Real-time polymerase chain reaction (PCR) is the current method of choice for detection and quantification of nucleic acids , especially for molecular diagnostics. Complementarity between primers and template is often crucial for PCR applications , as mismatches can severely reduce priming efficiency. However , little quantitative data on the effect of these mismatches is available. We quantitatively investigated the effects of primer-template mismatches within the 3-end primer region on real-time PCR using the 5-nuclease assay. Our results show that single mismatches instigate a broad variety of effects, ranging from minor (<1.5 cycle threshold , eg , A-C, C-A , T-G , G-T) to severe impact (>7.0 cycle threshold , eg , A-A , G-A , A-G , C-C) on PCR amplification. A clear relationship between specific mismatch types, position , and impact was found , which remained consistent for DNA versus RNA amplifications and Taq/Moloney murine leukemia virus versus rTth based amplifications. The overall size of the impact among the various master mixes used differed substantially (up to sevenfold) , and for certain master mixes a reverse or forward primer-specific impact was observed , emphasizing the importance of the experimental conditions used. Taken together these data suggest that mismatch impact follows a consistent pattern and enabled us to formulate several guidelines for predicting primer-template mismatch behavior when using specific 5-nuclease assay master mixes. Our study provides novel insight into mismatch behavior and should allow for more optimized development of real-time PCR assays involving primer-template mismatches.

Sensitive and reproducible molecular assays are essential for influenza virus diagnostics. This manuscript describes the design, validation, and evaluation of a set of real-time RT-PCR assays for quantification and subtyping of human influenza viruses from patient respiratory material. Four assays are included for detection of oseltamivir resistance mutations H275Y in prepandemic and pandemic influenza A/H1N1 and E119V and R292K in influenza A/H3N2 neuraminidase. The lower limits of detection of the quantification assay were determined to be 1.7 log(10) virus particles per milliliter (vp/mL) for influenza A and 2.2 log(10) vp/mL for influenza B virus. The lower limits of quantification were 2.1 and 2.3 log(10) vp/mL, respectively. The RT-PCR efficiencies and lower limits of detection of the quantification assays were only marginally affected when tested on the most dissimilar target sequences found in the GenBank database. Finally, the resistance RT-PCR assays detected at least 5% mutant viruses present in mixtures containing both wild-type and mutant viruses with approximated limits of detection of 2.4 log(10) vp/mL. Overall, this set of RT-PCR assays is a powerful tool for enhanced influenza virus surveillance.