Improved Sensitivity of Quantitative Polymerase Chain Reaction and Next Generation Sequencing for Detection of...

Detecting pathogens in environmental samples using molecular-based technologies can be challenging, particularly in low biomass environments or where pathogens represent a low percentage of the community. Multiple displacement amplification (MDA) is a whole genome amplification (WGA) method that has been developed for low biomass samples. However, there is a lack of information on how MDA could improve PCR and sequence-based detection and genomic characterization of pathogens in challenging environmental samples. In this study, MDA was evaluated on low template samples of the Salmonella LT2 isolate, a foodborne and waterborne environmental pathogen. MDA was also evaluated on a variety of low template mixed-microbial mock, environmental communities containing a range of Salmonella genome percentages to simulate different levels of Salmonella in the environment. Using MDA starting inputs of 1.8 × 10⁴-1.8 × 10¹ Salmonella LT2 genome copies, > 99% of the Salmonella genome was recovered following MDA at > 16X depth of coverage from as few as 500,000 merged, 250 bp paired-end reads. For the mock microbial communities, moderately high levels of genome abundance distortion were evident following MDA across all communities when compared to the expected compositions, which could not be attributed to either genome size or GC content alone. Overall, MDA may provide a useful method for increasing Salmonella detection sensitivity in low target environmental samples where downstream selective targeted applications such as real-time PCR or targeted amplicon sequencing are used, but MDA may not be appropriate for identification and detection of Salmonella when using untargeted, metagenomic sequencing.