Specific and quantitative detection of human polyomaviruses BKV and JCV by LightCycler real-time PCR.
BACKGROUND
BK virus (BKV) and JC virus (JCV) are the only two known human polyomavirus that typically establish subclinical persistent infections. In immunocompromised hosts reactivation of the JCV infection is the cause of the central nervous system disease progressive multifocal leucoencephalopathy (PML), while BKV may cause renal nephropathy and haemorrhagic cystitis.


OBJECTIVE
The goal of this study was to develop a specific quantification assay for each polyomavirus by LightCycler real-time polymerase chain reaction (PCR) based on SYBR Green I detection.


METHODS
DNA fragments of 138bp and 233bp from the "large T antigen" region of JCV and BKV, respectively, were amplified. The ability of the designed primer sets to separately quantify BKV DNA or JCV DNA and the PCR efficiency were assessed on reference DNA samples. Known amounts of cloned JCV DNA and BKV DNA from TEBU-BIO nv (Boechout, Belgium) were used to generate standard curves for the quantification assays. Species-specificity of the PCR was evaluated with cloned DNA and with DNA from patient samples.


RESULTS
The assay allowed a specific quantification over a 7log dynamic range. Seventeen copies each of the viral genes were reproducibly and accurately detected. The primer sets generated specific DNA fragments for each virus confirmed by agarose gel analysis and by cycle sequencing. The similarities of the amplified gene sequences by BLAST analysis were 99% and 100% for BKV and JCV, respectively. There was no cross-reactivity within the dynamic range of the standard dilutions.


CONCLUSIONS
We developed LightCycler real-time PCR assay based on SYBR Green I detection that provided rapid and specific quantification of polyomavirus load.
Sehbani
Laïla
L
Kabamba-Mukadi
Benoît
B
Vandenbroucke
Anne-Thérèse
AT
Bodéus
Monique
M
Goubau
Patrick
P
D001739:BK Virus / Q000235:genetics / Q000302:isolation & purification*; D052160:Benzothiazoles; D015169:Colony Count, Microbial / Q000379:methods*; D004279:DNA, Viral / Q000235:genetics; D003937:Diagnosis, Differential; D003959:Diamines; D006801:Humans; D007577:JC Virus / Q000235:genetics / Q000302:isolation & purification*; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods*; D027601:Polyomavirus Infections / Q000175:diagnosis / Q000821:virology*; D011804:Quinolines; D013045:Species Specificity; D014412:Tumor Virus Infections / Q000175:diagnosis / Q000821:virology*
D052160:Benzothiazoles; D004279:DNA, Viral; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I