Multiplex PCR coupled with direct amplicon sequencing for simultaneous detection of numerous waterborne pathogens

The current water quality monitoring and regulation approaches use fecal indicator bacteria (FIB) to indirectly assess health risks from fecal pathogens. Direct detection of waterborne pathogens is expected to provide more accurate and comprehensive risk assessment, which however has been hindered by the lack of methods for simultaneous detection of the numerous waterborne pathogens. This study aimed to develop a mPCR-NGS approach that uses the high sequencing depth of NGS and sequence-based detection to significantly increase the multiplex level of mPCR for direct pathogen detection in water. Individual PCR primers were designed for 16 target marker genes of nine different bacterial pathogens, and an optimal combination of primers with least primer complementarities was identified for the multiplex setting. Using an artificial tester sample, the mPCR system was optimized for annealing temperature and primer concentration, and bioinformatic procedures were developed to directly detect the target marker gene amplicons in NGS sequence reads, which showed simultaneous detection of 14 different target genes in one reaction. The effectiveness of the developed mPCR-NGS approach was subsequently demonstrated on DNA extracts from stream water samples and their counterparts that were spiked with various target pathogen DNA, and all target genes spiked into the environmental water samples were successfully detected. Several key issues for further improving the mPCR-NGS approach were also identified and discussed.