Analysis of Pseudomonas aeruginosa c-di-GMP High and Low Subpopulations Using Flow-assisted Cell Sorting (FACS) and Quantitative Reverse Transcriptase PCR (qRT-PCR)

The dissemination of prohibited species-specific central nervous system (CNS) tissue contamination in meat must be tracked to mitigate human health risk associated with bovine spongiform encephalopathy. The efficiency of compliance monitoring and risk control measures taken by concerned regulatory authorities at meat production facilities to avoid such contamination depends on the ability to detect CNS tissue with a reliable and adequately sensitive quantitative method. A rapid and convenient one-step real-time quantitative reverse transcriptase PCR (qRT-PCR) assay was developed based on the absolute quantification of glial fibrillary acidic protein (GFAP) mRNA as a marker for CNS tissue contamination in meat. The GFAP RNA quantity corresponding to a percentage of CNS tissue in artificially spiked meat was determined using an appropriate in vitro transcribed target GFAP RNA as a calibration standard in the assay. The assay had a linear dynamic range of 102 to 109 copies of target RNA and was able to detect 0.01% CNS contamination in meat. Further evaluation consisted of an analysis of 272 random meat cuts from carcasses and 109 ground meat samples received from a federally inspected abattoir and two meat processing facilities, respectively, over a 5-month period. The analyzed samples were all negative for CNS tissue contamination at an arbitrarily set lower threshold of 0.025%. Overall, the newly developed one-step qRT-PCR may be useful as an objective quantitative compliance monitoring tool and for setting an acceptable low tolerance threshold for such contamination in meat.

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Use of Propidium Monoazide in Reverse Transcriptase PCR To Distinguish between Infectious and Noninfectious Enteric Viruses in Water Samples

Applied and Environmental Microbiology ◽

10.1128/aem.02800-09 ◽

2010 ◽

Vol 76 (13) ◽

pp. 4318-4326 ◽

Cited By ~ 151

Author(s):

Sandhya Parshionikar ◽

Ian Laseke ◽

G. Shay Fout

Keyword(s):

Reverse Transcriptase ◽

Rna Extraction ◽

Enteric Viruses ◽

Health Concern ◽

Norwalk Virus ◽

Propidium Monoazide ◽

Rt Pcr ◽

Reverse Transcriptase Pcr ◽

Qrt Pcr ◽

Treated Drinking Water

ABSTRACT Human enteric viruses can be present in untreated and inadequately treated drinking water. Molecular methods, such as the reverse transcriptase PCR (RT-PCR), can detect viral genomes in a few hours, but they cannot distinguish between infectious and noninfectious viruses. Since only infectious viruses are a public health concern, methods that not only are rapid but also provide information on the infectivity of viruses are of interest. The intercalating dye propidium monoazide (PMA) has been used for distinguishing between viable and nonviable bacteria with DNA genomes, but it has not been used to distinguish between infectious and noninfectious enteric viruses with RNA genomes. In this study, PMA in conjunction with RT-PCR (PMA-RT-PCR) was used to determine the infectivity of enteric RNA viruses in water. Coxsackievirus, poliovirus, echovirus, and Norwalk virus were rendered noninfectious or inactivated by treatment with heat (72°C, 37°C, and 19°C) or hypochlorite. Infectious or native and noninfectious or inactivated viruses were treated with PMA. This was followed by RNA extraction and RT-PCR or quantitative RT-PCR (qRT-PCR) analysis. The PMA-RT-PCR results indicated that PMA treatment did not interfere with detection of infectious or native viruses but prevented detection of noninfectious or inactivated viruses that were rendered noninfectious or inactivated by treatment at 72°C and 37°C and by hypochlorite treatment. However, PMA-RT-PCR was unable to prevent detection of enteroviruses that were rendered noninfectious by treatment at 19°C. After PMA treatment poliovirus that was rendered noninfectious by treatment at 37°C was undetectable by qRT-PCR, but PMA treatment did not affect detection of Norwalk virus. PMA-RT-PCR was also shown to be effective for detecting infectious poliovirus in the presence of noninfectious virus and in an environmental matrix. We concluded that PMA can be used to differentiate between potentially infectious and noninfectious viruses under the conditions defined above.

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Rapid One-Step Quantitative Reverse Transcriptase PCR Assay with Competitive Internal Positive Control for Detection of Enteroviruses in Environmental Samples

Applied and Environmental Microbiology ◽

10.1128/aem.02291-05 ◽

2006 ◽

Vol 72 (6) ◽

pp. 3960-3967 ◽

Cited By ~ 87

Author(s):

Jason B. Gregory ◽

R. Wayne Litaker ◽

Rachel T. Noble

Keyword(s):

Reverse Transcriptase ◽

Fecal Contamination ◽

Genetic Material ◽

Positive Control ◽

Amplification Efficiency ◽

Specific Method ◽

Pcr Assay ◽

Reverse Transcriptase Pcr ◽

Qrt Pcr ◽

Internal Positive Control

ABSTRACT Human enteroviruses can serve as a more accurate indicator of human fecal contamination than conventional bacteriological fecal indicators. We describe here a quantitative reverse transcriptase PCR (qRT-PCR) assay specifically tailored to detect these viruses in environmental waters. The assay included a competitive internal positive control (CIPC) that allowed the inhibition of qRT-PCRs to be quantitatively assessed. Coamplification of the CIPC with enteroviral genetic material did not affect the sensitivity, specificity, or reproducibility of the enteroviral qRT-PCR assay. The assay is rapid (less than 5 h from sample to result), has a wide dynamic range (>3 logs), and is capable of detecting as few as 25 enteroviral genomes with an average amplification efficiency of 0.91. In samples with low or moderate inhibition, the delay in CIPC amplification was used to adjust enterovirus qRT-PCR concentrations to account for losses due to inhibition. Samples exhibiting significant inhibition were not corrected but instead diluted twofold and immediately assayed again. Using significantly inhibited samples, it was found that dilution relieved inhibition in 93% (25 of 27) of the samples. In addition, 15% (4 of 27) of these previously negative samples contained enteroviral genomes. The high-throughput format of the assay compared to conventional culture-based methods offers a fast, reliable, and specific method for detecting enteroviruses in environmental water samples. The ability of the assay to identify false negatives and provide improved quantitative assessments of enterovirus concentrations will facilitate the tracking of human fecal contamination and the assessment of potential public health risk due to enteroviruses in recreational and shellfish harvesting waters.

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Rapid Detection of Enteroviruses in Small Volumes of Natural Waters by Real-Time Quantitative Reverse Transcriptase PCR

Applied and Environmental Microbiology ◽

10.1128/aem.71.8.4523-4530.2005 ◽

2005 ◽

Vol 71 (8) ◽

pp. 4523-4530 ◽

Cited By ~ 75

Author(s):

Jed A. Fuhrman ◽

Xiaolin Liang ◽

Rachel T. Noble

Keyword(s):

Los Angeles ◽

Reverse Transcriptase ◽

Real Time ◽

Natural Waters ◽

Cost Effective ◽

Environmental Water ◽

Viral Contamination ◽

Reverse Transcriptase Pcr ◽

Qrt Pcr ◽

Field Samples

ABSTRACT Despite viral contamination of recreational waters, only bacterial, not viral, indicators are monitored routinely, due to a lack of rapid and cost-effective assays. We used negatively charged filters to capture enteroviruses from seawater and freshwater. Viral RNA was extracted using a commercial kit, and the viruses were quantified by real-time quantitative reverse transcriptase PCR (qRT-PCR). Poliovirus (6.6 to 330,000 virus particles/ml) was added to samples from watersheds in Los Angeles, California, and analysis showed that with 50-ml samples, a cellulose acetate/nitrate (HA) filter yielded final recovery of 51% (r 2 = 0.99) in fresh water and 23% (r 2 = 0.90) in seawater. However, for additions of low levels of virus (more likely to represent field samples; <104 enterovirus particles/ml), the recovery was lower and more variable, with HA being best in freshwater (17%, r 2 = 0.97) and the type GF/F glass filter having higher average recovery in seawater (GF/F, 17%; r 2 = 0.93; HA 12%, r 2 = 0.87). The optimized method was used with 1-liter field samples from two very different freshwater “creeks” that drain into Santa Monica Bay, California: Topanga Creek (TC), a relatively pristine mountain creek, and Ballona Creek (BC), a concrete-lined urban storm drain. One TC site out of 10 and 2 BC sites out of 7 tested significantly positive for enteroviruses, with higher enterovirus concentrations in BC than in TC (ca. 10 to 25 versus 1 equivalent enterovirus particle/ml). The presented filtration-qRT-PCR approach is fast (<8 h from sampling to results), sensitive, and cost efficient and is promising for monitoring viral contamination in environmental water samples.

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Comparison of Culture, Direct Immunofluorescence Assay, and Multiplex Reverse Transcriptase PCR for Detection of Respiratory Viruses

Laboratory Medicine Online ◽

10.3343/lmo.2011.1.4.8 ◽

2011 ◽

Vol 1 (4) ◽

pp. 221 ◽

Cited By ~ 1

Author(s):

Kui Hyun Yoon ◽

Ji Hyun Cho

Keyword(s):

Reverse Transcriptase ◽

Respiratory Viruses ◽

Immunofluorescence Assay ◽

Direct Immunofluorescence ◽

Reverse Transcriptase Pcr ◽

Direct Immunofluorescence Assay

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Sub-Inhibitory Concentrations of Ciprofloxacin Alone and Combinations with Plant-Derived Compounds against P. aeruginosa Biofilms and Their Effects on the Metabolomic Profile of P. aeruginosa Biofilms

Antibiotics ◽

10.3390/antibiotics10040414 ◽

2021 ◽

Vol 10 (4) ◽

pp. 414

Author(s):

Didem Kart ◽

Tuba Reçber ◽

Emirhan Nemutlu ◽

Meral Sagiroglu

Keyword(s):

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Inhibitory Concentration ◽

Molecular Level ◽

Metabolomic Profile ◽

Gene Expressions ◽

New Agents ◽

Qrt Pcr ◽

Metabolomic Approach

Introduction: Alternative anti-biofilm agents are needed to combat Pseudomonas aeruginosa infections. The mechanisms behind these new agents also need to be revealed at a molecular level. Materials and methods: The anti-biofilm effects of 10 plant-derived compounds on P. aeruginosa biofilms were investigated using minimum biofilm eradication concentration (MBEC) and virulence assays. The effects of ciprofloxacin and compound combinations on P. aeruginosa in mono and triple biofilms were compared. A metabolomic approach and qRT-PCR were applied to the biofilms treated with ciprofloxacin in combination with baicalein, esculin hydrate, curcumin, and cinnamaldehyde at sub-minimal biofilm inhibitory concentration (MBIC) concentrations to highlight the specific metabolic shifts between the biofilms and to determine the quorum sensing gene expressions, respectively. Results: The combinations of ciprofloxacin with curcumin, baicalein, esculetin, and cinnamaldehyde showed more reduced MBICs than ciprofloxacin alone. The quorum sensing genes were downregulated in the presence of curcumin and cinnamaldehyde, while upregulated in the presence of baicalein and esculin hydrate rather than for ciprofloxacin alone. The combinations exhibited different killing effects on P. aeruginosa in mono and triple biofilms without affecting its virulence. The findings of the decreased metabolite levels related to pyrimidine and lipopolysaccharide synthesis and to down-regulated alginate and lasI expressions strongly indicate the role of multifactorial mechanisms for curcumin-mediated P. aeruginosa growth inhibition. Conclusions: The use of curcumin, baicalein, esculetin, and cinnamaldehyde with ciprofloxacin will help fight against P. aeruginosa biofilms. To the best of our knowledge, this is the first study of its kind to define the effect of plant-based compounds as possible anti-biofilm agents with low MBICs for the treatment of P. aeruginosa biofilms through metabolomic pathways.

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