Recombinant plasmid-based quantitative Real-Time PCR analysis of Salmonella enterica serotypes and its application to milk samples

• Recombinant plasmids were used for Real-Time PCR analysis of Salmonella for the first time.
• pTZ57R/T plasmids containing invA and ttrRSBC genes were tested using milk samples.
• The developed method allowed much faster detection compared to traditional methods.
• The developed method also allowed economical quantification of Salmonella.
• Thus, the developed method has great potential to be used in routine analyses.

The aim of the current study was to develop, a new, rapid, sensitive and quantitative Salmonella detection method using a Real-Time PCR technique based on an inexpensive, easy to produce, convenient and standardized recombinant plasmid positive control. To achieve this, two recombinant plasmids were constructed as reference molecules by cloning the two most commonly used Salmonella-specific target gene regions, invA and ttrRSBC. The more rapid detection enabled by the developed method (21 h) compared to the traditional culture method (90 h) allows the quantitative evaluation of Salmonella (quantification limits of 101 CFU/ml and 100 CFU/ml for the invA target and the ttrRSBC target, respectively), as illustrated using milk samples. Three advantages illustrated by the current study demonstrate the potential of the newly developed method to be used in routine analyses in the medical, veterinary, food and water/environmental sectors: I – The method provides fast analyses including the simultaneous detection and determination of correct pathogen counts; II – The method is applicable to challenging samples, such as milk; III – The method's positive controls (recombinant plasmids) are reproducible in large quantities without the need to construct new calibration curves.