Dataset Information

An Improved Method for Quantification of Viable Fusarium Cells in Infected Soil Products by Propidium Monoazide Coupled with Real-Time PCR.

ABSTRACT: Fusarium is a soil-borne pathogen that causes root rot disease in cucumber. To date, quantitative real-time PCR (qPCR) is a common tool to detect the content of Fusarium in soil. However, qPCR cannot distinguish between viable and nonviable cells. The aim of this study was to develop a detection technique to pretreat tissue fluid with propidium monoazide (PMA) followed by extract DNA, and then to quantify viable Fusarium cells in contaminated soil. In this work, the specific primer pair F8-1/F8-2 was designed based on the translation elongation factor (EF) gene and a PMA-qPCR assay was established to amplify and quantify soils of viable Fusarium cells. The PMA pretreatment test was optimized, which indicated that the optimal PMA concentration and light exposure time were 50 mmol L^-1 and 15 min, respectively. The lowest limit of viable cells in suspension detected and soil by PMA-qPCR were 82 spore mL^-1 and 91.24 spore g^-1, respectively. For naturally contaminated soil, viable Fusarium cells were detected in eight of the 18 samples, and the Fusarium amount ranged from 10⁴ to 10⁶ spore g^-1. In conclusion, the PMA-qPCR method has the characteristics of high sensitivity, efficiency, and time saving, which could support nursery plants to avoid Fusarium infection and agro-industry losses.

SUBMITTER: Chen L

PROVIDER: S-EPMC9143521 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

An Improved Method for Quantification of Viable Fusarium Cells in Infected Soil Products by Propidium Monoazide Coupled with Real-Time PCR.

Chen Lida L Li Lei L Xie Xuewen X Chai Ali A Shi Yanxia Y Fan Tengfei T Xie Jianming J Li Baoju B

Microorganisms 20220517 5

Fusarium is a soil-borne pathogen that causes root rot disease in cucumber. To date, quantitative real-time PCR (qPCR) is a common tool to detect the content of Fusarium in soil. However, qPCR cannot distinguish between viable and nonviable cells. The aim of this study was to develop a detection technique to pretreat tissue fluid with propidium monoazide (PMA) followed by extract DNA, and then to quantify viable Fusarium cells in contaminated soil. In this work, the specific ...[more]

PMID: 35630479

Similar Datasets

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

An Improved Method for Quantification of Viable Fusarium Cells in Infected Soil Products by Propidium Monoazide Coupled with Real-Time PCR.

Publications

An Improved Method for Quantification of Viable <i>Fusarium</i> Cells in Infected Soil Products by Propidium Monoazide Coupled with Real-Time PCR.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets