Multiplex real-time PCR for the detection and quantification of latent and persistent viral genomes in cellular or plasma blood fractions

• Published in: Journal of virological methods Vol. 151; no. 1; pp. 47 - 54
• Main Authors: Compston, Lara Isobel, Sarkobie, Francis, Li, Chengyao, Candotti, Daniel, Opare-Sem, Ohene, Allain, Jean-Pierre
• Format: Journal Article
• Language: English
• Published: London Elsevier B.V 01.07.2008; Amsterdam Elsevier; New York, NY
• Subjects: Biological and medical sciences; Blood Donors; Cytomegalovirus; DNA, Viral - blood; Epstein-Barr virus; Fundamental and applied biological sciences. Psychology; GB virus C - classification; GB virus C - genetics; GB virus C - isolation & purification; GB virus C - physiology; GBV-C; Genome, Viral; HBV; Hepatitis B virus; Hepatitis B virus - classification; Hepatitis B virus - genetics; Hepatitis B virus - isolation & purification; Hepatitis B virus - physiology; Herpesviridae - classification; Herpesviridae - genetics; Herpesviridae - isolation & purification; Herpesviridae - physiology; Herpesviruses; Human herpesvirus 8; Humans; Microbiology; Multiplex PCR; Parvovirus B19; Parvovirus B19, Human - classification; Parvovirus B19, Human - genetics; Parvovirus B19, Human - isolation & purification; Parvovirus B19, Human - physiology; Polymerase Chain Reaction; Prevalence; Quantitative PCR; Reproducibility of Results; Sensitivity and Specificity; Techniques used in virology; Varicella-zoster virus; Viral Load; Viremia - diagnosis; Viremia - epidemiology; Viremia - virology; Virology; Virus Latency; Multiplex PCR; Herpesviruses; HBV; GBV-C; Quantitative PCR; Parvovirus B19; Microbiology; Orthohepadnavirus; Flavivirus; Virus B19; Blood plasma; Hepadnaviridae; Hepatitis B virus; Quantitative analysis; Hepatitis G virus; Multiplex polymerase chain reaction; Method; Erythrovirus; Real time; Virology; Virus; Polymerase chain reaction; Parvoviridae; Flaviviridae; Detection; Genome; Parvovirinae
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2008.03.023

In common with latent viruses such as herpesviruses, parvovirus B19, HBV and GBV-C are contained successfully by the immune response and persist in the host. When immune control breaks down, reactivation of both latent and persistent viruses occurs. Two multiplex assays were developed (B19, HBV, HHV-8), (EBV, CMV, VZV) for blood screening, and tested on blood donor samples from Ghana to determine baseline prevalence of viraemia in immunocompetent persons. Single-virus real-time quantitative PCR (qPCR) assays were optimised for viral load determination of positive initial screening. The qPCR method utilised was absolute quantification with external standards. Multiplex and single-virus qPCR assays had similar sensitivity, except for the B19 assay in which sensitivity was 100-fold lower. Assays were optimised for reproducibility and repeatability, with R 2 of 0.9 being obtained for most assays. With the exception of B19 and CMV, assays had 100% detection limit ranging between 10 1 and 10 2 copies, IU or arbitrary units under single-virus and multiplex assay conditions. The prevalence of viraemia was 1.6% HBV (0.8% DNA+/HBsAg−, 0.8% DNA+/HBsAg+), 0.8% parvovirus B19, and 3.3% GBV-C viraemia in the plasma fraction. The prevalence of four herpesviruses was 1.0% HHV-8, 0.85% CMV, and 8.3% EBV, and no detectable VZV viraemia.