title: Influence of reagents formulation on real-time PCR parameters.
abstract: Real-time polymerase chain reaction (PCR) techniques are increasingly used to quantify target sequences for diagnostic and research purposes. Due to its 'quantitative' character, it is very important to determine the variability of this technique correlating with several experimental conditions. The objective of this study was to analyse the effect of manufacturing lots of PCR reagents on two main PCR parameters, specificity and sensitivity. For this study, we used four different amplicons, using either mouse genomic DNA or viral DNA. Although a PCR product could be obtained in any of the conditions, we observed that there are relevant variations in sensitivity depending on the reagents formulation. We conclude that different lots of reagents may determine the analytical performance of PCR assays indicating that reagents testing are of special importance when the PCR protocol is used for quantitative purposes.
authors.affiliation: Centro de Biología Molecular Severo Ochoa - CSIC Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain. jburgos@cbm.uam.es
authors.forename: J S
authors.initials: JS
authors.lastname: Burgos
authors.forename: C
authors.initials: C
authors.lastname: Ramírez
authors.forename: R
authors.initials: R
authors.lastname: Tenorio
authors.forename: I
authors.initials: I
authors.lastname: Sastre
authors.forename: M J
authors.initials: MJ
authors.lastname: Bullido
mesh_terms: D000199:Actins / Q000235:genetics; D000818:Animals; D004247:DNA / Q000032:analysis; D004279:DNA, Viral / Q000032:analysis; D016678:Genome; D018259:Herpesvirus 1, Human / Q000235:genetics; D007202:Indicators and Reagents; D051379:Mice; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D000199:Actins; D004279:DNA, Viral; D007202:Indicators and Reagents; D004247:DNA