title: Evaluation of a viral microarray based on simultaneous extraction and amplification of viral nucleotide acid for detecting human herpesviruses and enteroviruses.
abstract: In this study, a viral microarray based assay was developed to detect the human herpesviruses and enteroviruses associated with central nervous system infections, including herpes simplex virus type 1, type 2 (HSV1 and HSV2), Epstein-Barr virus (EBV), cytomegalovirus (CMV), enterovirus 71 (EV71), coxsackievirus A 16 (CA16) and B 5(CB5). The DNA polymerase gene of human herpesviruses and 5'-untranslated region of enteroviruses were selected as the targets to design primers and probes. Human herpesviruses DNA and enteroviruses RNA were extracted simultaneously by using a guanidinium thiocyanate acid buffer, and were subsequently amplified through a biotinylated asymmetry multiplex RT-PCR with the specific primer of enteroviruses. In total, 90 blood samples and 49 cerebrospinal fluids samples with suspected systemic or neurological virus infections were investigated. Out of 139 samples, 66 were identified as positive. The specificities of this multiplex RT-PCR microarray assay were over 96% but the sensitivities were various from 100% for HSV1, HSV2, EV71 and CB5, 95.83% for CMV, 80% for EBV to 71.43% for CA16 in comparison with reference standards of TaqMan qPCR/qRT-PCR. The high Kappa values (&gt;0.90) from HSV1, HSV2, CMV, EV71 and CB5 were obtained, indicating almost perfect agreement in term of the 5 viruses detection. But lower Kappa values for EBV (0.63) and CA16 (0.74) displayed a moderate to substantial agreement. This study provides an innovation of simultaneous extraction, amplification, hybridization and detection of DNA viruses and RNA viruses with simplicity and specificity, and demonstrates a potential clinical utility for a variety of viruses' detection.
authors.affiliation: Pediatric Research Institute, Qilu Children's Hospital of Shandong University, Ji'nan, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of Pediatrics, Qilu Children's Hospital of Shandong University, Ji'nan, China.
authors.forename: Chunhong
authors.initials: C
authors.lastname: Duan
authors.affiliation: Shanghai Biochip National Engineering Research Center, Shanghai, China.
authors.forename: Chunxiu
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Pediatric Research Institute, Qilu Children's Hospital of Shandong University, Ji'nan, China.
authors.forename: Xiaomeng
authors.initials: X
authors.lastname: Yang
authors.affiliation: Clinical Central Laboratory, Maternal and Children's Health Care Institute of Jinan, Jinan, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Pediatric Research Institute, Qilu Children's Hospital of Shandong University, Ji'nan, China.
authors.forename: Rui
authors.initials: R
authors.lastname: Dong
authors.affiliation: Shanghai Biochip National Engineering Research Center, Shanghai, China.
authors.forename: Jiajing
authors.initials: J
authors.lastname: Zhou
authors.affiliation: Pediatric Research Institute, Qilu Children's Hospital of Shandong University, Ji'nan, China; Department of Pediatrics, Qilu Children's Hospital of Shandong University, Ji'nan, China.
authors.forename: Zhongtao
authors.initials: Z
authors.lastname: Gai
mesh_terms: D002460:Cell Line; D002648:Child; D004279:DNA, Viral / Q000235:genetics* / Q000302:isolation & purification*; D004770:Enterovirus / Q000235:genetics*; D005240:Feasibility Studies; D006564:Herpesviridae / Q000235:genetics*; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012367:RNA, Viral / Q000235:genetics* / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D004279:DNA, Viral; D012367:RNA, Viral