scholarly journals Rapid and Accurate Detection of Bacteriophage Activity againstEscherichia coliO157:H7 by Propidium Monoazide Real-Time PCR

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Hui Liu ◽  
Yan D. Niu ◽  
Jinquan Li ◽  
Kim Stanford ◽  
Tim A. McAllister
Keyword(s):  
Cell Surface ◽  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Extracellular Dna ◽  
Bacterial Cells ◽  
Propidium Monoazide ◽  
Direct Plating ◽  
E Coli ◽  
Polymerase Chain ◽  
Phage Activity

Conventional methods to determine the efficacy of bacteriophage (phage) for biocontrol ofE. colirequire several days, due to the need to culture bacteria. Furthermore, cell surface-attached phage particles may lyse bacterial cells during experiments, leading to an overestimation of phage activity. DNA-based real-time quantitative polymerase chain reaction (qPCR) is a fast, sensitive, and highly specific means of enumerating pathogens. However, qPCR may underestimate phage activity due to its inability to distinguish viable from nonviable cells. In this study, we evaluated the suitability of propidium monoazide (PMA), a microbial membrane-impermeable dye that inhibits amplification of extracellular DNA and DNA within dead or membrane-compromised cells as a means of using qPCR to identify only intactE. colicells that survive phage exposure.Escherichia coliO157:H7 strain R508N and 4 phages (T5-like, T1-like, T4-like, and O1-like) were studied. Results compared PMA-qPCR and direct plating and confirmed that PMA could successfully inhibit amplification of DNA from compromised/damaged cellsE. coliO157:H7. Compared to PMA-qPCR, direct plating overestimated (P< 0.01) phage efficacy as cell surface-attached phage particles lysedE. coliO157:H7 during the plating process. Treatment of samples with PMA in combination with qPCR can therefore be considered beneficial when assessing the efficacy of bacteriophage for biocontrol ofE. coliO157:H7.

Use of real-time PCR with propidium monoazide for enumeration of viable Escherichia coli in anaerobic digestion

2016 ◽  
Vol 74 (5) ◽  
pp. 1243-1254 ◽  
Author(s):  
Wataru Ruike ◽  
Atsushi Higashimori ◽  
Junichi Yaguchi ◽  
Yu-you Li
Keyword(s):  
Escherichia Coli ◽  
Anaerobic Digestion ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Polymerase Chain Reaction ◽  
Fecal Coliforms ◽  
Most Probable Number ◽  
Propidium Monoazide ◽  
Probable Number ◽  
E Coli

A combination of propidium monoazide (PMA) with real-time quantitative polymerase chain reaction (PMA-qPCR) was optimized to enumerate only viable Escherichia coli in anaerobic digestion processes. Repeating the PMA treatment twice and a final concentration of 100 μM resulted in an effective exclusion of DNA from heat-treated E. coli cells. In three anaerobic digestion processes, real-time PCR, PMA-qPCR, and the most probable number method (MPN) were used to estimate the numbers of total, viable, and culturable E. coli cells, respectively. Culturable concentrations of fecal coliforms were also measured by the membrane filter method. For thermophilic digestion, the reductions in total and viable E. coli cells from the digester influent to the effluent were significantly lower than those in culturable cells and fecal coliforms by two to four orders of magnitude. For mesophilic digestion, the differences in the reductions in E. coli and fecal coliforms counts were less than two orders of magnitude. Based on the measurements of viable E. coli determined by the PMA-qPCR method, the microbial quality of digester effluents was discussed for agricultural application, and pasteurization after anaerobic digestion was suggested for the destruction of viable pathogens.

scholarly journals Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Yang Zhang ◽  
Chunyang Dai ◽  
Huiyan Wang ◽  
Yong Gao ◽  
Tuantuan Li ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Highly Sensitive ◽  
One Step ◽  
Polymerase Chain

Abstract Background Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30 to 60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. Method In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. Result The limit of detection (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI: 363.23–1145.69) for ORF1ab and 528.1 (95% CI: 347.7–1248.7) for N, 401.8 (95% CI: 284.8–938.3) for ORF1ab and 336.8 (95% CI: 244.6–792.5) for N, and 194.74 (95% CI: 139.7–430.9) for ORF1ab and 189.1 (95% CI: 130.9–433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. Conclusion In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

Quantification of Common Wheat Adulteration of Durum Wheat Pasta Using Real-Time Quantitative Polymerase Chain Reaction (PCR)

2002 ◽  
Vol 79 (4) ◽  
pp. 553-558 ◽  
Author(s):  
Rémi Alary ◽  
Arnaud Serin ◽  
Marie-Pierre Duviau ◽  
Philippe Jourdrier ◽  
Marie-Françoise Gautier
Keyword(s):  
Polymerase Chain Reaction ◽  
Durum Wheat ◽  
Real Time ◽  
Common Wheat ◽  
Quantitative Polymerase Chain Reaction ◽  
Chain Reaction ◽  
Polymerase Chain

Real-time activity assays of β-lactamases in living bacterial cells: application to the inhibition of antibiotic-resistant E. coli strains

2017 ◽  
Vol 13 (11) ◽  
pp. 2323-2327 ◽  
Author(s):  
Ying Ge ◽  
Ya-Jun Zhou ◽  
Ke-Wu Yang ◽  
Yi-Lin Zhang ◽  
Yang Xiang ◽  
...  
Keyword(s):  
Real Time ◽  
Biological Systems ◽  
Bacterial Cells ◽  
Antibiotic Resistant ◽  
Time Activity ◽  
E Coli ◽  
Living Bacteria

A UV-Vis approach is reported for activity assays and inhibition of β-lactamases in complex biological systems of living bacteria.

scholarly journals Eliminating false positives in a qPCR assay for the detection of the uidA gene in Escherichia coli

2013 ◽  
Vol 11 (3) ◽  
pp. 382-386 ◽  
Author(s):  
Richard Kibbee ◽  
Natalie Linklater ◽  
Banu Örmeci
Keyword(s):  
Escherichia Coli ◽  
Quantitative Polymerase Chain Reaction ◽  
Qpcr Assay ◽  
Uida Gene ◽  
Chain Reaction ◽  
Taq Polymerase ◽  
E Coli ◽  
Gene Copies ◽  
Polymerase Chain ◽  
Low Levels

Due to contaminant Escherichia coli DNA present in recombinant Taq polymerase reagents, it is not possible to reliably detect low levels of E. coli in samples using the quantitative polymerase chain reaction (qPCR) assay. Native Taq polymerase was successfully used in this study to detect five uidA gene copies (5 fg of genomic DNA) of the uidA gene.

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