated as the mean plus 2 standard deviations [0.107 + 2(0.026)] following replicate analysis of 20 B19-DNA-negative specimens. A level of 8 IU of B19 DNA was reliably detectable following proteinase K extraction. Amplicon detection by agarose gel electrophoresis detection is also shown (inset). It can be seen that a minimum of 200 IU of B19 DNA only can be detected by this approach. PD, primer dimer.

FIG. 2.

Qualitative detection of B19 DNA in 30 pooled plasma specimens by PCR-ELISA. Five pools (PS1 to PS5) were found to have high levels of B19 DNA, the levels of which were subsequently quantified in terms of B19 DNA (in international units per milliliter) (see text). Eleven pools (PS6 to PS15) appeared to contain low levels of B19 DNA. In 14 pools (PS17 to PS30), any B19 DNA present was below the PCR-ELISA cutoff of 1.6 × 10³ IU/ml.