title: Multiplex real-time PCR assay for simultaneous quantification of BK polyomavirus, JC polyomavirus, and adenovirus DNA.
abstract: 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.
authors.affiliation: Department of Urology, Nagoya University Graduate School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya 466-8550, Japan.
authors.forename: Yasuhito
authors.initials: Y
authors.lastname: Funahashi
authors.forename: Seiko
authors.initials: S
authors.lastname: Iwata
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Ito
authors.forename: Seiji
authors.initials: S
authors.lastname: Kojima
authors.forename: Tetsushi
authors.initials: T
authors.lastname: Yoshikawa
authors.forename: Ryohei
authors.initials: R
authors.lastname: Hattori
authors.forename: Momokazu
authors.initials: M
authors.lastname: Gotoh
authors.forename: Yukihiro
authors.initials: Y
authors.lastname: Nishiyama
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kimura
mesh_terms: 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
chemical_list: D004279:DNA, Viral