A multiplex PCR/LDR assay for simultaneous detection and identification of the NIAID category B bacterial food and water-borne pathogens

Abstract

Enteric pathogens that cause gastroenteritis remain a major global health concern. The goal of this study was to develop a multiplex PCR/ligation detection reaction (LDR) assay for the detection of all NIAID category B bacterial food and water-borne pathogens directly from stool specimens. To validate the PCR/LDR assay, clinical isolates of Campylobacter spp., Vibrio spp., Shigella spp., Salmonella spp., Listeria monocytogenes, Yersinia enterocolitica, and diarrheagenic Escherichia coli were tested. The sensitivity and specificity of the assay were assessed using a large number of seeded culture-negative stool specimens and a smaller set of clinical specimens from Haiti. The overall sensitivity ranged from 91% to 100% (median 100%) depending on the species. For the majority of organisms, the sensitivity was 100%. The overall specificity based on initial testing ranged from 98% to 100% depending on the species. After additional testing of discordant samples, the lowest specificity was 99.4%. PCR/LDR detected additional category B agents (particularly diarrheagenic E. coli) in 11/40 specimens from Haiti that were culture-positive for V. cholerae and in approximately 1% of routine culture-negative stool specimens from a hospital in New York. This study demonstrated the ability of the PCR/LDR assay to detect a large comprehensive panel of category B enteric bacterial pathogens as well as mixed infections. This type of assay has the potential to provide earlier warnings of possible public health threats and more accurate surveillance of food and water-borne pathogens.

Keywords: Enteric pathogens; Infectious disease; Molecular diagnostics; Multiplex.

Figure 1

Schematic of the PCR/LDR assay with molecular readout using the BeadXpress and Veracode platform. Gene-specific PCR primer pairs were designed to amplify their distinct genetic targets if present. Each PCR amplicon ranged in size between 400 and 600 base pairs. Within each PCR amplicon, we then designed LDR primer pairs to identify single base variants at multiple locations to allow us to distinguish between different pathogenic bacteria. At any given single base variant, the allele specific upstream LDR primers are designed to ligate to the downstream primer only if there is a perfect match at the junction point. The upstream LDR primers bear zipcode complement sequences and amino blocking groups on the 5′-end, while the downstream LDR primers have either a Cy3 or Cy5 fluorescent label. Ligation of the LDR primers results in fluorescently labeled products that are subsequently hybridized to zipcode addresses attached to veracode beads. Each bead contains a unique barcode that can be scanned by the BeadXpress reader. Detection of the bead and fluorescent signal scores for the presence of the microbial pathogen.