An Assay Combining Droplet Digital PCR With Propidium Monoazide Treatment for the Accurate Detection of Live Cells of Vibrio vulnificus in Plasma Samples

Abstract

Vibrio vulnificus (V. vulnificus) is one of the most common pathogenic Vibrio species to humans; therefore, the establishment of timely and credible detection methods has become an urgent requirement for V. vulnificus illness surveillance. In this study, an assay combining droplet digital PCR (ddPCR) with propidium monoazide (PMA) treatment was developed for detecting V. vulnificus. The primers/probes targeting the V. vulnificus hemolysin A (vvhA) gene, amplification procedures, and PMA processing conditions involved in the assay were optimized. Then, we analyzed the specificity, sensitivity, and ability to detect live cell DNA while testing the performance of PMA-ddPCR in clinical samples. The optimal concentrations of primers and probes were 1.0 and 0.3 μM, respectively. The annealing temperature achieving the highest accuracy in ddPCR assay was 60°C. With an initial V. vulnificus cell concentration of 108 CFU/mL (colony-forming units per milliliter), the optimal strategy to distinguish live cells from dead cells was to treat samples with 100 μM PMA for 15 min in the dark and expose them to LED light with an output wavelength of 465 nm for 10 min. The specificity of the PMA-ddPCR assay was tested on 27 strains, including seven V. vulnificus strains and 20 other bacterial strains. Only the seven V. vulnificus strains were observed with positive signals in specificity analysis. Comparative experiments on the detection ability of PMA-ddPCR and PMA-qPCR in pure cultures and plasma samples were performed. The limit of detection (LOD) and the limit of quantitation (LOQ) in pure culture solutions of V. vulnificus were 29.33 and 53.64 CFU/mL in PMA-ddPCR, respectively. For artificially clinical sample tests in PMA-ddPCR, V. vulnificus could be detected at concentrations as low as 65.20 CFU/mL. The sensitivity of the PMA-ddPCR assay was 15- to 40-fold more sensitive than the PMA-qPCR in this study. The PMA-ddPCR assay we developed provides a new insight to accurately detect live cells of V. vulnificus in clinical samples, which is of great significance to enhance public health safety and security capability and improve the emergency response level for V. vulnificus infection.

Evaluation of the diagnostic performance of an immunochromatographic test for Chlamydia trachomatis

“…In addition, reagents E, F, and G failed to detect positive results in clinical samples. A similar situation was reported by a study that evaluated the diagnostic performance of an assay combining droplet digital PCR with propidium monoazide treatment for Vibrio vulnificus and showed that the LOD in simulated clinical samples was 2.2-fold that in pure culture samples [ 13 ]. Since the dead microbial DNA also remained in the clinical samples for a long time, routine PCR assays cannot distinguish the target DNA coming from live or dead bacteria in the actual samples, which results in an overestimation of the concentration of pathogens.…”

A Novel RAA Combined Test Strip Method Based on Dual Gene Targets for Pathogenic Vibrio vulnificus in Aquatic Products

“…In addition, the sensitive detection of V. vulnificus in oysters was realized by RAA-TS-DTL. As shown in Table 5, the classical qPCR, multiple PCR and droplet digital PCR (ddPCR) methods for V. vulnificus had LODs ranging from 10 to 100 CFU/mL [8,22,23]. The RAA-TS-DTL developed on the basis of the gyrB gene could detect V. vulnificus in oyster at levels as low as 6 CFU/mL, which was 15 times more sensitive than that of the qPCR method established by D'Souza et al (100 CFU/mL) [8].…”

Public health aspects of Vibrio spp. related to the consumption of seafood in the EU

“…Loop‐mediated isothermal amplification (LAMP) assays have also been developed and successfully applied to detect V. parahaemolyticus and V. vulnificus from environmental water and aquatic products (Liu et al., 2017 ; Tian et al., 2022 ). Recent FBO investigations involving digital droplet PCR (Hu et al., 2022 ; Zhao et al., 2022 ; Zhou et al., 2023 ) or combining next generation sequencing (NGS) and digital PCR (Li et al., 2019 ) showed that these methods provide new fast tools for accurate detection and quantification of pathogenic Vibrio species. Finally, viability PCR showed to be a promising approach to overcome the underestimation of microorganisms in the VBNC state by culture‐based methods, and different assays have been developed to detect and quantify VBNC V. parahaemolyticus and V. cholerae , although, to date, their application has been mostly on experimentally contaminated seafood (Liu et al., 2018 ; Wu et al., 2015 ).…”