Cargando…
Measurement of Epstein-Barr virus DNA load using a novel quantification standard containing two EBV DNA targets and SYBR Green I dye
BACKGROUND: Reactivation of Epstein-Barr virus (EBV) infection may cause serious, life-threatening complications in immunocompromised individuals. EBV DNA is often detected in EBV-associated disease states, with viral load believed to be a reflection of virus activity. Two separate real-time quantit...
Autores principales: | , , , , , |
---|---|
Formato: | Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2010
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2958162/ https://www.ncbi.nlm.nih.gov/pubmed/20860842 http://dx.doi.org/10.1186/1743-422X-7-252 |
_version_ | 1782188308214841344 |
---|---|
author | Lay, Meav-Lang J Lucas, Robyn M Ratnamohan, Mala Taylor, Janette Ponsonby, Anne-Louise Dwyer, Dominic E |
author_facet | Lay, Meav-Lang J Lucas, Robyn M Ratnamohan, Mala Taylor, Janette Ponsonby, Anne-Louise Dwyer, Dominic E |
author_sort | Lay, Meav-Lang J |
collection | PubMed |
description | BACKGROUND: Reactivation of Epstein-Barr virus (EBV) infection may cause serious, life-threatening complications in immunocompromised individuals. EBV DNA is often detected in EBV-associated disease states, with viral load believed to be a reflection of virus activity. Two separate real-time quantitative polymerase chain reaction (QPCR) assays using SYBR Green I dye and a single quantification standard containing two EBV genes, Epstein-Barr nuclear antigen-1 (EBNA-1) and BamHI fragment H rightward open reading frame-1 (BHRF-1), were developed to detect and measure absolute EBV DNA load in patients with various EBV-associated diseases. EBV DNA loads and viral capsid antigen (VCA) IgG antibody titres were also quantified on a population sample. RESULTS: EBV DNA was measurable in ethylenediaminetetraacetic acid (EDTA) whole blood, peripheral blood mononuclear cells (PBMCs), plasma and cerebrospinal fluid (CSF) samples. EBV DNA loads were detectable from 8.0 × 10(2 )to 1.3 × 10(8 )copies/ml in post-transplant lymphoproliferative disease (n = 5), 1.5 × 10(3 )to 2.0 × 10(5 )copies/ml in infectious mononucleosis (n = 7), 7.5 × 10(4 )to 1.1 × 10(5 )copies/ml in EBV-associated haemophagocytic syndrome (n = 1), 2.0 × 10(2 )to 5.6 × 10(3 )copies/ml in HIV-infected patients (n = 12), and 2.0 × 10(2 )to 9.1 × 10(4 )copies/ml in the population sample (n = 218). EBNA-1 and BHRF-1 DNA were detected in 11.0% and 21.6% of the population sample respectively. There was a modest correlation between VCA IgG antibody titre and BHRF-1 DNA load (rho = 0.13, p = 0.05) but not EBNA-1 DNA load (rho = 0.11, p = 0.11). CONCLUSION: Two sensitive and specific real-time PCR assays using SYBR Green I dye and a single quantification standard containing two EBV DNA targets, were developed for the detection and measurement of EBV DNA load in a variety of clinical samples. These assays have application in the investigation of EBV-related illnesses in immunocompromised individuals. |
format | Text |
id | pubmed-2958162 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2010 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-29581622010-10-21 Measurement of Epstein-Barr virus DNA load using a novel quantification standard containing two EBV DNA targets and SYBR Green I dye Lay, Meav-Lang J Lucas, Robyn M Ratnamohan, Mala Taylor, Janette Ponsonby, Anne-Louise Dwyer, Dominic E Virol J Methodology BACKGROUND: Reactivation of Epstein-Barr virus (EBV) infection may cause serious, life-threatening complications in immunocompromised individuals. EBV DNA is often detected in EBV-associated disease states, with viral load believed to be a reflection of virus activity. Two separate real-time quantitative polymerase chain reaction (QPCR) assays using SYBR Green I dye and a single quantification standard containing two EBV genes, Epstein-Barr nuclear antigen-1 (EBNA-1) and BamHI fragment H rightward open reading frame-1 (BHRF-1), were developed to detect and measure absolute EBV DNA load in patients with various EBV-associated diseases. EBV DNA loads and viral capsid antigen (VCA) IgG antibody titres were also quantified on a population sample. RESULTS: EBV DNA was measurable in ethylenediaminetetraacetic acid (EDTA) whole blood, peripheral blood mononuclear cells (PBMCs), plasma and cerebrospinal fluid (CSF) samples. EBV DNA loads were detectable from 8.0 × 10(2 )to 1.3 × 10(8 )copies/ml in post-transplant lymphoproliferative disease (n = 5), 1.5 × 10(3 )to 2.0 × 10(5 )copies/ml in infectious mononucleosis (n = 7), 7.5 × 10(4 )to 1.1 × 10(5 )copies/ml in EBV-associated haemophagocytic syndrome (n = 1), 2.0 × 10(2 )to 5.6 × 10(3 )copies/ml in HIV-infected patients (n = 12), and 2.0 × 10(2 )to 9.1 × 10(4 )copies/ml in the population sample (n = 218). EBNA-1 and BHRF-1 DNA were detected in 11.0% and 21.6% of the population sample respectively. There was a modest correlation between VCA IgG antibody titre and BHRF-1 DNA load (rho = 0.13, p = 0.05) but not EBNA-1 DNA load (rho = 0.11, p = 0.11). CONCLUSION: Two sensitive and specific real-time PCR assays using SYBR Green I dye and a single quantification standard containing two EBV DNA targets, were developed for the detection and measurement of EBV DNA load in a variety of clinical samples. These assays have application in the investigation of EBV-related illnesses in immunocompromised individuals. BioMed Central 2010-09-22 /pmc/articles/PMC2958162/ /pubmed/20860842 http://dx.doi.org/10.1186/1743-422X-7-252 Text en Copyright ©2010 Lay et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Methodology Lay, Meav-Lang J Lucas, Robyn M Ratnamohan, Mala Taylor, Janette Ponsonby, Anne-Louise Dwyer, Dominic E Measurement of Epstein-Barr virus DNA load using a novel quantification standard containing two EBV DNA targets and SYBR Green I dye |
title | Measurement of Epstein-Barr virus DNA load using a novel quantification standard containing two EBV DNA targets and SYBR Green I dye |
title_full | Measurement of Epstein-Barr virus DNA load using a novel quantification standard containing two EBV DNA targets and SYBR Green I dye |
title_fullStr | Measurement of Epstein-Barr virus DNA load using a novel quantification standard containing two EBV DNA targets and SYBR Green I dye |
title_full_unstemmed | Measurement of Epstein-Barr virus DNA load using a novel quantification standard containing two EBV DNA targets and SYBR Green I dye |
title_short | Measurement of Epstein-Barr virus DNA load using a novel quantification standard containing two EBV DNA targets and SYBR Green I dye |
title_sort | measurement of epstein-barr virus dna load using a novel quantification standard containing two ebv dna targets and sybr green i dye |
topic | Methodology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2958162/ https://www.ncbi.nlm.nih.gov/pubmed/20860842 http://dx.doi.org/10.1186/1743-422X-7-252 |
work_keys_str_mv | AT laymeavlangj measurementofepsteinbarrvirusdnaloadusinganovelquantificationstandardcontainingtwoebvdnatargetsandsybrgreenidye AT lucasrobynm measurementofepsteinbarrvirusdnaloadusinganovelquantificationstandardcontainingtwoebvdnatargetsandsybrgreenidye AT ratnamohanmala measurementofepsteinbarrvirusdnaloadusinganovelquantificationstandardcontainingtwoebvdnatargetsandsybrgreenidye AT taylorjanette measurementofepsteinbarrvirusdnaloadusinganovelquantificationstandardcontainingtwoebvdnatargetsandsybrgreenidye AT ponsonbyannelouise measurementofepsteinbarrvirusdnaloadusinganovelquantificationstandardcontainingtwoebvdnatargetsandsybrgreenidye AT dwyerdominice measurementofepsteinbarrvirusdnaloadusinganovelquantificationstandardcontainingtwoebvdnatargetsandsybrgreenidye |