scholarly journals Validation of bacterial markers to discriminate against the source of nitrate contamination: a promising application within the EU Nitrates Directive

2021 ◽  
Vol 4 ◽  
Author(s):  
Daniela Losacco ◽  
Marina Tumolo ◽  
Roberto Mauro ◽  
Barbara Casale ◽  
Carmine Massarelli ◽  
...  
Keyword(s):  
Real Time ◽  
Enterococcus Faecalis ◽  
Real Time Pcr ◽  
Nitrate Contamination ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Environmental Matrices ◽  
Groundwater Samples ◽  
Nitrates Directive

The European Community (EC) Nitrates Directive (ND) (Council Directive 1991/676/EEC) aims to avoid water pollution by nitrates from poor farming practices. Under the ND, Nitrate Vulnerable Zones (NVZs) have been defined as areas where the concentration of agricultural nitrates in surface or groundwater exceeds 50 mg/L. Groundwater is one of the main sources of drinking water in Europe, so ensuring its quality is of significant importance. Monitoring of water parameters, the identification of pollution, the development of good agricultural practice codes is included in ND as measures and action programs. ND provides for the attribution of areas affected by nitrate pollution to the NVZ in which farmers must comply with measures necessary to reduce nitrogen in excess and protect the quality of environmental matrices. However, in some cases the quality of water bodies remains poor, suggesting that some action measures are ineffective or need improvements. Nevertheless, nitrates in excess in groundwater might be related to industrial inputs, untreated waste discharge, and sewage spilling. Due to this, identifying non-agricultural contributions to nitrate groundwater pollution can be of great interest. With this aim, the employment of biomarkers such as microbes associated with the intestinal tract of a specific host is under study for identifying fecal pollution. This strategy is known, in scientific literature, as Microbial Source Tracking (MST)(Furtula et al. 2011)The amplification of 16S rRNA genes of host-specific Bacteroidales allows discriminating against human and livestock fecal sources in samples from nitrate polluted environment. The aim of this work is to evaluate the sensitivity and specificity of these intestinal biomarkers in groundwater samples collected along with the Apulian Region (Southern Italy) in order to assess the applicability of the MST in the employ of ND. A preliminary study was carried out to validate the performance of seven markers for MST using two different approaches: Polimerase Chain Reaction (PCR) and quantitative Real-time PCR (qPCR) assays. For both PCR and qPCR experiments selected primer sets were checked using fecal samples of known origin as positive controls. PCR assay was used for human (HF74 and Enterococcus faecalis) (Bernhard and Field 2000, Jackson et al. 2004), cattle (CF123) (Bernhard and Field 2000), equine (HoF597) (Dick et al. 2005), and pig (Pig163) (Dick et al. 2005) markers screening. Two additional markers were tested through Real-time PCR: human (Human- Bacteroides) (Seurinck et al. 2005) and zootechnical (BacPre I) (Kobayashi et al. 2012). The results indicate that biomarkers can be considered reliable in distinguishing human from animal pollution. Nevertheless, our studies show that the tests conducted with the human Enterococcus faecalis biomarker do not discriminate the zootechnical source from the human one. In some cases, PCR-tested biomarkers cannot determine the source of contamination in environmental matrices due to the detection limit. Animal and human fecal markers were widely detected on eleven groundwater samples through Real Time PCR, highlighting the prevalent source of contamination in the environmental matrix. This research provides evidence that MST technology is a valid tool for local authorities to identify the source of nitrate contamination and review uncertainties during the NVZs definition and the action program development required by the European Nitrate Directive.

Monitoring of Microbial Community Inhabiting a Low-Grade Copper Sulphide Ore by Quantitative Real-Time PCR Analysis of 16S rRNA Genes

2007 ◽  
Vol 20-21 ◽  
pp. 539-542 ◽  
Author(s):  
Francisco Remonsellez ◽  
F. Galleguillos ◽  
Sonestie Janse van Rensburg ◽  
G.F. Rautenbach ◽  
Pedro A. Galleguillos ◽  
...  
Keyword(s):  
16S Rrna ◽  
Real Time ◽  
Real Time Pcr ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Low Grade ◽  
Copper Sulphide ◽  
Quantitative Real Time Pcr ◽  
Sulphide Ore ◽  
Heap Bioleaching

Microbial heap bioleaching is being used as an industrial process to recover copper from low grade ores. It is known that a consortium of different microorganisms participates in this process. Therefore identification and quantification of communities inhabiting heap bioleaching operations is a key step for understanding the dynamics and role of these microorganisms in the process. A quantitative real-time PCR approach was used to investigate the microbial dynamics in this process. To study the microbial population inhabiting a low-grade copper sulphide ore bioleaching industrial heap process at Escondida Mine in Chile, 16S rRNA genetic libraries were constructed using bacterial and archaeal universal primers. Phylogenetic analyses of sequences retrieved from genetic libraries showed that the community is mainly composed by microoganisms related to Acidithiobacillus ferrooxidans (2 strains), Acidithiobacillus thiooxidans, Leptospirillum ferrooxidans, Leptospirillum ferriphilum and the archaea Ferroplasma. Specific primers for real-time PCR determination were designed and tested to amplify each of the sequences obtained by cloning. Standard curves for real time PCR were performed using plasmid DNA from selected clones. This methodology is actually being used to monitor relevant microorganisms inhabiting this low-grade copper sulphide ore bioleaching industrial heap.

Quantification of ammonia-oxidizing bacteria populations in full-scale sewage activated sludge systems and assessment of system variables affecting their performance

2006 ◽  
Vol 54 (1) ◽  
pp. 91-99 ◽  
Author(s):  
T. Limpiyakorn ◽  
F. Kurisu ◽  
O. Yagi
Keyword(s):  
Activated Sludge ◽  
Real Time ◽  
Real Time Pcr ◽  
Full Scale ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Ammonia Oxidizing Bacteria ◽  
Primer Sets ◽  
Activated Sludge Systems ◽  
Pcr Primer

This study carried out quantification of ammonia-oxidizing bacteria (AOB) populations in 12 full-scale sewage activated sludge systems that were different in ammonia removals and treatment processes during three different seasons. Experiment was divided into 3 parts: 1) analysis of AOB communities by PCR-DGGE-cloning-sequencing of 16S rRNA genes; 2) development of four real-time PCR primer sets for quantification of the particular AOB of interest; and 3) quantification of AOB populations by using the newly developed real-time PCR primer sets. The results suggested that all the primer sets gave good reproducibility and specificity for PCR amplification with the detection limits of 102 copies/PCR reaction. Although the 12 systems were different in several aspects, one of the identified sequence types of Nitrosomonas oligotropha cluster was the dominant AOB in every system and every season studied. However, the other sequence type of this cluster was not significantly involved in ammonia removals in the systems. The occurrence of N. communis cluster in the systems seemed to depend on the remaining oxygen concentrations in the sludge floc and thus the activity of aerobic heterotrophs in the aeration tanks. N. europaea–Nitrosococcus. mobilis solely existed in one A2O system of which the influent contained twice the chloride concentrations than those of other systems.

scholarly journals Real-Time PCR Quantitation of Clostridia in Feces of Autistic Children

2004 ◽  
Vol 70 (11) ◽  
pp. 6459-6465 ◽  
Author(s):  
Yuli Song ◽  
Chengxu Liu ◽  
Sydney M. Finegold
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Late Onset ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Pcr Analysis ◽  
Bacterial Cells ◽  
Autistic Children ◽  
Bacterial Strains ◽  
And Control

ABSTRACT Based on the hypothesis that intestinal clostridia play a role in late-onset autism, we have been characterizing clostridia from stools of autistic and control children. We applied the TaqMan real-time PCR procedure to detect and quantitate three Clostridium clusters and one Clostridium species, C. bolteae, in stool specimens. Group- and species-specific primers targeting the 16S rRNA genes were designed, and specificity of the primers was confirmed with DNA from related bacterial strains. In this procedure, a linear relationship exists between the threshold cycle (CT ) fluorescence value and the number of bacterial cells (CFU). The assay showed high sensitivity: as few as 2 cells of members of cluster I, 6 cells of cluster XI, 4 cells of cluster XIVab, and 0.6 cell of C. bolteae could be detected per PCR. Analysis of the real-time PCR data indicated that the cell count differences between autistic and control children for C. bolteae and the following Clostridium groups were statistically significant: mean counts of C. bolteae and clusters I and XI in autistic children were 46-fold (P = 0.01), 9.0-fold (P = 0.014), and 3.5-fold (P = 0.004) greater than those in control children, respectively, but not for cluster XIVab (2.6 � 108 CFU/g in autistic children and 4.8 � 108 CFU/g in controls; respectively). More subjects need to be studied. The assay is a rapid and reliable method, and it should have great potential for quantitation of other bacteria in the intestinal tract.

scholarly journals Real-Time PCR Screening for 16S rRNA Mutations Associated with Resistance to Tetracycline in Helicobacter pylori

2005 ◽  
Vol 49 (8) ◽  
pp. 3166-3170 ◽  
Author(s):  
Erik Glocker ◽  
Marco Berning ◽  
Monique M. Gerrits ◽  
Johannes G. Kusters ◽  
Manfred Kist
Keyword(s):  
Helicobacter Pylori ◽  
16S Rrna ◽  
Real Time ◽  
Real Time Pcr ◽  
Tetracycline Resistance ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Pcr Screening ◽  
H Pylori

ABSTRACT The effectiveness of recommended first-line therapies for Helicobacter pylori infections is decreasing due to the occurrence of resistance to metronidazole and/or clarithromycin. Quadruple therapies, which include tetracycline and a bismuth salt, are useful alternative regimens. However, resistance to tetracycline, mainly caused by mutations in the 16S rRNA genes (rrnA and rrnB) affecting nucleotides 926 to 928, are already emerging and can impair the efficacies of such second-line regimens. Here, we describe a novel real-time PCR for the detection of 16S rRNA gene mutations associated with tetracycline resistance. Our PCR method was able to distinguish between wild-type strains and resistant strains exhibiting single-, double, or triple-base-pair mutations. The method was applicable both to DNA extracted from pure cultures and to DNA extracted from fresh or frozen H. pylori-infected gastric biopsy samples. We therefore conclude that this real-time PCR is an excellent method for determination of H. pylori tetracycline resistance even when live bacteria are no longer available.

scholarly journals Novel Sensitive Real-Time PCR for Quantification of Bacterial 16S rRNA Genes in Plasma of HIV-Infected Patients as a Marker for Microbial Translocation

2011 ◽  
Vol 49 (10) ◽  
pp. 3691-3693 ◽  
Author(s):  
M. Kramski ◽  
A. J. Gaeguta ◽  
G. F. Lichtfuss ◽  
R. Rajasuriar ◽  
S. M. Crowe ◽  
...  
Keyword(s):  
16S Rrna ◽  
Real Time ◽  
Real Time Pcr ◽  
Microbial Translocation ◽  
16S Rrna Genes ◽  
Rrna Genes

scholarly journals Development and Assessment of a Real-Time PCR Assay for Rapid and Sensitive Detection of a Novel Thermotolerant Bacterium, Lactobacillus thermotolerans, in Chicken Feces

2005 ◽  
Vol 71 (8) ◽  
pp. 4214-4219 ◽  
Author(s):  
Abu Sadeque Md Selim ◽  
Piyanuch Boonkumklao ◽  
Teruo Sone ◽  
Apinya Assavanig ◽  
Masaru Wada ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Real Time ◽  
Real Time Pcr ◽  
Pure Culture ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Pcr Assay ◽  
Chicken Feces

ABSTRACT A new real-time PCR assay was successfully developed using a TaqMan fluorescence probe for specific detection and enumeration of a novel bacterium, Lactobacillus thermotolerans, in chicken feces. The specific primers and probe were designed based on the L. thermotolerans 16S rRNA gene sequences, and these sequences were compared to those of all available 16S rRNA genes in the GenBank database. The assay, targeting 16S rRNA gene, was evaluated using DNA from a pure culture of L. thermotolerans, DNA from the closely related bacteria Lactobacillus mucosae DSM 13345T and Lactobacillus fermentum JCM 1173T, and DNA from other lactic acid bacteria in quantitative experiments. Serial dilutions of L. thermotolerans DNA were used as external standards for calibration. The minimum detection limit of this technique was 1.84 × 103 cells/ml of an L. thermotolerans pure culture. The assay was then applied to chicken feces in two different trials. In the first trial, the cell population was 104 cells/g feces on day 4 and 105 cells/g feces on days 11 to 18. However, cell populations of 106 to 107 cells/g feces were detected in the second trial. The total bacterial count, measured by 4′,6-diamidino-2-phenylindole (DAPI) staining, was approximately 1011 cells/g feces. These results suggest that in general, L. thermotolerans is a normal member of the chicken gut microbiota, although it is present at relatively low levels in the feces.

Detection of Non-Tuberculosis Mycobacteria by Real Time PCR

2021 ◽  
Vol 19 ◽  
Author(s):  
Mina Ahmadi ◽  
Pegah Shakib ◽  
Mohammad Reza Zolfaghari
Keyword(s):  
16S Rrna ◽  
Real Time ◽  
Internal Control ◽  
Real Time Pcr ◽  
Respiratory Infections ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Beta Actin ◽  
Pcr Method ◽  
Slow Growing

Background: Identification of non-tuberculosis mycobacteria by culture and phenotypic description is commonly used; however, it takes 4 to 6 weeks or even a longer time for slow growing species as well as for identification of some species that may be missed by biochemical characteristics methods. This study aimed to evaluate Real Time PCR for Detection of NTM by Amplification of Internal Transcribed Spacer (ITS) and 16S rRNA. Methods: In our investigation, using Real Time PCR and two pairs of unique primers targeted to ITS and 16S rRNA genes as well as Beta- actin as an internal control, Non tuberculosis mycobacteria species were detected. Results: Real time PCR was performed on the prepared dilutions. In addition, the threshold of sensitivity in this study was 10pg. To test the specificity, the genome of several bacteria responsible for respiratory infections was used, in which only the test response related to the non-tuberculosis mycobacterium genome and internal control was positive. Conclusion: In this research, an effective and up-to-date Real Time PCR method was used to design a diagnostic kit from all aspects. To avoid any error or mistake and to minimize the false results, internal control was used. The ability to design diagnostic kits allows us to increase efficiency, minimize mistakes, and save a considerable amount of time and cost.

Real-Time PCR Detection of 16S rRNA Genes Speeds Most-Probable-Number Enumeration of Foodborne Listeria monocytogenes

2007 ◽  
Vol 70 (7) ◽  
pp. 1650-1655 ◽  
Author(s):  
ELAINE CRISTINA PEREIRA DE MARTINIS ◽  
ROBERT E. DUVALL ◽  
ANTHONY D. HITCHINS
Keyword(s):  
Listeria Monocytogenes ◽  
16S Rrna ◽  
Real Time ◽  
Real Time Pcr ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Most Probable Number ◽  
Food Ingredient ◽  
Specific Sequence ◽  
Probable Number

Quantifying foodborne pathogens at concentrations of 0.1 to 1,000 CFU/g of food generally involves most-probable-number (MPN) enumeration, which takes at least 4 days. A real-time PCR assay (RTi-PCR) was developed to accelerate MPN enumeration of foodborne Listeria monocytogenes. Foods were spiked from 70 to 110 CFU/g, and triplicate subportions from 0.0001 to 1 g were selectively enriched for 48 h at 30°C. For standard MPN enumeration, the enrichments were subcultured on Oxford agar (48 h at 35°C) to isolate Listeria. For RTi-PCR MPN, the L. monocytogenes cells from the same enrichments were washed and resuspended in 2 ml of sterile water. DNA was extracted by boiling for 10 min. The DNA in the extract's supernatant was targeted with published oligonucleotide primers for amplifying an Lmo-specific sequence of 16S rRNA genes. Amplification was continuously monitored with SYBR Green. The resulting amplicon was characterized by its melting temperature. The L. monocytogenes specificity of the primers was confirmed by testing L. monocytogenes (15 strains), Listeria innocua (11 strains), and Listeria welshimeri, Listeria seeligeri, Listeria ivanovii, and Listeria grayi (1 strain each). Quantitatively spiked milk, lettuce, smoked salmon, Brie cheese, ice cream, pork pâté, salami, ready-to-eat shrimp, raw ground beef, and fresh soft cheese were enumerated by both the standard and the PCR MPN method. The paired results from the two MPN methods agreed well, except for the fresh cheese. For some foods, 1-g samples required a decimal dilution for a positive test result, suggesting concentration-dependent food ingredient interference with the RTi-PCR. This RTi-PCR method reduced the time necessary for the MPN enumeration of foodborne L. monocytogenes from 4 to 2 days.

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