BACKGROUND: The detailed characterization of virus DNA is a challenge, and the genotyping that has been achieved to date has only been possible because researchers have sent a great deal of time and effort to do so. Instead of the simultaneous detection of hundreds of viruses on a single high-density DNA-chip at very high costs per chip, we present here an alternative approach using a well-designed and tailored microarray which can establish whether or not a handful of viral genes are present in a clinical sample. METHODS: In this study we applied a new concept of microarray-based, optimized and robust biochemistry for molecular diagnostics of the herpesviruses. For comparison, all samples were genotyped using standard procedures. RESULTS: The biochemical procedure of a knowledge-based, low-density microarray was established based on the molecular diagnostics of human herpes viruses: herpes simplex virus (HSV) HSV-1, HSV-2, varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and HHV-6. The study attempted to optimize parameters of microarray design, surface chemistry, oligonucleotide probe spotting, sample labeling and DNA hybridization to the developed DNA microarray. The results of 12 900 hybridization reactions on about 150 configured herpes virus microarrays showed that the established microarray-based typing procedure was reproducible, virus-specific and sufficiently sensitive with a lower limit of 100 viral copies per mL sample. CONCLUSIONS: The developed method utilizes low-fluorescence background coverslips, epoxy surface chemistry, standardized oligonucleotide probe spotting, PCR-labeling with Cy3 of isolated DNA, array hybridization, and detecting of specific spot fluorescence by an automatic microarray reader. We expect the configured microarray approach to be the method for high-throughput associated studies on human herpes viruses.
BACKGROUND: The detailed characterization of virus DNA is a challenge, and the genotyping that has been achieved to date has only been possible because researchers have sent a great deal of time and effort to do so. Instead of the simultaneous detection of hundreds of viruses on a single high-density DNA-chip at very high costs per chip, we present here an alternative approach using a well-designed and tailored microarray which can establish whether or not a handful of viral genes are present in a clinical sample. METHODS: In this study we applied a new concept of microarray-based, optimized and robust biochemistry for molecular diagnostics of the herpesviruses. For comparison, all samples were genotyped using standard procedures. RESULTS: The biochemical procedure of a knowledge-based, low-density microarray was established based on the molecular diagnostics of human herpes viruses: herpes simplex virus (HSV) HSV-1, HSV-2, varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and HHV-6. The study attempted to optimize parameters of microarray design, surface chemistry, oligonucleotide probe spotting, sample labeling and DNA hybridization to the developed DNA microarray. The results of 12 900 hybridization reactions on about 150 configured herpes virus microarrays showed that the established microarray-based typing procedure was reproducible, virus-specific and sufficiently sensitive with a lower limit of 100 viral copies per mL sample. CONCLUSIONS: The developed method utilizes low-fluorescence background coverslips, epoxy surface chemistry, standardized oligonucleotide probe spotting, PCR-labeling with Cy3 of isolated DNA, array hybridization, and detecting of specific spot fluorescence by an automatic microarray reader. We expect the configured microarray approach to be the method for high-throughput associated studies on human herpes viruses.
Authors: David Wang; Laurent Coscoy; Maxine Zylberberg; Pedro C Avila; Homer A Boushey; Don Ganem; Joseph L DeRisi Journal: Proc Natl Acad Sci U S A Date: 2002-11-12 Impact factor: 11.205
Authors: P Markoulatos; A Georgopoulou; N Siafakas; E Plakokefalos; G Tzanakaki; J Kourea-Kremastinou Journal: J Clin Microbiol Date: 2001-12 Impact factor: 5.948
Authors: M Luppi; P Barozzi; T F Schulz; G Setti; K Staskus; R Trovato; F Narni; A Donelli; A Maiorana; R Marasca; S Sandrini; G Torelli Journal: N Engl J Med Date: 2000-11-09 Impact factor: 91.245