Multiplex PCR-based DNA array for simultaneous detection of three human herpesviruses, EVB, CMV and KSHV.
Human lymphotropic herpesviruses, Epstein-Barr virus (EBV), cytomegalovirus (CMV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are responsible for a wide variety of human diseases. Due to an increase in diseased states associated with immunosuppression, more instances of co-morbid infections with these herpesviruses have resulted in viral reactivations that have caused numerous fatalities. Therefore, the development of rapid and accurate method to detect these viruses in immunocompromised patients is vital for immediate treatment with antiviral prophylactic drugs. In this study, we developed a new multiplex PCR method coupled to DNA array hybridization, which can simultaneously detect all three human herpesviruses in one single cell sample. Multiplex PCR primers were designed to amplify specific regions of the EBV (EBER1), CMV (IE) and KSHV (LANA) viral genomes. Pre-clinical application of this method revealed that this approach is capable of detecting as few as 1 copy of the viral genomes for KSHV and CMV and 100 copies of the genome for EBV. Furthermore, this highly sensitive test showed no cross-reactivity among the three viruses and is capable of detecting both KSHV and EBV viral genomes simultaneously in the lymphoblastoid cells that have been double infected with both viruses. Thus, this array-based approach serves as a rapid and reliable diagnostic tool for clinical applications.
Fujimuro
Masahiro
M
Department of Biochemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo 060-0812, Japan. fuji2@pharm.hokudai.ac.jp
Nakaso
Kazuhiro
K
Nakashima
Kenji
K
Sadanari
Hidetaka
H
Hisanori
Inoue
I
Teishikata
Yasuhiro
Y
Hayward
S Diane
SD
Yokosawa
Hideyoshi
H
D003587:Cytomegalovirus / Q000235:genetics* / Q000302:isolation & purification*; D015342:DNA Probes / Q000235:genetics; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics; D005347:Fibroblasts / Q000821:virology; D016679:Genome, Viral / Q000235:genetics; D004854:Herpesvirus 4, Human / Q000235:genetics* / Q000302:isolation & purification*; D019288:Herpesvirus 8, Human / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D015342:DNA Probes; D004279:DNA, Viral