DNA microarray technology for simultaneous detection and species identification of seven human herpes viruses

Abstract

The aim of the study was to develop a multiplex PCR-based DNA microarray technology for simultaneous detection and species identification of seven human herpes viruses, namely herpes simplex virus type 1, type 2 (HSV-1, HSV-2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and human herpes virus 6 (HHV-6A, HHV-6B), and to apply this technology to accurate diagnosis of herpesvirus-associated diseases. Primers and oligonucleotide probes were designed and synthesized based on the highly conserved regions of the DNA polymerase gene in human herpes viruses. DNA microarrays were made by printing the oligonucleotide probes onto special glass slides. After amplification and labeling with CY5, the PCR products were hybridized with the DNA microarrays and species identified. Sixty-one cerebrospinal fluid (CSF) and 132 blood specimens were analyzed by this technique, and the results were compared with those of TaqMan PCR. Several specimens were sequenced further after cloning. The PCR products of the seven human herpes viruses ranged from 224 to 252 bp, and could be species identified with DNA microarrays. The detection limits were 10(1) copies/microl for each virus. And the test showed no cross-reaction to DNA extracted from S. aureus, E. coli, hepatitis B virus, Cryptococcus neoformans, Candida albicans and human genome. Among 132 blood and 61 CSF specimens, 55 were tested positive for human herpes virus DNA. Compared with the results of TaqMan PCR, the sensitivity and specificity of the DNA microarray technology was 96.2% and 99.3%, respectively. This multiplex PCR-based DNA microarray technology, which is rapid, specific and sensitive, serves as an effective technique for simultaneous detection and species identification of seven human herpes viruses.

Rapid and simultaneous detection of 6 types of human herpes virus (herpes simplex virus, varicella-zoster virus, Epstein-Barr virus, cytomegalovirus, human herpes virus 6A/B, and human herpes virus 7) by multiplex PCR assay

“…and specialized analysis processes such as southern blot hybridization and microarray methods (28,29). Moreover, if 6 types of human herpes virus are to be detected, PCR with multiple tubes and requiring expensive equipment must be performed (22).…”

“…The conventional PCR method requires the individual examination of each virus, which leads to problems with respect to time and cost. Although PCR techniques that can simultaneously detect human herpes viruses have been reported, amplification product analysis requires complicated methods such as southern blot hybridization (28) and microarray technology (29) to detect many viruses. Furthermore, there are few reports of the 6 Extraction of DNA.…”

Quantitative and Qualitative RNA-Seq-Based Evaluation of Epstein-Barr Virus Transcription in Type I Latency Burkitt's Lymphoma Cells

“…Microarrays have been used to assess the levels of EpsteinBarr virus (EBV) gene expression in experimental and clinical settings (1,7,(16)(17)(18). Nevertheless, this analysis typically requires the use of custom arrays with user-specified probes against each EBV gene of interest.…”

Rapid Etiologic Diagnosis of Herpes Virus Infections in Children

“…Total DNA was extracted by using Axyprep Blood Genomic DNA Miniprep Kit (Axygen, Shanghai, China). This technology has been described previously in detail (13,14). First, multiplex PCR was performed.…”