Establishment And Application Of PCR-microarray Technology For Simultaneous Detection And Species Identification Of Seven Human Herpes Viruses

Human herpes viruses, particularly 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), are common pathogens in clinic. The protean manifestations of herpesvirus-mediated infections make early diagnosis of great importance. However, the clinical signs and symptoms resulting from these human herpes viruses, such as fever, jaundice, hepato-splenomegaly, encephalitis or meningitis, are often non-specific and indistinguishable from other viral infections, which makes the laboratory approaches contribute a lot to the early diagnosis of human herpes viral infection and prompt onset of treatment. In this study, a multiplex PCR-based DNA microarray technology for simultaneous detection and species identification of seven human herpes viruses was described. 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 amplified and labeled with CY5, the PCR products of specimens werehybridized with the DNA microarrays. Then the hybridization results were scanned and read by laser-scanner. Sixty- one cerebrospinal fluid (CSF) and 132 blood specimens of children were analyzed by this technique. The results indicate that this multiplex PCR-based DNA microarray technology proves to be an effective way for detecting and species identification of seven human herpes viruses. The study was composed of two parts—part one and part two.Part one: Establishment of PCR- microarray technology for simultaneous detection and species identification of seven human herpes viruses;Part two: Application of the PCR- microarray technology for clinical specimens.Part OneEstablishment of PCR- microarray technology for simultaneous detection and species identification of seven human herpes virusesObjective: To establish 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).Methods: Based on the highly conserved regions of the DNA polymerase gene in human herpes viruses, primers were designed to simultaneously amplify seven human herpes viruses, namely HSV-1, HSV-2, VZV, EBV, CMV, HHV-6A and HHV-6B. Within the sequences of each PCR amplicon, specific oligonucleotide probes were designed and synthesized, and were printed onto special glass slides to make DNA microarrays. After amplified and labeled with CY5, the PCR products of the seven human herpes viruses were hybridized with the DNA microarrays. Then the hybridization results were scanned and read by laser-scanner. Results: The products of the seven human herpes viruses after PCR amplification rangedfrom 224bp to 252bp (232bp for HSV-1 and HSV-2, 224bp for VZV, 227bp for EBV, 235bp for CMV, 252bp for HHV-6A and HHV-6B, respectively), and could be species identified with DNA microarrays. No fragment of the same length was detected and no cross-reaction was shown to DNA derived from S. aureus, E. coli, HBV, C. neoformans, Candidaalbicans and human genome in the test. The detection limits were 10~1 copy/μl for HSV-1, HSV-2, VZV, EBV, CMV, HHV-6A and HHV-6B, respectively. Conclusions: This multiplex PCR-based DNA microarray technology, which is rapid, specific and sensitive in simultaneous detection and species identification of seven human herpes viruses, will serve as an effective way for early diagnosis of herpesvirus-associated diseases.Part TwoApplication of the PCR- microarray technology for clinicalspecimensObjective: To apply the PCR-microarray technology to early and rapid diagnosis of herpesvirus-associated diseases.Methods: A total of 132 blood specimens from children with suspected virus infections and 61 cerebrospinal fluid (CSF) specimens from children with suspected meningitis and encephalitis were collected and analyzed by this PCR-microarray technology. The results were compared with those of TaqMan PCR. Some of the specimens positive for human herpes virus DNA were further confirmed by sequencing after cloning. In addition, thirty five blood specimens from healthy children and thirty CSF specimens from children with clinical manifestations unrelated to herpes virus infections (eight with encephalon tumor and twenty-two with purulent meningitis) served as negative controls. Results: Among 132 blood specimens from children with suspected virus infections, 49 were tested positive for human herpes virus DNA by PCR-microarray technology, namely one for HSV-1, one for HSV-2, sixteen for EBV, twenty-two for CMV, one for HHV-6A, three for HHV-6B, and two showed EBV/CMV coinfections, one showed VZV/CMV, oneshowed HSV-2/CMV, one showed EBV/HHV-6B. Among sixty-one CSF specimens from children with suspected meningitis and encephalitis, 6 were tested positive for human herpes virus DNA by PCR-microarray technology, namely one for HSV-1, two for HSV-2, one for EBV, and one showed HSV-2/CMV coinfection, one showed EBV/HHV-6B. Compared with the results of TaqMan PCR, the sensitivity and specificity of DNA microarray technology for HSV-1, HSV-2, EBV and CMV was 96.2% and 99.3%, respectively. Seven specimens which were positive for VZV, HHV-6A or HHV-6B were sequenced after cloning. And the results of sequencing were aligned with the sequences of standard virus strains. The homology of all was higher than 98%. None of the 35 control blood and 30 control CSF was positive for human herpes virus DNA by this PCR-microarray technology. The clinical symptoms as well as the results of other laboratory tests of most cases supported the diagnosis of PCR-microarray technology quite well. For those which were not clearly or definitely diagnosed, PCR-microarray technology provided an effective way for making clear the cause of diseases.Conclusions: This PCR-microarray technology, which is rapid, specific and sensitive, serves as an effective technique for simultaneous detection and species identification of seven human herpes viruses in clinical cerebrospinal fluid and blood specimens, and has a considerable value in early diagnosis of human herpes viral infection in clinic.