Analysis of real time PCR amplification efficiencies from three genomic region of dengue virus

• Published in: Investigación clínica 2013-03, Vol.54 (1), p.5-19
• Main Authors: Odreman-Macchioli, María, Vielma, Silvana, Atchley, Daniel, Comach, Guillermo, Ramirez, Alvaro, Pérez, Saberio, Téllez, Luis, Quintero, Beatriz, Hernández, Erick, Muñoz, Maritza, Mendoza, José
• Format: Article
• Language: English
• Subjects: 3' Untranslated Regions - genetics; Antibodies, Viral - blood; Capsid Proteins - genetics; Dengue - blood; Dengue - virology; Dengue Virus - classification; Dengue Virus - genetics; Dengue Virus - immunology; Dengue Virus - isolation & purification; Early Diagnosis; Enzyme-Linked Immunosorbent Assay; Genome, Viral; Humans; Immunoglobulin M - blood; MEDICINE, RESEARCH & EXPERIMENTAL; Organic Chemicals; Real-Time Polymerase Chain Reaction - methods; Reproducibility of Results; RNA, Viral - analysis; Sensitivity and Specificity; Serotyping; Taq Polymerase; Viral Nonstructural Proteins - genetics; Virus Cultivation
• ISSN: 0535-5133

Early diagnosis of dengue virus (DENV) infection represents a key factor in preventing clinical complications attributed to the disease. The aim of this study was to evaluate the amplification efficiencies of an in-house quantitative real time-PCR (qPCR) assay of DENV, using the non-structural conserved genomic region protein-5 (NS5) versus two genomic regions usually employed for virus detection, the capsid/pre-membrane region (C-prM) and the 3'-noncoding region (3'NC). One-hundred sixty seven acute phase serum samples from febrile patients were used for validation purposes. Results showed that the three genomic regions had similar amplification profiles and correlation coefficients (0.987-0.999). When isolated viruses were used, the NS5 region had the highest qPCR efficiencies for the four serotypes (98-100%). Amplification from acute serum samples showed that 41.1% (67/167) were positive for the universal assay by at least two of the selected genomic regions. The agreement rates between NS5/C-prM and NS5/3'NC regions were 56.7% and 97%, respectively. Amplification concordance values between C-prM/NS5 and NS5/3'NC regions showed a weak (kappa = 0.109; CI 95%) and a moderate (kappa = 0.489; CI 95%) efficiencies in amplification, respectively. Serotyping assay using a singleplex NS5-TaqMan format was much more sensitive than the C-prM/SYBR Green I protocol (76%). External evaluation showed a high sensitivity (100%), specificity (78%) and high agreement between the assays. According to the results, the NS5 genomic region provides the best genomic region for optimal detection and typification of DENV in clinical samples.