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

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 x 10 0 to 1.24 x 10 5 copies/reaction for Salmonella and 1.9 x 10 0 to 1.84 x 10 5 copies/reaction for Shigella . When analyzed with spiked stool samples, the limit of detection and limit of quantification were 550 and 5500 colonyforming units per mL of stool sample for Shigella , respectively, whereas both were 1.0 x 10 4 colonyforming 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 cutoff value was 1.25 x 10 4 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.(c) 2022 The Author(s). Published by Elsevier Ltd on behalf of International Society for Infectious Diseases.This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ )

Automated summary

This study evaluated a duplex droplet digital polymerase chain reaction (ddPCR) assay for detecting Salmonella fimY and Shigella ipaH genes. The ddPCR assay showed good linearity and high sensitivity and quantification accuracy for detecting Salmonella and Shigella in stool samples. Compared to quantitative real-time PCR (qPCR), ddPCR detected more positive samples. Salmonella load was higher in diarrheal samples compared to non-diarrheal samples. An optimal cutoff value was determined to distinguish between symptomatic and asymptomatic Salmonella infections.