A duplex droplet digital PCR assay for Salmonella and Shigella and its application in diarrheal and non-diarrheal samples

Abstract

Objectives: To evaluate a duplex droplet digital polymerase chain reaction (ddPCR) assay targeting Salmonella fimY and Shigella ipaH genes. Methods: The linear range, precision, analytical sensitivity, and analytical specificity of the ddPCR assay were analyzed. The ddPCR assay was compared with quantitative real-time PCR (qPCR) using 362 stool samples from 187 children with mild diarrhea and 175 children without diarrhea. Results: The duplex ddPCR assay showed good linearity in the range of 5.3 × 100 to 1.24 × 105 copies/reaction for Salmonella and 1.9 × 100 to 1.84 × 105 copies/reaction for Shigella. When analyzed with spiked stool samples, the limit of detection and limit of quantification were 550 and 5500 colony-forming units per mL of stool sample for Shigella, respectively, whereas both were 1.0 × 104 colony-forming units per mL of stool sample for Salmonella. Among 362 stool samples, more samples were detected as positive by ddPCR than by qPCR. Salmonella load was significantly higher in diarrheal samples than in non-diarrheal samples. Determined by receiver-operating characteristic analysis, the optimal cut-off value was 1.25 × 104 copies/mL of stool sample to distinguish between symptomatic and asymptomatic Salmonella infections. Conclusion: Salmonella and Shigella prevalence may have been underestimated in the past.

Keywords

• Salmonella
• Shigella
• Droplet digital PCR
• Real-time PCR
• Children with diarrhea
• Children without diarrhea