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.

We obtained the full genome of Middle East respiratory syndrome coronavirus (MERS-CoV) from a camel in Qatar. This virus is highly similar to the human England/Qatar 1 virus isolated in 2012. The MERS-CoV from the camel efficiently replicated in human cells, providing further evidence for the zoonotic potential of MERS-CoV from camels.

Two of the earliest Middle East respiratory syndrome (MERS) cases were men who had visited the Doha central animal market and adjoining slaughterhouse in Qatar. We show that a high proportion of camels presenting for slaughter in Qatar show evidence for nasal MERS-CoV shedding (62/105). Sequence analysis showed the circulation of at least five different virus strains at these premises, suggesting that this location is a driver of MERS-CoV circulation and a high-risk area for human exposure. No correlation between RNA loads and levels of neutralizing antibodies was observed, suggesting limited immune protection and potential for reinfection despite previous exposure.

NCT02401828.