Dataset Information

Selective detection of viable seed-borne Acidovorax citrulli by real-time PCR with propidium monoazide.

ABSTRACT: In recent years, use of the DNA-intercalating dye propidium monoazide (PMA) in real-time PCR has been reported as a novel method to detect viable bacteria in different types of samples, such as food, environmental, and microbiological samples. In this study, viable cells of Acidovorax citrulli, the causal agent of bacterial seedling blight and fruit blotch, were selectively detected and differentiated from dead cells by real-time fluorescent polymerase chain reaction amplification after the bacterial solution was treated with the DNA-binding dye PMA. The primers and TaqMan probe were based on the A. citrulli genome (Aave_1909, Gene ID: 4669443) and were highly specific for A. citrulli. The detection threshold of this assay was 10³ colony-forming units per mL (CFU/mL) in pure cell suspensions containing viable and dead cells and infected watermelon seeds. Application of this assay enables the selective detection of viable cells of A. citrulli and facilitates monitoring of the pathogen in watermelon and melon seeds.

SUBMITTER: Tian Q

PROVIDER: S-EPMC5064318 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Selective detection of viable seed-borne Acidovorax citrulli by real-time PCR with propidium monoazide.

Tian Qian Q Feng Jian-Jun JJ Hu Jie J Zhao Wen-Jun WJ

Scientific reports 20161014

In recent years, use of the DNA-intercalating dye propidium monoazide (PMA) in real-time PCR has been reported as a novel method to detect viable bacteria in different types of samples, such as food, environmental, and microbiological samples. In this study, viable cells of Acidovorax citrulli, the causal agent of bacterial seedling blight and fruit blotch, were selectively detected and differentiated from dead cells by real-time fluorescent polymerase chain reaction amplification after the bact ...[more]

PMID: 27739469

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Project description:IntroductionToxigenic Vibrio cholerae serogroup O1 and O139 are the pathogens responsible for the global cholera epidemic. V. cholerae can settle in the water and spread via the fecal-oral route. Rapid and accurate monitoring of live V. cholerae in environmental water has become an important strategy to prevent and control cholera transmission. Conventional plate counting is widely used to detect viable bacteria but requires time and effort.MethodsThis study aims to develop a new assay that combines triplex droplet digital PCR (ddPCR) with propidium monoazide (PMA) treatment for quantitatively detecting live V. cholerae O1/O139 and cholera enterotoxin. Specific primers and probes were designed according to the conserved regions of gene rfb O1, rfb O139, and ctxA. The amplification procedures and PMA treatment conditions were optimized. The specificity, sensitivity, and ability of PMA-ddPCR to detect viable bacteria-derived DNA were evaluated in simulated seawater samples.Results and discussionThe results revealed that the optimal primer concentrations of rfb O1, rfb O139, and ctxA were 1 μM, while the concentrations of the three probes were 0.25, 0.25, and 0.4 μM, respectively. The best annealing temperature was 58°C to obtain the most accurate results. The optimal strategy for distinguishing dead and live bacteria from PMA treatment was incubation at the concentration of 20 μM for 15 min, followed by exposure to a 650-W halogen lamp for 20 min. In pure culture solutions, the limit of detection (LODs) of V. cholerae O1 and O139, and ctxA were 127.91, 120.23 CFU/mL, and 1.5 copies/reaction in PMA-triplex ddPCR, respectively, while the LODs of the three targets were 150.66, 147.57 CFU/mL, and 2 copies/reaction in seawater samples. The PMA-ddPCR sensitivity was about 10 times higher than that of PMA-qPCR. When detecting spiked seawater samples with live bacterial concentrations of 1.53 × 102 and 1.53 × 105 CFU/mL, the assay presented a higher sensitivity (100%, 16/16) than qPCR (50.00%, 8/16) and a perfect specificity (100%, 9/9). These results indicate that the developed PMA-triplex ddPCR is superior to the qPCR regarding sensitivity and specificity and can be used to rapidly detect viable toxigenic V. cholerae O1 and O139 in suspicious seawater samples.

Dataset Information

Selective detection of viable seed-borne Acidovorax citrulli by real-time PCR with propidium monoazide.

Publications

Selective detection of viable seed-borne Acidovorax citrulli by real-time PCR with propidium monoazide.

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