Selective PCR: a novel internal amplification control strategy for enhanced sensitivity in Salmonella diagnosis

Abstract

Aims: The aim of this study was to develop a novel strategy that permits the independent amplification of internal amplification control (IAC) and target sequence using the same set of primers, to improve the sensitivity of diagnostic PCR assays. Methods and Results: The method described here is a Salmonella specific PCR test targeting the quorum sensing gene sdiA. It is based on a large size difference between the IAC and the target and consequently on their different extension time. The results indicate the enhanced sensitivity of this test when compared with the competitive IAC strategy. This is demonstrated through parallel testing of artificially contaminated human faecal samples. Conclusions: Utilizing this method, the concentration of the IAC, which often leads to false negative results if the target is present in extremely low concentration owing to competition, does not constitute a critical parameter for the detection limit of a PCR assay. Significance and Impact of the Study: To our knowledge, this is the first report of using extension time as a critical parameter for the sensitivity of a PCR test. A different approach for the construction of an IAC, based on inverse PCR, has also been introduced.

Detection of Live Salmonella sp. Cells in Produce by a TaqMan-Based Quantitative Reverse Transcriptase Real-Time PCR Targeting invA mRNA

“…Numerous molecular assays have been developed for the detection of Salmonella in different matrices (8,10,23,28,32). Some of these are based on conventional PCR, and some others are based on qPCR technology, whether using SYBR green I or molecular probes (e.g., TaqMan).…”

Rapid and Sensitive Detection of Staphylococcus aureus in Food Using Selective Enrichment and Real-Time PCR Targeting a New Gene Marker

“…Nevertheless, the use of the internal amplification control in each reaction is considered necessary for diagnostic purposes (RodriguezLazaro et al 2004;Oikonomou et al 2008) to eliminate of false-negative results.…”

Development of a recombinase polymerase amplification assay forVibrio parahaemolyticusdetection with an internal amplification control

“…However, a competitive IAC can co-amplify simultaneously with the target sequence, and hence effectively indicate false-negative results. Moreover, by using the competitive strategy, both the target sequence and the IAC are amplified with one common set of primers under the same conditions, which eliminates the risk of undesired interactions among multiple primers and which may not become sub-efficient for one or both reactions after optimization (Oikonomou et al 2008). In this study, the target gene and the IAC were easily differentiated by agarose gel electrophoresis due to differences in RPA product D r a f t sizes.…”