Comparison of real-time PCR and hemagglutination assay for quantitation of human polyomavirus JC.
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.
Chapagain
Moti L
ML
Department of Tropical Medicine, Medical Microbiology and Pharmacology, Asia-Pacific Institute of Tropical Medicine and Infectious Diseases, John A. Burns of School of Medicine, University of Hawaii, Honolulu, Hawaii 96813, USA. moti@hawaii.edu
Nguyen
Taylor
T
Bui
Thomas
T
Verma
Saguna
S
Nerurkar
Vivek R
VR
D004279:DNA, Viral / Q000032:analysis; D018628:Gene Dosage; D006386:Hemagglutination Tests / Q000379:methods*; D006801:Humans; D007577:JC Virus / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*
D004279:DNA, Viral