scholarly journals Real-Time PCR Detection of Pathogenic Microorganisms in Roof-Harvested Rainwater in Southeast Queensland, Australia

2008 ◽  
Vol 74 (17) ◽  
pp. 5490-5496 ◽  
Author(s):  
W. Ahmed ◽  
F. Huygens ◽  
A. Goonetilleke ◽  
T. Gardner
Keyword(s):  
Real Time ◽  
Legionella Pneumophila ◽  
Real Time Pcr ◽  
Microbiological Quality ◽  
Poor Correlation ◽  
Pathogenic Microorganisms ◽  
Fecal Indicators ◽  
Binary Logistic Regression Model ◽  
Southeast Queensland

ABSTRACT In this study, the microbiological quality of roof-harvested rainwater was assessed by monitoring the concentrations of Escherichia coli, enterococci, Clostridium perfringens, and Bacteroides spp. in rainwater obtained from tanks in Southeast Queensland, Australia. Samples were also tested using real-time PCR (with SYBR Green I dye) for the presence of potential pathogenic microorganisms. Of the 27 rainwater samples tested, 17 (63%), 21 (78%), 13 (48%), and 24 (89%) were positive for E. coli, enterococci, C. perfringens, and Bacteroides spp., respectively. Of the 27 samples, 11 (41%), 7 (26%), 4 (15%), 3 (11%), and 1 (4%) were PCR positive for the Campylobacter coli ceuE gene, the Legionella pneumophila mip gene, the Aeromonas hydrophila lip gene, the Salmonella invA gene, and the Campylobacter jejuni mapA gene. Of the 21 samples tested, 4 (19%) were positive for the Giardia lamblia β-giardin gene. The binary logistic regression model indicated a positive correlation (P < 0.02) between the presence/absence of enterococci and A. hydrophila. In contrast, the presence/absence of the remaining potential pathogens did not correlate with traditional fecal indicators. The poor correlation between fecal indicators and potential pathogens suggested that fecal indicators may not be adequate to assess the microbiological quality of rainwater and consequent health risk.

scholarly journals Implications of faecal indicator bacteria for the microbiological assessment of roof-harvested rainwater quality in southeast Queensland, Australia

2010 ◽  
Vol 56 (6) ◽  
pp. 471-479 ◽  
Author(s):  
W. Ahmed ◽  
A. Goonetilleke ◽  
T. Gardner
Keyword(s):  
Clostridium Perfringens ◽  
Legionella Pneumophila ◽  
Microbiological Quality ◽  
Indicator Bacteria ◽  
Microbiological Analysis ◽  
Pathogenic Microorganisms ◽  
Faecal Indicator Bacteria ◽  
E Coli ◽  
Faecal Indicators

The study aimed to evaluate the suitability of Escherichia coli , enterococci, and Clostridium perfringens for assessing the microbiological quality of roof-harvested rainwater and assessing whether the concentrations of these faecal indicators can be used to predict the presence or absence of specific zoonotic bacterial or protozoan pathogens. From a total of 100 samples tested, 58%, 83%, and 46% of samples were found to be positive for, respectively, E. coli, enterococci, and Clostridium perfringens spores, as determined by traditional culture-based methods. Additionally, in the samples tested, 7%, 19%, 1%, 8%, 17%, and 15% were PCR positive for Aeromonas hydrophila lip, Campylobacter coli ceuE, Campylobacter jejuni mapA, Legionella pneumophila mip, Salmonella invA, and Giardia lamblia β-giardin genes, respectively. However, none of the samples was positive for E. coli O157 lipopolysaccharide, verocytotoxin 1, and verocytotoxin 2 and Cryptosporidium parvum oocyst wall protein genes. The presence or absence of these potential pathogens did not correlate with any of the faecal indicator bacterial concentrations as determined by a binary logistic regression model. The roof-harvested rainwater samples tested in this study appeared to be of poor microbiological quality, and no significant correlation was found between the concentration of faecal indicators and pathogenic microorganisms. The use of faecal indicator bacteria raises questions regarding their reliability in assessing the microbiological quality of water and particularly their poor correlation with pathogenic microorganisms. The presence of one or more zoonotic pathogens suggests that the microbiological analysis of water should be performed and that appropriate treatment measures should be undertaken, especially in tanks where the water is used for drinking.

Molecular Tracing of the Origin of Six Different Plant Species in Bee Honey Using Real-Time PCR

2017 ◽  
Vol 100 (3) ◽  
pp. 744-752 ◽  
Author(s):  
Yajun Wu ◽  
Yange Yang ◽  
Mingchang Liu ◽  
Bin Wang ◽  
Meige Li ◽  
...  
Keyword(s):  
Real Time ◽  
Plant Species ◽  
Real Time Pcr ◽  
Sensitivity Testing ◽  
Foreign Pollen ◽  
Geographic Regions ◽  
Fair Market ◽  
Chaste Tree ◽  
Parallel Tests

Abstract The quality of honey is significantly influenced by floralorigin. Mislabeling floral species occurs frequently in bee honey products. To protect consumers from economic fraud and maintain a fair market environment, methods to identify floralspecies in honey are necessary. In our study, real-time PCRs were established, targeting six honey types mainly produced in China (canola, Chinese milkvetch, Chinese chaste tree, locust tree, litchi, and longan). Sensitivity testing on DNA fromplant tissues exhibited LODs of about 0.5–5 pg/μL. For DNA extracts of pollen sediments from different honeyspecies, LODs ranged from 13.6 to 403.2 pg/μL. In an experiment to determine the practical LODs of honey in which adulterant honey was spiked in the genuine honey, adulterant honey as low as about 0.1–0.5% was detected in 90–100% in 10 parallel tests. Additionally, pollen was spiked in the honey and stored under various conditions to investigate the migration of pollen DNA into the honey supernatant. Finally, the efficiency of our method was investigated by testing honey samples of unknown compositions from different geographic regions. Of the 159 honey samples that were supposed tobe monofloral that had been collected in five provinces, a small portion were found to be contaminated with foreign pollen(7%). The methods proved to be specific, sensitive, and reliable in identifying the six plant species in honey, which would be a useful tool during the market supervision and QC of honey products.

scholarly journals Necrotrophic Growth of Legionella pneumophila

2006 ◽  
Vol 72 (6) ◽  
pp. 4323-4328 ◽  
Author(s):  
R. Temmerman ◽  
H. Vervaeren ◽  
B. Noseda ◽  
N. Boon ◽  
W. Verstraete
Keyword(s):  
Flow Cytometry ◽  
Real Time ◽  
Legionella Pneumophila ◽  
Real Time Pcr ◽  
Water Systems ◽  
Saccharomyces Boulardii ◽  
Wall Structure ◽  
Lower Growth ◽  
Average Growth ◽  
Microbial Cells

ABSTRACT This study examined whether Legionella pneumophila is able to thrive on heat-killed microbial cells (necrotrophy) present in biofilms or heat-treated water systems. Quantification by means of plate counting, real-time PCR, and flow cytometry demonstrated necrotrophic growth of L. pneumophila in water after 96 h, when at least 100 dead cells are available to one L. pneumophila cell. Compared to the starting concentration of L. pneumophila, the maximum observed necrotrophic growth was 1.89 log units for real-time PCR and 1.49 log units for plate counting. The average growth was 1.57 � 0.32 log units (n = 5) for real-time PCR and 1.14 � 0.35 log units (n = 5) for plate counting. Viability staining and flow cytometry showed that the fraction of living cells in the L. pneumophila population rose from the initial 54% to 82% after 96 h. Growth was measured on heat-killed Pseudomonas putida, Escherichia coli, Acanthamoeba castellanii, Saccharomyces boulardii, and a biofilm sample. Gram-positive organisms did not result in significant growth of L. pneumophila, probably due to their robust cell wall structure. Although necrotrophy showed lower growth yields compared to replication within protozoan hosts, these findings indicate that it may be of major importance in the environmental persistence of L. pneumophila. Techniques aimed at the elimination of protozoa or biofilm from water systems will not necessarily result in a subsequent removal of L. pneumophila unless the formation of dead microbial cells is minimized.

scholarly journals Evaluation of Microbiological and Free-Living Protozoa Contamination in Dental Unit Waterlines

2019 ◽  
Vol 16 (15) ◽  
pp. 2648 ◽  
Author(s):  
Anna Maria Spagnolo ◽  
Marina Sartini ◽  
David Di Cave ◽  
Beatrice Casini ◽  
Benedetta Tuvo ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Tap Water ◽  
Microbiological Quality ◽  
Coliform Bacteria ◽  
Dental Staff ◽  
Plate Counts ◽  
Dental Unit Waterlines ◽  
Heterotrophic Plate Counts

Studies conducted over the last 40 years have demonstrated that the water output from dental unit waterlines (DUWLs) is often contaminated with high densities of microorganisms. It has been monitored the microbiological quality of the water in 30 public dental facilities in northern Italy in order to assess the health risk for patients and dental staff. In each facility, samples of water both from taps and from DUWLs were analyzed in order to evaluate heterotrophic plate counts (HPCs) at 22 °C and 36 °C, and to detect coliform bacteria, Pseudomonas aeruginosa, Legionella pneumophila and amoebae. In 100% of the samples taken from the DUWLs, the concentration of HPCs was above the threshold as determined by the Ministère de la Santé et des Solidarités (2007). The concentration of P. aeruginosa was greater than the indicated threshold in 16.67% of the hand-pieces analyzed. A total of 78.33% of samples were contaminated by L. pneumophila, while in the samples taken from the DUWLs alone, this percentage rose to 86.67%. Amoebae were detected in 60% of the samples taken from hand-pieces; all belonging to the species V. vermiformis. This study documented the presence of various microorganisms, including Legionella spp., at considerably higher concentrations in water samples from DUWLs than in samples of tap water in the same facilities, confirming the role of the internal DUWLs in increasing microbial contamination, especially in the absence of proper management of waterborne health risks.

scholarly journals Quantitative Detection of Legionella pneumophila in Water Samples by Immunomagnetic Purification and Real-Time PCR Amplification of the dotA Gene

2005 ◽  
Vol 71 (7) ◽  
pp. 3433-3441 ◽  
Author(s):  
M. A. Yáñez ◽  
C. Carrasco-Serrano ◽  
V. M. Barberá ◽  
V. Catalán
Keyword(s):  
Cell Culture ◽  
Real Time ◽  
Legionella Pneumophila ◽  
Water Samples ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Immunomagnetic Separation ◽  
Pcr Analysis ◽  
Quantitative Detection ◽  
Specific Sequence

ABSTRACT A new real-time PCR assay was developed and validated in combination with an immunomagnetic separation system for the quantitative determination of Legionella pneumophila in water samples. Primers that amplify simultaneously an 80-bp fragment of the dotA gene from L. pneumophila and a recombinant fragment including a specific sequence of the gyrB gene from Aeromonas hydrophila, added as an internal positive control, were used. The specificity, limit of detection, limit of quantification, repetitivity, reproducibility, and accuracy of the method were calculated, and the values obtained confirmed the applicability of the method for the quantitative detection of L. pneumophila. Moreover, the efficiency of immunomagnetic separation in the recovery of L. pneumophila from different kinds of water was evaluated. The recovery rates decreased as the water contamination increased (ranging from 59.9% for distilled water to 36% for cooling tower water), and the reproducibility also decreased in parallel to water complexity. The feasibility of the method was evaluated by cell culture and real-time PCR analysis of 60 samples in parallel. All the samples found to be positive by cell culture were also positive by real-time PCR, while only eight samples were found to be positive only by PCR. Finally, the correlation of both methods showed that the number of cells calculated by PCR was 20-fold higher than the culture values. In conclusion, the real-time PCR method combined with immunomagnetic separation provides a sensitive, specific, and accurate method for the rapid quantification of L. pneumophila in water samples. However, the recovery efficiency of immunomagnetic separation should be considered in complex samples.

Quality of UHT goat's milk in Poland evaluated by real-time PCR

2010 ◽  
Vol 94 (1-3) ◽  
pp. 32-37 ◽  
Author(s):  
A. Dąbrowska ◽  
E. Wałecka ◽  
J. Bania ◽  
M. Żelazko ◽  
M. Szołtysik ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Goat’S Milk ◽  
Goat's Milk

scholarly journals Development of qualitative methods of bifidobacterium bifidum in probiotic with Real-time PCR method

2018 ◽  
Vol 1 (1) ◽  
pp. 13-17
Author(s):  
Trong Pham Nhu ◽  
Long Le Thanh ◽  
Trung Nguyen Thanh ◽  
Yen Ta Thi ◽  
Loan Pham Thi ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Dairy Products ◽  
Food Additives ◽  
Bifidobacterium Bifidum ◽  
Pcr Assay ◽  
Accurate Identification ◽  
Powdered Milk ◽  
Pcr Method

Bifidobacterium strains with probiotic effects have been widely used in dairy products, food additives and pharmaceuticals. Especially, Bifidobacterium bifidum (B. bifidum) is usually presented into food products such as functional food. However, it is difficult to detect, and quantify B. bifidum in a sample with a combination of different probiotics. In Vietnam, there is no official standard method to identify and quantify B. bifidum in the sample with the mix of probiotic species. To fulfil the requirements of a robust quality management, we have developed a quantitative real-time PCR assay based on groEL gene for accurate identification and quantification of Bifidobacterium bifidum. The developed assay allows an unambiguous speciesspecific detection. We built the real-time PCR method to detect and identify B. bifidum in functional and supplemented food with specific up to 100% and reproducibility (SR&lt;0.25) suitable with Annex F AOAC: 2016. This real-time PCR method is rapidly and effectively than conventional method. It takes only 24 hours to detect and identify B. bifidum in compare with at least a period of 3-5 days for conventional methods. The low quantitative limit is 105 CFU/g/mL, which is consistent with probiotic and powdered milk products with a declared quality of more than 106 CFU/g/mL.

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