Development and evaluation of a multiplex droplet digital polymerase chain reaction method for simultaneous detection of five biothreat pathogens

Biothreat agents pose a huge threat to human and public health, necessitating the development of rapid and highly sensitive detection approaches. This study establishes a multiplex droplet digital polymerase chain reaction (ddPCR) method for simultaneously detecting five high-risk bacterial biothreats: Yersinia pestis, Bacillus anthracis, Brucella spp., Burkholderia pseudomallei, and Francisella tularensis. Unlike conventional multiplex real-time PCR (qPCR) methods, the multiplex ddPCR assay was developed using two types of probe fluorophores, allowing the assay to perform with a common two-color ddPCR system. After optimization, the assay performance was evaluated, showing a lower limit of detection (LOD) (0.1-1.0 pg/mu L) and good selectivity for the five bacteria targets. The multiplex assay's ability to simultaneously detect two or more kinds of targets in a sample was also demonstrated. The assay showed strong sample tolerance when testing simulated soil samples; the LOD for bacteria in soil was 2 x 10(2)-2 x 10(3) colony-forming unit (CFU)/100 mg soil (around 5-50 CFU/reaction), which was 10-fold lower than that of the single-target qPCR method. When testing simulated soil samples at bacterial concentrations of 2 x 10(3)-2 x 10(4) CFU/100 mg soil, the assay presented a higher sensitivity (100%, 35/35) than that of the qPCR method (65.71%, 23/35) and a good specificity (100%, 15/15). These results suggest that the developed 5-plex ddPCR method is more sensitive than conventional qPCR methods and is potentially suitable for rapidly detecting or screening the five selected bacterial biothreats in suspicious samples.

Automated summary

Biothreat agents pose a huge threat to human and public health. This study establishes a multiplex droplet digital polymerase chain reaction (ddPCR) method for simultaneously detecting five high-risk bacterial biothreats. The developed ddPCR method is more sensitive than conventional methods and is potentially suitable for rapidly detecting or screening the selected bacterial biothreats in suspicious samples.