Multiplex real-time PCR assay for simultaneous quantification of BK polyomavirus, JC polyomavirus, and adenovirus DNA.
In recent years, virus-induced nephropathy caused mainly by BK polyomavirus (BKPyV), JC polyomavirus (JCPyV), and adenovirus has emerged as a problem in renal transplant patients. In the present study, we developed a multiplex real-time PCR assay to quantify the viral load of BKPyV, JCPyV, and adenovirus simultaneously. The dynamic range covered at least 6 orders of magnitude. This system was specific and reproducible, even in the presence of large amounts of DNA of other viruses. To validate this assay, urine samples from 124 renal transplant patients and serum samples from 18 hemorrhagic cystitis patients after hematopoietic stem cell transplantation were examined. In the urine samples from renal transplant patients, BKPyV was detected in 28 patients (22.6%), JCPyV was detected in 51 patients (41.1%), and adenovirus was detected in 2 patients (1.6%). The maximum amounts of each virus detected were 2.7 x 10(9), 8.7 x 10(8), and 1.2 x 10(2) copies/ml, respectively. Decoy cells were observed in 31 patients. The quantities of both BKPyV and JCPyV DNA were greater in samples with decoy cells. Two patients whose BKPyV viral loads exceeded 10(8) copies/ml had elevated serum creatinine levels and were diagnosed with BKPyV nephropathy based on graft biopsies. In serum samples from hemorrhagic cystitis patients, BKPyV, JCPyV, and adenovirus was detected in six, two, and three patients, respectively. Strong correlations were observed between the viral DNA copy numbers determined in the multiplex assays and those determined in single assays. Since this new assay is rapid, sensitive, and specific, it can be used for quantitative analyses of viruses in urine and serum samples after transplantation.
Funahashi
Yasuhito
Y
Department of Urology, Nagoya University Graduate School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya 466-8550, Japan.
Iwata
Seiko
S
Ito
Yoshinori
Y
Kojima
Seiji
S
Yoshikawa
Tetsushi
T
Hattori
Ryohei
R
Gotoh
Momokazu
M
Nishiyama
Yukihiro
Y
Kimura
Hiroshi
H
D000260:Adenoviruses, Human / Q000235:genetics / Q000302:isolation & purification*; D000293:Adolescent; D000328:Adult; D000368:Aged; D001739:BK Virus / Q000235:genetics / Q000302:isolation & purification*; D002648:Child; D002675:Child, Preschool; D019411:Clinical Laboratory Techniques / Q000379:methods*; D004279:DNA, Viral / Q000235:genetics; D006801:Humans; D007577:JC Virus / Q000235:genetics / Q000302:isolation & purification*; D016030:Kidney Transplantation; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014412:Tumor Virus Infections / Q000473:pathology / Q000821:virology*; D014556:Urine / Q000821:virology; D019562:Viral Load; D055815:Young Adult
D004279:DNA, Viral