Zastosowanie metody real-time PCR do wykrywania DNA ludzkiego poliomawirusa BK
[Real-time PCR test for detection and quantification of human polyomavirus BK (BKV) DNA]

Streszczenie

Celem pracy było adaptowanie i zbadanie użyteczności metody real-time PCR do diagnostyki zakażeń ludzkim poliomawirusem BK, zwłaszcza u pacjentów poddanych immunosupresji po przeszczepieniu komórek krwiotwórczych oraz przeszczepach nerek.

Abstract

The human polyomavirus BK (BKV) is wide-spread pathogen, associated with urogenital tract disorders or even nephropathy in immunosuppressed patients. Nowadays molecular detection by real-time PCR (qPCR) is recognized as a method-of-choice for detecting human polyomaviruses in clinical samples. The aim of the study was development of real-time PCR assay for detection and quantification of polyomavirus BK DNA in clinical samples, using specific primers targeting a viral
DNA VP3 gene and a TaqMan hydrolyzing probe. The analytical sensitivity of assay was tested using
serial dilutions of BKV DNA in range between 13500 and 15 copies/ml. 27 urine samples and 23 plasma samples taken from a group of 22 adult recipients of allogeneic HSCT were tested for the presence of polyomavirus BK in the LightCycler® system. Described in-house real-time PCR assay detected BKV DNA in 8 specimens (6 urine and 2 plasma). Detected average viral load was 170 copies/ml for plasma and 1250 copies/ml for urine samples, respectively. The results of this study show that developed TaqMan-based probe qPCR assay is very reliable and valuable for detection and quantification of BKV DNA, both in urine and plasma samples. These data, combined with its rapid turnaround time for results and decreased hands-on time, make the LightCycler PCR assay highly suitable for the rapid diagnostics of polyomavirus BK infections in the clinical laboratory.