Development and validation of multiplex real-time PCR assays for rapid detection of cytomegalovirus, Epstein-Barr virus, and polyomavirus BK in whole blood from transplant candidates

Kyung-Ah Hwang; Ji Hoon Ahn; Jae-Hwan Nam

doi:10.1007/s12275-018-8273-2

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Development and validation of multiplex real-time PCR assays for rapid detection of cytomegalovirus, Epstein-Barr virus, and polyomavirus BK in whole blood from transplant candidates: Kyung-Ah Hwang ^1,2, Ji Hoon Ahn ², Jae-Hwan Nam ¹; Journal of Microbiology 2018;56(8):593-599.
DOI: https://doi.org/10.1007/s12275-018-8273-2
Published online: July 25, 2018

¹Department of Biotechnology, The Catholic University of Korea, Bucheon 14662, Republic of Korea, ²Department of Research and Development, Genetree Research, Seoul 06745, Republic of Korea¹Department of Biotechnology, The Catholic University of Korea, Bucheon 14662, Republic of Korea, ²Department of Research and Development, Genetree Research, Seoul 06745, Republic of Korea

Corresponding author: Jae-Hwan Nam , Tel: +82-2-2164-4852,

Received: 18 May 2018 • Revised: 18 June 2018 • Accepted: 19 June 2018

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Abstract

Transplant recipients are more susceptible to bacterial and viral infections. Cytomegalovirus (CMV), Epstein-Barr virus (EBV), and polyomavirus BK (BK) are risk factors for graft dysfunction. All three of them are latent viruses that can cause serious disease in immunocompromised patients. Mainly qualitative PCR tests are required for diagnosis and quantitative monitoring, which are used to follow the response to transplantation. We developed a multiplex real-time PCR (qPCR)
method
to detect these viruses during blood screenings of transplant recipients. We also validated analytical and clinical performance tests using the developed multiplex qPCR. The limit of detection (LOD) was 100, 125, and 183 copies/ml for CMV, EBV, and BK, respectively. These results had high linearity (R2 = 0.997) and reproducibility (CV range, 0.95– 2.38%, 0.52–3.32%, and 0.31–2.45%, respectively). Among 183 samples, we detected 8 samples that were positive for CMV, while only 6 were positive for EBV, and 3 were positive for BK. Therefore, the viral infection prevalence in transplant candidates was 4.40% for CMV, 3.29% for EBV, and 1.64% for BK. This multiplex qPCR method should be used widely for diagnosing and monitoring latent viral infections in transplant recipients.

Keywords: cytomegalovirus;Epstein-Barr virus;polyomavirus BK;multiplex qPCR;transplant recipients

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Development and validation of multiplex real-time PCR assays for rapid detection of cytomegalovirus, Epstein-Barr virus, and polyomavirus BK in whole blood from transplant candidates

J. Microbiol. 2018;56(8):593-599. Published online July 25, 2018

DOI: https://doi.org/10.1007/s12275-018-8273-2

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