Comparison of droplet digital PCR and quantitative PCR for the detection of Salmonella and its application for river sediments Gulshan Singh, Ayanda Sithebe, Abimbola M. Enitan, Sheena Kumari, Faizal Bux and Thor Axel Stenström ABSTRACT Despite advances in microbial detection that quantitative polymerase chain reaction (qPCR) has led to, complex environmental samples, such as sediments, remain a challenge due to presence of PCR inhibitors. Aquatic sediments accumulate particle-bound microbial contaminants and thereby reflect a cumulative microbial load over time. The relatively new droplet digital PCR (ddPCR) has emerged as a direct quantitative method, highly tolerant to PCR inhibitors and relinquishing the necessity for calibration/standard curves. Information is virtually absent where ddPCR has been applied to detect pathogenic organisms in aquatic sediments. This study compared the efficacy of ddPCR with qPCR, for quantification of Salmonella in sediments from the Palmiet River near an informal settlement in Durban, South Africa. ddPCR significantly improved both analytical sensitivity and detection of low concentrations of Salmonella as compared to qPCR. The expected copy numbers measured from both qPCR and ddPCR showed good R 2 values (0.999 and 0.994, respectively). The site mostly affected by the informal settlements exhibited Salmonella in the range of 255 ± 37 and 818 ± 30 Salmonella/g ( p  0.0001) in qPCR and ddPCR, respectively. The improved detection of Salmonella in sediments with ddPCR makes it a promising technical method for the quantification of Salmonella in multifarious environmental samples. Gulshan Singh (corresponding author) Ayanda Sithebe Abimbola M. Enitan Sheena Kumari Faizal Bux Thor Axel Stenström SARChI Chair, Institute for Water and Wastewater Technology (IWWT), Durban University of Technology, P.O. Box 1334, Durban 4000, South Africa E-mail: GulshanS@dut.ac.za Key words | ddPCR, qPCR, Salmonella, sediments INTRODUCTION Pathogenic bacteria can survive longer in aquatic sediments than in the overlying water column (Luna et al. ; Vignar- oli et al. ) and will represent the particle-associated fraction, accumulating over time. The occurrence and quantification of human pathogenic bacteria in environ- mental regimes, like surface water or bottom sediments, still to a large extent rely on quantification on selective media or enrichment. The direct quantification of specific target genes, representing the pathogen in question with quantitative polymerase chain reaction (qPCR) has, how- ever, progressively been accepted as the gold standard and been applied for the detection and quantification of patho- gens in water environmental samples (Li & Chen ; Singh et al. ). In general, qPCR has several advantages as compared to classical bacteriological cultivation methods and identification schemes, in terms of speed, detection limit, potential for automation, and cost. The application of qPCR in sediment samples is a chal- lenge mainly due to the presence of PCR inhibitory substances. Even a small quantity of PCR inhibitors can delay the C q (threshold cycles) of complex samples in qPCR, causing erroneously low estimates of the template 505 © IWA Publishing 2017 Journal of Water and Health | 15.4 | 2017 doi: 10.2166/wh.2017.259 Downloaded from https://iwaponline.com/jwh/article-pdf/15/4/505/393309/jwh0150505.pdf by guest on 13 June 2020