Rapid and sensitive detection of 68 unique varicella zoster virus gene transcripts in five multiplex reverse transcription-polymerase chain reactions.
Varicella zoster virus (VZV) becomes latent in ganglionic neurons along the entire neuraxis. Although all predicted VZV open reading frames (ORFs) have been detected by macroarray and microarray analysis in virus-infected cells in culture where virus gene expression is abundant, array technology does not detect all VZV gene transcripts in latently infected human ganglia, where the abundance of ganglionic RNA is low and VZV gene transcription is highly variable. Using reverse transcription-polymerase chain reaction (RT-PCR) and the GenomeLab Genetic Analysis System (GeXPS), transcripts mapping to all 68 predicted unique VZV ORFs were detected in VZV-infected MeWo cells. Oligonucleotide primers contained both VZV- and cell-specific sequences linked to universal DNA sequences such that PCR amplification products were of predetermined sizes. Amplification products were resolved by capillary gel electrophoresis and detected by fluorescence spectrophotometry. Serial dilutions of total RNA extracted from VZV-infected MeWo cells were analyzed in parallel by GeXPS multiplex RT-PCR and real-time RT-PCR. GeXPS technology detected as few as 20 copies of VZV gene-specific transcripts. Only five multiplex RT-PCR assays were needed to analyze the entire VZV transcriptome. This technology will allow rapid analysis of all VZV genes transcribed during latency in human ganglia.
Nagel
Maria A
MA
Department of Neurology, University of Colorado Denver School of Medicine, 12700 East 19th Avenue, Mail Stop B182, Aurora, CO 80045, USA.
Gilden
Don
D
Shade
Ted
T
Gao
Bifeng
B
Cohrs
Randall J
RJ
D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D019075:Electrophoresis, Capillary; D020869:Gene Expression Profiling / Q000379:methods*; D014645:Herpesvirus 3, Human / Q000235:genetics*; D006801:Humans; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics; D012367:RNA, Viral / Q000096:biosynthesis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence
D017931:DNA Primers; D012333:RNA, Messenger; D012367:RNA, Viral