Duplex realtime PCR method for Epstein–Barr virus and human DNA quantification: its application for post-transplant lymphoproliferative disorders detection

Authors
Fellner, María Dolores; Durand, Karina; Rodríguez, Marcelo; Irazu, Lucía; Alonio, Lidia Virginia; Picconi, María Alejandra
Publication Year
2014
Language
English
Format
article
Status
Published version
Description
INTRODUCTION: The quantification of circulating Epstein-Barr virus (EBV) DNA is used to monitor transplant patients as an early marker of Post-Transplant Lymphoproliferative Disorders (PTLD). So far no standardized methodology exists for such determination. OBJECTIVE: Our purpose was to develop and validate a real-time PCR assay to quantify EBV DNA in clinical samples from transplant recipients. METHODS: A duplex real-time PCR method was developed to amplify DNA from EBV and from a human gene. The EBV load was determined in peripheral blood mononuclear cells (PBMC), plasma and oropharyngeal tissue from 64 non-transplanted patients with lymphoid-hypertrophy (Non-Tx), 47 transplant recipients without PTLD (Tx), 54 recipients with PTLD (Tx-PTLD), and 66 blood donors (BD). WinPEPI, version 11.14 software was used for statistical analysis. RESULTS: Analytical validation: the intra and inter-assays variation coefficients were less than 4.5% (EBV-reaction) and 3% (glyceraldehyde 3-phosphate dehydrogenase - GAPDH reaction). Linear ranges comprised 10(7)-10 EBV genome equivalents (gEq) (EBV-reaction) and 500,000-32 human gEq (GAPDH-reaction). The detection limit was 2.9 EBV gEq (EBV-reaction). Both reactions showed specificity. Application to clinical samples: higher levels of EBV were found in oropharyngeal tissue from transplanted groups with and without PTLD, compared to Non-Tx (p<0.05). The EBV load in PBMC from the groups of BD, Non-Tx, Tx and Tx-PTLD exhibited increasing levels (p<0.05). In BD, PBMC and plasma, EBV loads were undetectable. CONCLUSIONS: The performance of the assay was suitable for the required clinical application. The assay may be useful to monitor EBV infection in transplant patients, in particular in laboratories from low-income regions that cannot afford to use commercial assays.
Fil: Fellner, María Dolores. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud; Argentina
Fil: Durand, Karina. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud; Argentina
Fil: Rodríguez, Marcelo. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud; Argentina
Fil: Irazu, Lucía. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud; Argentina
Fil: Alonio, Lidia Virginia. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Picconi, María Alejandra. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud; Argentina
Subject
EBV
Real time PCR
Quantification
Transplant
Viral load
PTLD
Salud Ocupacional
Ciencias de la Salud
CIENCIAS MÉDICAS Y DE LA SALUD
Access level
Open access
License
https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
Repository
CONICET Digital (CONICET)
Institution
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identifier
oai:ri.conicet.gov.ar:11336/33358