| Summary: | <p>Abstract</p> <p>Human polyomavirus JC (JCV), the etiological agent of the disease progressive multifocal leukoencephalopathy (PML) affects immunocompromised patients particularly patients with AIDS. <it>In vitro </it>studies of JCV infection are hampered by the lack of sensitive JCV quantitation tests. Although the hemagglutination (HA) assay has been routinely employed for <it>in vitro </it>quantitation of JCV, its sensitivity is severely limited. We have employed a real-time PCR assay which compares favorably with the HA assay for the <it>in vitro </it>quantitation of JCV. JCV(Mad1), propagated in primary human fetal glial (PHFG) cells in two independent laboratories, was purified and quantitated by the HA assay. Both batches of purified JCV(Mad1) were then serially diluted in Dulbecco's Modified Eagle's Medium to obtain HA titers ranging from 64 to 0.001 HA units (HAU) per 100 μL of virus suspension. DNA was extracted from 100 μL of virus suspension and eluted in 50 μL of buffer, and DNA amplification and quantitation were performed in the Bio-Rad iCycler iQ Multicolor Real-Time PCR Detection System using T-antigen as the target gene. Real-time PCR for quantitation of JCV was sensitive and consistently detected 1.8 × 10<sup>1 </sup>copies of JCV DNA, and as low as 0.001 HAU equivalent of JCV. Moreover, there was a strong linear correlation between the HA assay and the DNA copy number of JCV(Mad1). The intra-run and inter-run coefficients of variation for the JCV standard curve were 0.06% to 4.8% and 2.6% to 5.2%, respectively. Based on these data, real-time PCR can replace the less-sensitive HA assay for the reliable detection, quantitation and monitoring of <it>in vitro </it>JCV replication.</p>
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