title: Analysis of real time PCR amplification efficiencies from three genomic region of dengue virus.
abstract: 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.
authors.affiliation: Laboratorio de Microbiología y Salud Pública del Estado Mérida, Universidad de Los Andes, Mérida, Venezuela.
authors.forename: María
authors.initials: M
authors.lastname: Odreman-Macchioli
authors.forename: Silvana
authors.initials: S
authors.lastname: Vielma
authors.forename: Daniel
authors.initials: D
authors.lastname: Atchley
authors.forename: Guillermo
authors.initials: G
authors.lastname: Comach
authors.forename: Alvaro
authors.initials: A
authors.lastname: Ramirez
authors.forename: Saberio
authors.initials: S
authors.lastname: Pérez
authors.forename: Luis
authors.initials: L
authors.lastname: Téllez
authors.forename: Beatriz
authors.initials: B
authors.lastname: Quintero
authors.forename: Erick
authors.initials: E
authors.lastname: Hernández
authors.forename: Maritza
authors.initials: M
authors.lastname: Muñoz
authors.forename: José
authors.initials: J
authors.lastname: Mendoza
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics*; D000914:Antibodies, Viral / Q000097:blood; D052160:Benzothiazoles; D036022:Capsid Proteins / Q000235:genetics*; D003715:Dengue / Q000097:blood / Q000821:virology*; D003716:Dengue Virus / Q000145:classification / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D003959:Diamines; D042241:Early Diagnosis; D004797:Enzyme-Linked Immunosorbent Assay; D016679:Genome, Viral; D006801:Humans; D007075:Immunoglobulin M / Q000097:blood; D009930:Organic Chemicals; D011804:Quinolines; D012367:RNA, Viral / Q000032:analysis*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012703:Serotyping; D019914:Taq Polymerase; D017361:Viral Nonstructural Proteins / Q000235:genetics*; D014776:Virus Cultivation
chemical_list: D020413:3' Untranslated Regions; D000914:Antibodies, Viral; D052160:Benzothiazoles; D036022:Capsid Proteins; D003959:Diamines; D007075:Immunoglobulin M; C582223:NS5 protein, dengue virus; D009930:Organic Chemicals; D011804:Quinolines; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C098022:SYBR Green I; D019914:Taq Polymerase