Simultaneous quantification of Epstein-Barr virus, cytomegalovirus, and human herpesvirus 6 DNA in samples from transplant recipients by multiplex real-time PCR assay.
We developed a multiplex real-time PCR assay using 6-carboxyfluorescein, 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein, and carbocyanine 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 blood is more suitable for the quantification of EBV DNA in transplant patients. However, a cutoff value with clinical relevance still needs to be determined.
Wada
Kaoru
K
Department of Virology, Nagoya University Graduate School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya 466-8550, Japan.
Kubota
Naomi
N
Ito
Yoshinori
Y
Yagasaki
Hiroshi
H
Kato
Koji
K
Yoshikawa
Tetsushi
T
Ono
Yasuyuki
Y
Ando
Hisami
H
Fujimoto
Yasuhiro
Y
Kiuchi
Tetsuya
T
Kojima
Seiji
S
Nishiyama
Yukihiro
Y
Kimura
Hiroshi
H
D000293:Adolescent; D000328:Adult; D000956:Antigens, Viral / Q000097:blood; D002648:Child; D002675:Child, Preschool; D003587:Cytomegalovirus / Q000235:genetics / Q000302:isolation & purification*; D003586:Cytomegalovirus Infections / Q000175:diagnosis / Q000635:transmission; D004279:DNA, Viral / Q000097:blood*; D020031:Epstein-Barr Virus Infections / Q000175:diagnosis / Q000635:transmission; D005260:Female; D018380:Hematopoietic Stem Cell Transplantation / Q000009:adverse effects*; D004854:Herpesvirus 4, Human / Q000235:genetics / Q000302:isolation & purification*; D015654:Herpesvirus 6, Human / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D007223:Infant; D016031:Liver Transplantation / Q000009:adverse effects*; D008297:Male; D016133:Polymerase Chain Reaction; D019349:Roseolovirus Infections / Q000175:diagnosis / Q000635:transmission; D012680:Sensitivity and Specificity; D019562:Viral Load
D000956:Antigens, Viral; D004279:DNA, Viral