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 (>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.

Integrated DNA and RNA extraction using magnetic beads from viral pathogens causing acute respiratory infections

“…The main chemical constituents for the co-extraction of RNA and DNA are GTC, DTT, sodium dodecyl sulfate (SDS), and β-mercaptoethanol121315. In the 1990s, I. Casas, L established the “GuSCN-DNA/RNA” extraction method to extract viral RNA and DNA from cerebrospinal fluid, this method was the basis for most subsequent co-extraction methods15.…”

“…The prerequisite to implement these technologies is the extraction of high quality nucleic acids12. Viral nucleic acids are primarily divided into deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)13, which share equally important statuses in ARIs11. For example, RSV (RNA virus) and ADV (DNA virus) are both common viruses that cause pneumonia in children.…”

S‐S‐PEG‐COOH Self‐Assembled Monolayer on Gold Surface Enabled a Combined Assay for Serological EBV Antibody Isotypes

“…Biochip technology holds a variety of advantages with simultaneous detection of multiple analysis, reduction in sample and reagent volumes, and higher output of test results over conventional assays . Although gene biochips were used in EBV detection,various EBV genotypes within the lesion and wrong gene probe hybridizations as a result of point mutation, deletion, or rearrangement of the genome, could contribute to false results . On the other hand, serological assay for EBV infection are clinically applied.…”

Rapid Etiologic Diagnosis of Herpes Virus Infections in Children

“…1, 10 0 copy/mL for HSV-1; 2, 10 1 copy/mL for HSV-1; 3, 10 2 copy/mL for HSV-1; 4, 10 3 copy/mL for HSV-1; 5, 10 4 copy/mL for HSV-1; 6, 10 5 copy/mL for HSV-1; 7, 10 6 copy/mL for HSV-1; 8, 10 7 copy/mL for HSV-1. vous system and organs, leading to encephalitis, hepatitis, and Hodgkin lymphoma (2,16). Among herpes viruses, EBV and CMV are more prevalent in the world and are commonly contracted during infancy (17,18).…”