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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.

SUBMITTER: Funahashi Y

PROVIDER: S-EPMC2832414 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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Multiplex real-time PCR assay for simultaneous quantification of BK polyomavirus, JC polyomavirus, and adenovirus DNA.

Funahashi Yasuhito Y Iwata Seiko S Ito Yoshinori Y Kojima Seiji S Yoshikawa Tetsushi T Hattori Ryohei R Gotoh Momokazu M Nishiyama Yukihiro Y Kimura Hiroshi H

Journal of clinical microbiology 20100106 3

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 th ...[more]

PMID: 20053854

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Project description:Classification of clinical symptoms and diagnostic microbiology are essential to effectively employ antimicrobial therapy for lower respiratory tract infections (LRTIs) in a timely manner. Empirical antibiotic treatment without microbial identification hinders the selective use of narrow-spectrum antibiotics and effective patient treatment. Thus, the development of rapid and accurate diagnostic procedures that can be readily adopted by the clinic is necessary to minimize non-essential or excessive use of antibiotics and accelerate patient recovery from LRTI-induced damage. We developed and validated a multiplex real-time polymerase chain reaction (mRT-PCR) assay with good analytical performance and high specificity to simultaneously detect four bacterial pathogens causing pneumonia: Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and Moraxella catarrhalis. The analytical performance of mRT-PCR against target pathogens was evaluated by the limit of detection (LOD), specificity, and repeatability. Two hundred and ten clinical specimens from pneumonia patients were processed using an automatic nucleic acid extraction system for the "respiratory bacteria four" (RB4) mRT-PCR assay, and the results were directly compared to references from bacterial culture and/or Sanger sequencing. The RB4 mRT-PCR assay detected all target pathogens from sputum specimens with a coefficient of variation ranging from 0.29 to 1.71 and conservative LOD of DNA corresponding to 5 × 102 copies/reaction. The concordance of the assay with reference-positive specimens was 100%, and additional bacterial infections were detected from reference-negative specimens. Overall, the RB4 mRT-PCR assay showed a more rapid turnaround time and higher performance that those of reference assays. The RB4 mRT-PCR assay is a high-throughput and reliable tool that assists decision-making assessment and outperforms other standard methods. This tool supports patient management by considerably reducing the inappropriate use of antibiotics.

Dataset Information

Multiplex real-time PCR assay for simultaneous quantification of BK polyomavirus, JC polyomavirus, and adenovirus DNA.

Publications

Multiplex real-time PCR assay for simultaneous quantification of BK polyomavirus, JC polyomavirus, and adenovirus DNA.

Similar Datasets

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