scholarly journals Quantification of Viable Cells of Pseudomonas syringae pv. tomato in Tomato Seed Using Propidium Monoazide and a Real-Time PCR Assay

Plant Disease ◽  
2020 ◽  
Vol 104 (8) ◽  
pp. 2225-2232
Author(s):  
A-li Chai ◽  
Hai-yan Ben ◽  
Wei-tao Guo ◽  
Yan-xia Shi ◽  
Xue-wen Xie ◽  
...  
Keyword(s):  
Real Time ◽  
Pseudomonas Syringae ◽  
Real Time Pcr ◽  
Light Exposure ◽  
Qpcr Assay ◽  
Bacterial Suspension ◽  
Propidium Monoazide ◽  
Chromosomal Dna ◽  
Tomato Seed ◽  
Viable Cells

Pseudomonas syringae pv. tomato is a seedborne pathogen that causes bacterial speck disease in tomato. P. syringae pv. tomato is typically detected in tomato seed using quantitative real-time PCR (qPCR) but the inability of qPCR to distinguish between viable and nonviable cells might lead to an overestimation of viable P. syringae pv. tomato cells. In the present study, a strategy involving a propidium monoazide (PMA) pretreatment followed by a qPCR (PMA-qPCR) assay was developed for quantifying viable P. syringae pv. tomato cells in contaminated tomato seed. PMA could selectively bind to the chromosomal DNA of dead bacterial cells and, therefore, block DNA amplification of qPCR. The primer pair Pst3F/Pst3R was designed based on gene hrpZ to specifically amplify and quantify P. syringae pv. tomato by qPCR. The PMA pretreatment protocol was optimized for selectively detecting viable P. syringae pv. tomato cells, and the optimal PMA concentration and light exposure time were 10 μmol liter−1 and 10 min, respectively. In the sensitivity test, the detection limit of PMA-qPCR for detecting viable cells in bacterial suspension and artificially contaminated tomato seed was 102 CFU ml−1 and 11.86 CFU g−1, respectively. For naturally contaminated tomato seed, viable P. syringae pv. tomato cells were quantified in 6 of the 19 samples, with infestation levels of approximately 102 to 104 CFU g−1. The results indicated that the PMA-qPCR assay is a suitable tool for quantifying viable P. syringae pv. tomato cells in tomato seed, which could be useful for avoiding the potential risks of primary inoculum sources from contaminated seed.

Enumeration of viable Escherichia coli by real-time PCR with propidium monoazide

2012 ◽  
Vol 66 (10) ◽  
pp. 2065-2073 ◽  
Author(s):  
N. Yokomachi ◽  
J. Yaguchi
Keyword(s):  
Escherichia Coli ◽  
Wastewater Treatment ◽  
Real Time ◽  
Real Time Pcr ◽  
Wastewater Treatment Plants ◽  
Propidium Monoazide ◽  
E Coli ◽  
Cell Counts ◽  
Viable Cells ◽  
Heat Treated

A photo-inducible DNA-binding dye, propidium monoazide (PMA), was used to distinguish viable and dead Escherichia coli cells. Microscopic observations using a combination of the dyes 4′,6-diamidino-2-phenylindole and PMA indicated that PMA stained only dead cells, with membrane damage, red. Mixtures of viable and heat-treated E. coli cells were subjected to real-time polymerase chain reaction (PCR) with PMA treatment. Viable cell counts were linearly related to real-time PCR threshold cycle values for PMA-treated cells in the mixtures of viable and heat-treated cells, as long as the ratio of dead cells to viable cells was no greater than 10. In the wastewater treatment plants, total, viable and culturable E. coli were enumerated by real-time PCR, real-time PCR coupled with PMA treatment and the most probable number method using EC-MUG medium, respectively. The concentrations of viable E. coli in the wastewater treatment plants were much higher than those of culturable cells. In addition, viable cells were even more chlorine resistant than culturable ones.

Application of real-time polymerase chain reaction (PCR) coupled with ethidium monoazide treatment for selective quantification of viable bacteria in aquatic environment

2008 ◽  
Vol 58 (5) ◽  
pp. 1107-1112 ◽  
Author(s):  
D. Inoue ◽  
H. Tsutsui ◽  
Y. Yamazaki ◽  
K. Sei ◽  
S. Soda ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Aquatic Environment ◽  
Pathogenic Bacteria ◽  
Light Exposure ◽  
Coli Strain ◽  
Ethidium Monoazide ◽  
Viable Cells ◽  
Halogen Light ◽  
Viable Population

Ethidium monoazide (EMA) was used to quantify DNA selectively from viable cells with healthy membrane/cell wall system, but not from dead cells, of a target bacterium in the aquatic environment using real-time PCR. Spiking experiments to determine the EMA treatment conditions showed that EMA treatment with EMA at 10–25 μg/ml and subsequent halogen light exposure for 2 min was suitable for selective quantification of DNA from viable cells in an aquatic sample using real-time PCR coupled with EMA treatment (real-time EMA-PCR). Optimized real-time EMA-PCR was applied in combination with culture-based method and conventional real-time PCR without EMA treatment to elucidate the behavior of an Escherichia coli strain inoculated into a pond water microcosm. Quantification results obtained using real-time EMA-PCR were lower than those by conventional real-time PCR without EMA treatment and higher than those by culture-based method. The results suggest that quantification by real-time EMA-PCR seemed to represent the viable population, which would partly include viable but non-culturable state bacteria. Real-time EMA-PCR optimized here can be a useful tool for selective monitoring of the viable population of a target bacterium in the aquatic environment, and thereby contribute to assessment of potential microbial risks generated from waterborne pathogenic bacteria.

Evaluation of droplet digital PCR for the detection of black canker disease in tomato

Plant Disease ◽  
2021 ◽  
Author(s):  
Li Wang ◽  
Tian Qian ◽  
Pei Zhou ◽  
Wenjun Zhao ◽  
Xianchao Sun
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Plant Pathogens ◽  
Plant Protection ◽  
Detection Threshold ◽  
Digital Pcr ◽  
Taqman Probe ◽  
Tomato Seed ◽  
Canker Disease ◽  
Probe Set

Clavibacter michiganensis subsp. michiganensis (Cmm), the cause of bacterial canker disease, is one of the most destructive pathogens in greenhouse and field tomato. The pathogen is now present in all main production areas of tomato and is quite widely distributed in the EPPO（European and Mediterranean Plant Protection Organization）region. The inspection and quarantine of the plant pathogens relies heavily on accurate detection tools. Primers and probes reported in previous studies do not distinguish the Cmm pathogen from other closely related subspecies of C. michiganensis, especially the non-pathogenic subspecies that were identified from tomato seeds recently. Here, we have developed a droplet digital polymerase chain reaction (ddPCR) method for the identification of this specific bacterium with primers/TaqMan probe set designed based on the pat-1 gene of Cmm. This new primers/probe set has been evaluated by qPCRthe real time PCR（qPCR） and ddPCR. The detection results suggest that the ddPCR method established in this study was highly specific for the target strains. The result showed the positive amplification for all 5 Cmm strains，and no amplification was observed for the other 43 tested bacteria, including the closely related C. michiganensis strains. The detection threshold of ddPCR was 10.8 CFU/mL for both pure Cmm cell suspensions and infected tomato seed, which was 100 times-fold more sensitive than that of the real-time PCR (qPCR ) performed using the same primers and probe. The data obtained suggest that our established ddPCR could detect Cmm even with low bacteria load, which could facilitate both Cmm inspection for pathogen quarantine and the routine pathogen detection for disease control of black canker in tomato.

scholarly journals Distinction between intact and antibiotic-inactivated bacteria by real-time PCR after treatment with propidium monoazide

2010 ◽  
Vol 28 (9) ◽  
pp. 1245-1251 ◽  
Author(s):  
Hideo Kobayashi ◽  
Margret Oethinger ◽  
Marion J. Tuohy ◽  
Gerri S. Hall ◽  
Thomas W. Bauer
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Propidium Monoazide

scholarly journals Improved HF183 Quantitative Real-Time PCR Assay for Characterization of Human Fecal Pollution in Ambient Surface Water Samples

2014 ◽  
Vol 80 (10) ◽  
pp. 3086-3094 ◽  
Author(s):  
Hyatt C. Green ◽  
Richard A. Haugland ◽  
Manju Varma ◽  
Hana T. Millen ◽  
Mark A. Borchardt ◽  
...  
Keyword(s):  
Real Time ◽  
Surface Waters ◽  
Real Time Pcr ◽  
Fecal Pollution ◽  
Qpcr Assay ◽  
Taqman Assay ◽  
Reference Database ◽  
Quantitative Real Time Pcr ◽  
Ambient Surface

ABSTRACTQuantitative real-time PCR (qPCR) assays that target the human-associated HF183 bacterial cluster within members of the genusBacteroidesare among the most widely used methods for the characterization of human fecal pollution in ambient surface waters. In this study, we show that a current TaqMan HF183 qPCR assay (HF183/BFDrev) routinely forms nonspecific amplification products and introduce a modified TaqMan assay (HF183/BacR287) that alleviates this problem. The performance of each qPCR assay was compared in head-to-head experiments investigating limits of detection, analytical precision, predicted hybridization to 16S rRNA gene sequences from a reference database, and relative marker concentrations in fecal and sewage samples. The performance of the modified HF183/BacR287 assay is equal to or improves upon that of the original HF183/BFDrev assay. In addition, a qPCR chemistry designed to combat amplification inhibition and a multiplexed internal amplification control are included. In light of the expanding use of PCR-based methods that rely on the detection of extremely low concentrations of DNA template, such as qPCR and digital PCR, the new TaqMan HF183/BacR287 assay should provide more accurate estimations of human-derived fecal contaminants in ambient surface waters.

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