A strategy to increase the specificity of Syber Green I qRT-PCR in hepatitis A detection.
Various methods for the recovery and detection of HAV have been suggested, and molecular tests have recently provided an effective replacement for the traditional methods. Real-time RT-PCR technology offers many advantages over conventional RT-PCR in terms of rapidity and specificity. Most procedures are based on the TaqMan chemistry, but some researchers have used the SYBR Green I approach, which is less expensive and simpler to carry out. However the formation of primer-dimers needs to be distinguished from specific products through a melting curve analysis. This study focused on a strategy to increase the specificity of Syber Green I chemistry, thus nullifying the primer-dimers interference. To this end, forward and reverse primers were specially designed for hairpin loop formation, a strategy widely used to improve the specificity and the efficiency of PCR. Two different concentrations of primers were assayed (200 nM and 400 nM) in a one-step, real-time RT-PCR procedure, evaluating the specificity of the amplicons and the optimization of the real-time protocol. We demonstrated that this approach can increase the specificity of the Syber Green I qRT-PCR performance with a good reproducibility of the method. Because of the simplicity of the assay and the lower costs involved, this procedure could be a valid alternative to HAV monitoring from environmental matrices.
Donia
Domenica
D
Department Public Health, School of Medicine, University of Tor Vergata, Faculty of Medicine, Rome, Italy. donia@med.uniroma2.it
Petrinca
Anna Rita
AR
Divizia
Maurizio
M
D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D005456:Fluorescent Dyes / Q000737:chemistry; D006506:Hepatitis A / Q000821:virology*; D030041:Hepatitis A virus / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D017931:DNA Primers; D005456:Fluorescent Dyes