Comparison of real-time PCR methods for detection of Salmonella enterica and Escherichia coli O157:H7, and introduction of a general internal amplification control.
The objectives of this study were to compare different real-time PCR-based methods for detection of either Salmonella spp. or E. coli O157:H7 with respect to sensitivity, precision and accuracy. In addition, a general internal amplification control (IAC) is presented, allowing prevention of false negative results. The IAC allows insight in amplification efficiency and enables a more accurate quantification with the evaluated real-time PCR methods. Implementation of the IAC with the different PCR methods did not affect the precision of the methods, but the sensitivity was reduced 10-fold. Introduction of an IAC with the Salmonella enterica specific detection method showed a shift in Ct-value (increase of target Ct-value with 0.45+/-0.17 cycles), while with the method to detect E. coli O157:H7 no influence of IAC co-amplification was observed. The quantification threshold of the methods in which the IAC was included was determined at 1 pg of target DNA (equal to 200 CFU) per reaction. Qualitative detection was feasible down to 10 fg of target DNA per reaction using both methods in which the IAC was incorporated. The adjusted methods have the potential to provide fast and sensitive detection of Salmonella spp. or E. coli O157:H7, enabling accurate quantification and preventing false negative results by using the general IAC.
Wageningen University and Research Centre, Plant Research International BV, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands. Michel.Klerks@wur.nl
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D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D019453:Escherichia coli O157 / Q000235:genetics / Q000302:isolation & purification*; D005188:False Negative Reactions; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D019779:Salmonella enterica / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D019914:Taq Polymerase / Q000378:metabolism
D004269:DNA, Bacterial; C485192:green fluorescent protein, Aequorea victoria; D049452:Green Fluorescent Proteins; D019914:Taq Polymerase