Evaluation of the performance of quantitative detection of the Listeria monocytogenes prfA locus with droplet digital PCR

Abstract

Fast and reliable pathogen detection is an important issue for human health. Since conventional microbiological methods are rather slow, there is growing interest in detection and quantification using molecular methods. The droplet digital polymerase chain reaction (ddPCR) is a relatively new PCR method for absolute and accurate quantification without external standards. Using the Listeria monocytogenes specific prfA assay, we focused on the questions of whether the assay was directly transferable to ddPCR and whether ddPCR was suitable for samples derived from heterogeneous matrices, such as foodstuffs that often included inhibitors and a non-target bacterial background flora. Although the prfA assay showed suboptimal cluster formation, use of ddPCR for quantification of L. monocytogenes from pure bacterial cultures, artificially contaminated cheese, and naturally contaminated foodstuff was satisfactory over a relatively broad dynamic range. Moreover, results demonstrated the outstanding detection limit of one copy. However, while poorer DNA quality, such as resulting from longer storage, can impair ddPCR, internal amplification control (IAC) of prfA by ddPCR, that is integrated in the genome of L. monocytogenes ΔprfA, showed even slightly better quantification over a broader dynamic range. Graphical AbstractEvaluating the absolute quantification potential of ddPCR targeting Listeria monocytogenes prfA Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-016-9861-9) contains supplementary material, which is available to authorized users.

A Systematic Investigation of Parameters Influencing Droplet Rain in the Listeria monocytogenes prfA Assay - Reduction of Ambiguous Results in ddPCR

“…In the case of the prfA assay, we showed that direct (one-to-one) transfer resulted in strongly biased Ct values when using the chemistry (mastermix) required for ddPCR [16]. This bias was not as pronounced in the Δ prfA assay.…”

“…However, when only one or two droplets are positive, results can be interpreted as either false positive or classified as negative, because in negative controls up to three droplets are regularly found with intermediate (and high) fluorescence [2,16,19]. Thus, improved cluster separation reduces the number of ambiguous samples that must be repeated for confirmation and improved quantification.…”

“…Slower ramp rate is recommended for ddPCR and was previously shown to be important for ddPCR [16], but it cannot be changed when using the gradient program. Thus, the ramp rate is probably responsible for the stronger rain in this experiment.…”

“…In a previous study, we showed that quantification with the Bio-Rad ddPCR system was sufficient precise, despite suboptimal cluster formation [16]. However, samples with one single droplet with intermediate fluorescence could not be clearly defined.…”

Comparison of next-generation droplet digital PCR with quantitative PCR for enumeration of Naegleria fowleri in environmental water and clinical samples

“…Because of a substantially higher ratio between target DNA to PCR reagents than in qPCR, it is less affected by PCR inhibitors and therefore, more robust against inhibition , is more sensitive and accurate than qPCR (Sanders et al 2013;Yang et al 2014;Cao et al 2015). In the light of these potential advantages, ddPCR has already shown its potential utility in the accurate quantitation of pathogens in clinical microbiology laboratories (Whale et al 2012;Verhaegen et al 2016;Witte et al 2016;Zhao et al 2016). Accurate detecting of N. fowleri in drinking and surface waters has been an important issue for water utilities because of its critical impact on human health.…”