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

Gokduman, Kurtulus
Cakiris, Aris
Ustek, Duran
Gültekin, Güzin Candan
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 10(1) CFU/ml and 10(0) 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.


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Orman, Mehmet Ali; Çalık, Pınar; Department of Chemical Engineering (2007)
In this study, the effects of bioprocess operation parameters on recombinant human growth hormone (rhGH) production by P. pastoris were systematically investigated. In this frame, first, for the extracellular expression and purification of human growth hormone by recombinant P. pastoris the cDNA of hGH, fused with a polyhistidine tag and also fused with a target site for the Factor Xa protease in which cleavage produces a mature N- and C- termini of rhGH, was cloned into pPICZαA plasmid and the constructed ...
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A microarray was developed to simultaneously screen Escherichia coli and Salmonella enterica for multiple genetic traits. The final array included 203 60-mer oligonucleotide probes, including 117 for resistance genes, 16 for virulence genes, 25 for replicon markers, and 45 other markers. Validity of the array was tested by assessing inter-laboratory agreement among four collaborating groups using a blinded study design. Internal validation indicated that the assay was reliable (area under the receiver-opera...
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The main objective of this study was to investigate the effects of high hydrostatic pressure on the stability of blood constituents for the purpose of an effective reduction of viral and bacterial count. The effect of HHP treatment on the several blood constituents were analyzed at different HHP levels at 25 0C for 5 minutes. The bovine blood as the model material was separated into two major parts; namely, serum and blood cells by centrifugation. Erythrocytes were found to be mostly stable up to 220 MPa pr...
Fast fluorometric enumeration of E. coli using passive chip
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Pediococcus pentosaceus Pep1 is a vacuum-packaged Turkish sausage isolate which produces a potentially novel bacteriocin of the pediocin (anti-Listeria) family of peptides designated as pediocin P. Curing experiments and plasmid profile analysis indicated that both bacteriocin immunity and production determinants were linked and encoded by 9.0 MDa plasmid, pHD1.0. Attempts to transform purified plasmid pHD1.0 into recipient Escherichia coli JM109 cells by electroporation were successful but none of the E. c...
Citation Formats
K. Gokduman, M. D. AVŞAROĞLU ERKAN, A. Cakiris, D. Ustek, and G. C. Gültekin, “Recombinant plasmid-based quantitative Real-Time PCR analysis of Salmonella enterica serotypes and its application to milk samples,” JOURNAL OF MICROBIOLOGICAL METHODS, pp. 50–58, 2016, Accessed: 00, 2020. [Online]. Available: