Specific and quantitative detection of human polyomaviruses BKV, JCV, and SV40 by real time PCR.
BACKGROUND
The polyomaviruses that infect humans, BK virus (BKV), JC virus (JCV), and simian virus 40 (SV40), typically establish subclinical persistent infections. However, reactivation of these viruses in immunocompromised hosts is associated with renal nephropathy and hemorrhagic cystitis (HC) caused by BKV and with progressive multifocal leukoencephalopathy (PML) caused by JCV. Additionally, SV40 is associated with several types of human cancers including primary brain and bone cancers, mesotheliomas, and non-Hodgkin's lymphoma. Advancements in detection of these viruses may contribute to improved diagnosis and treatment of affected patients.


OBJECTIVE
To develop sensitive and specific real time quantitative polymerase chain reaction (RQ-PCR) assays for the detection of T-antigen DNA sequences of the human polyomaviruses BKV, JCV, and SV40 using the ABI Prism 7000 Sequence Detection System.


METHODS
Assays for absolute quantification of the viral T-ag sequences were designed and the sensitivity and specificity were evaluated. A quantitative assay to measure the single copy human RNAse P gene was also developed and evaluated in order to normalize viral gene copy numbers to cell numbers.


RESULTS
Quantification of the target genes is sensitive and specific over a 7 log dynamic range. Ten copies each of the viral and cellular genes are reproducibly and accurately detected. The sensitivity of detection of the RQ-PCR assays is increased 10- to 100-fold compared to conventional PCR and agarose gel protocols. The primers and probes used to detect the viral genes are specific for each virus and there is no cross reactivity within the dynamic range of the standard dilutions. The sensitivity of detection for these assays is not reduced in human cellular extracts; however, different DNA extraction protocols may affect quantification.


CONCLUSIONS
These assays provide a technique for rapid and specific quantification of polyomavirus genomes per cell in human samples.
McNees
Adrienne L
AL
Department of Molecular Virology and Microbiology, Baylor College of Medicine, One Baylor Plaza, MS BCM-385, Houston, TX 77030, USA.
White
Zoe S
ZS
Zanwar
Preeti
P
Vilchez
Regis A
RA
Butel
Janet S
JS
D001739:BK Virus / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D017124:Conserved Sequence; D017931:DNA Primers; D005784:Gene Amplification; D016679:Genome, Viral; D006801:Humans; D007577:JC Virus / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010957:Plasmids; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D013539:Simian virus 40 / Q000235:genetics / Q000302:isolation & purification*
D017931:DNA Primers; D015345:Oligonucleotide Probes