Simultaneous quantification of Epstein-Barr virus, cytomegalovirus, and human herpesvirus 6 DNA in samples from transplant recipients by multiplex real-time PCR assay

Kaoru Wada, Naomi Kubota, Yoshinori Ito, Hiroshi Yagasaki, Koji Kato, Tetsushi Yoshikawa, Yasuyuki Ono, Hisami Ando, Yasuhiro Fujimoto, Tetsuya Kiuchi, Seiji Kojima, Yukihiro Nishiyama, Hiroshi Kimura

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Abstract

We developed a multiplex real-time PCR assay using 6-carboxyfluorescein, 6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein, and carbecyanine 5-labeled probes to simultaneously quantify Epstein-Barr virus (EBV), cytomegalovirus (CMV), and human herpesvirus 6 (HHV-6) DNA. When previously tested and stored DNA samples were examined, results of the multiplex real-time PCR assay were as sensitive and specific as those of a single real-time PCR assay. The multiplex assay was used to quantify the EBV, CMV, and HHV-6 DNA in 46 transplant recipients. A total of 303 whole-blood and plasma specimens were collected and analyzed. According to the results of the multiplex assay, the detection rates for viral DNA in whole blood and plasma were 23.8% and 5.9% for EBV, 11.2% and 5.3% for CMV, and 12.5% and 2.0% for HHV-6, respectively. All forms of viral DNA were detected more frequently in whole blood than in plasma. During the symptomatic period, EBV DNA was detected in all whole-blood specimens but not in all plasma specimens. Furthermore, the EBV DNA load in whole blood was higher during the symptomatic period than during the asymptomatic period, whereas the EBV DNA load in plasma was similar for both periods. These results demonstrate that whole Wood is more suitable for the quantification of EBV DNA in transplant patients. However, a cutoff value with clinical relevance still needs to be determined.

Original languageEnglish
Pages (from-to)1426-1432
Number of pages7
JournalJournal of Clinical Microbiology
Volume45
Issue number5
DOIs
Publication statusPublished - May 2007

All Science Journal Classification (ASJC) codes

  • Microbiology (medical)

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    Wada, K., Kubota, N., Ito, Y., Yagasaki, H., Kato, K., Yoshikawa, T., Ono, Y., Ando, H., Fujimoto, Y., Kiuchi, T., Kojima, S., Nishiyama, Y., & Kimura, H. (2007). Simultaneous quantification of Epstein-Barr virus, cytomegalovirus, and human herpesvirus 6 DNA in samples from transplant recipients by multiplex real-time PCR assay. Journal of Clinical Microbiology, 45(5), 1426-1432. https://doi.org/10.1128/JCM.01515-06