PCR-Stop analysis as a new tool for qPCR assay validation.

Progressively more qPCR assays have been developed in recent years in numerous fields of application. These assays are routinely validated using calibration curves, but essential validation per se such as Poisson analysis is frequently neglected. However, validation is crucial for determination of resolution and quantitative and qualitative limits. The new test method PCR-Stop analysis presented in this work investigates assay performance during initial qPCR cycles. PCRs with one to five pre-runs are performed while the subsequent main qPCR runs reflect pre-run replication rates. Ideally, DNA doubles according to pre-runs, there is no variation between replicates and qPCR starts immediately at the first cycle with its average efficiency. This study shows two exemplary qPCR assays, both with suitable calibration curves and efficiencies. We demonstrated thereby the benefits of PCR-Stop analysis revealing quantitative and qualitative resolution of both assays, the limits of one of those assays and thus avoiding misinterpretations in qPCR analysis. Furthermore, data displayed that a well performing assay starts indeed with its average efficiency.

Studying the effect of single mismatches in primer and probe binding regions on amplification curves and quantification in real-time PCR.

Comparison of a broad range of characteristics of real-time PCR amplification curves yielded only slight alterations for low numbers of mismatches in the primer binding regions, resulting in a quantification error up to 63.12%. The effects were more pronounced for mismatches in the probe binding region and resulted in a quantification error up to 33%.

Rapid growth of planktonic Vibrio cholerae non-O1/non-O139 strains in a large alkaline lake in Austria: dependence on temperature and dissolved organic carbon quality.

Vibrio cholerae non-O1/non-O139 strains have caused several cases of ear, wound, and blood infections, including one lethal case of septicemia in Austria, during recent years. All of these cases had a history of local recreational activities in the large eastern Austrian lake Neusiedler See. Thus, a monitoring program was started to investigate the prevalence of V. cholerae strains in the lake over several years. Genetic analyses of isolated strains revealed the presence of a variety of pathogenic genes, but in no case did we detect the cholera toxin gene or the toxin-coregulated pilus gene, both of which are prerequisites for the pathogen to be able to cause cholera. In addition, experiments were performed to elucidate the preferred ecological niche of this pathogen. As size filtration experiments indicated and laboratory microcosms showed, endemic V. cholerae could rapidly grow in a free-living state in natural lake water at growth rates similar to those of the bulk natural bacterial population. Temperature and the quality of dissolved organic carbon had a highly significant influence on V. cholerae growth. Specific growth rates, growth yield, and enzyme activity decreased markedly with increasing concentrations of high-molecular-weight substances, indicating that the humic substances originating from the extensive reed belt in the lake can inhibit V. cholerae growth.

Development of matrix lysis for concentration of gram positive bacteria from food and blood.

The development of a fast, reliable and inexpensive protocol for the concentration of bacteria from food by the removal of fat, carbohydrates and proteins that is compatible with downstream alternative DNA-based quantification methods is described. The protocol was used for dairy products, cooked and smoked fish and meat, carbohydrate-rich cooked products, ready-to-eat sauces, egg and blood. Lysis resulted in pellets of reasonable size for further processing. Starch, plant materials, fungi, tissues such as sinew, and chalaza could not be dissolved. Using L. monocytogenes, S. aureus and B. cereus as model organisms, microscopic analysis of the remaining bacterial pellets revealed that the recovered bacteria remained physically intact, albeit that the viability of the cells was compromised. Using real-time PCR, 7.3 CFU of L. monocytogenes were detected in artificially contaminated ultra-high temperature treated (UHT) milk and raw milk.