Comparison of real-time PCR and hemagglutination assay for quantitation of human polyomavirus JC

Abstract

Human polyomavirus JC (JCV), the etiological agent of the disease progressive multifocal leukoencephalopathy (PML) affects immunocompromised patients particularly patients with AIDS. In vitro studies of JCV infection are hampered by the lack of sensitive JCV quantitation tests. Although the hemagglutination (HA) assay has been routinely employed for in vitro 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 in vitro 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 microL of virus suspension. DNA was extracted from 100 microL of virus suspension and eluted in 50 microL 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 x 10(1) 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 in vitro JCV replication.

Figure 1

A – C: Analysis of HA and quantitative real-time PCR data employed for the determination of JC viral load. JCV (Mad1), propagated in Dr. Walker's laboratory (I) or propagated at the RRL (II) was serially diluted with DMEM to make 100 μl of viral suspension containing 64 HAU to 0.001 HAU of JCV. DNA was extracted using a QIAprep Miniprep Spin Kit and eluted in 50 μl of the elution buffer. Two μl of the 1:10 diluted template DNA was used for PCR in a final volume of 20 μl of PCR mixture. Copies of JCV(Mad1) T antigen gene were calculated from the standard curve and were expressed as copies of viral DNA per 100 μl of suspension. A) Amplification plots of relative fluorescence units (RFU) vs. cycle number of the JCV T antigen gene in known amounts of JCV plasmid DNA, ranging from 10 pg to 1 fg in decreasing 1:10 serial dilutions. B) Standard curve plot of the log of plasmid copy number against cycle threshold (Ct), where Ct is defined as the first cycle in which amplification signal is detected over mean baseline signal. The slope was -3.32 and r² = 1.00. C) The data representative of two independent experiments with samples tested in triplicate for each run of real-time PCR. Standard error bars are represented as standard deviation. The slopes were Y = 3 × 10⁶X - 350714 and Y = 4 × 10⁶X + 1 × 10⁷ and the r² were 1.0 and 0.95 for JCV(Mad1) virus stocks I and II, respectively.