scholarly journals Detection of Giardia intestinalis in water samples collected from natural water reservoirs and wells in northern and north-eastern Poland using LAMP, real-time PCR and nested PCR

2017 ◽  
Vol 15 (5) ◽  
pp. 775-787 ◽  
Author(s):  
Anna Lass ◽  
Beata Szostakowska ◽  
Krzysztof Korzeniewski ◽  
Panagiotis Karanis
Keyword(s):  
Surface Water ◽  
Real Time ◽  
Water Samples ◽  
Real Time Pcr ◽  
Nested Pcr ◽  
Lamp Assay ◽  
Giardia Intestinalis ◽  
Detection Methods ◽  
Rrna Gene ◽  
Water Reservoirs

Giardia intestinalis is a protozoan parasite, transmitted to humans and animals by the faecal–oral route, mainly through contaminated water and food. Knowledge about the distribution of this parasite in surface water in Poland is fragmentary and incomplete. Accordingly, 36 environmental water samples taken from surface water reservoirs and wells were collected in Pomerania and Warmia-Masuria provinces, Poland. The 50 L samples were filtered and subsequently analysed with three molecular detection methods: loop-mediated isothermal amplification (LAMP), real-time polymerase chain reaction (real-time PCR) and nested PCR. Of the samples examined, Giardia DNA was found in 15 (42%) samples with the use of LAMP; in 12 (33%) of these samples, Giardia DNA from this parasite was also detected using real-time PCR; and in 9 (25%) using nested PCR. Sequencing of selected positive samples confirmed that the PCR products were fragments of the Giardia intestinalis small subunit rRNA gene. Genotyping using multiplex real-time PCR indicated the presence of assemblages A and B, with the latter predominating. The results indicate that surface water in Poland, as well as water taken from surface wells, may be a source of Giardia strains which are potentially pathogenic for humans. It was also demonstrated that LAMP assay is more sensitive than the other two molecular assays.

Rapid and specific detection of Salmonella in water samples using real-time PCR and High Resolution Melt (HRM) curve analysis

2011 ◽  
Vol 64 (12) ◽  
pp. 2453-2459 ◽  
Author(s):  
G. N. van Blerk ◽  
L. Leibach ◽  
A. Mabunda ◽  
A. Chapman ◽  
D. Louw
Keyword(s):  
Surface Water ◽  
High Resolution ◽  
Real Time ◽  
Water Samples ◽  
Real Time Pcr ◽  
Curve Analysis ◽  
Pcr Assay ◽  
High Resolution Melt ◽  
Gene Target ◽  
Enrichment Step

A real-time PCR assay combined with a pre-enrichment step for the specific and rapid detection of Salmonella in water samples is described. Following amplification of the invA gene target, High Resolution Melt (HRM) curve analysis was used to discriminate between products formed and to positively identify invA amplification. The real-time PCR assay was evaluated for specificity and sensitivity. The assay displayed 100% specificity for Salmonella and combined with a 16–18 h non-selective pre-enrichment step, the assay proved to be highly sensitive with a detection limit of 1.0 CFU/ml for surface water samples. The detection assay also demonstrated a high intra-run and inter-run repeatability with very little variation in invA amplicon melting temperature. When applied to water samples received routinely by the laboratory, the assay showed the presence of Salmonella in particularly surface water and treated effluent samples. Using the HRM based assay, the time required for Salmonella detection was drastically shortened to less than 24 h compared to several days when using standard culturing methods. This assay provides a useful tool for routine water quality monitoring as well as for quick screening during disease outbreaks.

scholarly journals A critical assessment of two real-time PCR assays targeting the (SSU) rRNA and gdh genes for the molecular identification of Giardia intestinalis in a clinical laboratory

2014 ◽  
Vol 67 (9) ◽  
pp. 811-816 ◽  
Author(s):  
Samuel Boadi ◽  
Spencer D Polley ◽  
Sally Kilburn ◽  
Graham A Mills ◽  
Peter L Chiodini
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Clinical Laboratory ◽  
Protozoan Parasite ◽  
Molecular Techniques ◽  
Giardia Intestinalis ◽  
Diagnostic Sensitivity ◽  
Rrna Gene ◽  
Ssu Rrna ◽  
Pcr Assays

IntroductionGiardiasis is an intestinal diarrhoeal illness caused by the flagellate protozoan parasite Giardia intestinalis. Molecular techniques for the identification of G. intestinalis have generally been shown to offer a better detection rate of the parasite than the traditional faecal concentration and microscopy techniques.AimThe aim of this study was to critically assess the performance of a commercial and a published real-time PCR assay for their potential use as frontline tests for the diagnosis of giardiasis.MethodsA composite reference standard of enzyme immunoassay and rapid membrane test was used in a diagnostic accuracy study to assess the performance of Primerdesign's, and Verweij et alG. intestinalis real-time PCR assays, comparing them with the traditional ova, cysts and parasite microscopy test (OCP-M).ResultsThe Verweij real-time PCR used primers for the (SSU) rRNA gene, and produced a diagnostic sensitivity of 93.4% (95% CI 88.30% to 98.50%) and an efficiency of 100%. Primerdesign's real-time PCR used primers for the glutamate dehydrogenase gene and produced a diagnostic sensitivity of 61.5% (95% CI 51.50% to 71.50%) and an efficiency of 203%. The OCP-M sensitivity was 83.5% (95% CI 75.87% to 91.13%).ConclusionsThe Verweij real-time PCR was robust and the most sensitive assay suited for use as a first-line diagnostic test for giardiasis.

scholarly journals Estimation of Pig Fecal Contamination in a River Catchment by Real-Time PCR Using Two Pig-Specific Bacteroidales 16S rRNA Genetic Markers

2009 ◽  
Vol 75 (10) ◽  
pp. 3045-3054 ◽  
Author(s):  
Sophie Mieszkin ◽  
Jean-Pierre Furet ◽  
G�rard Corthier ◽  
Mich�le Gourmelon
Keyword(s):  
16S Rrna ◽  
Real Time ◽  
Water Samples ◽  
Real Time Pcr ◽  
Fecal Contamination ◽  
Fecal Pollution ◽  
Rrna Gene ◽  
River Catchment ◽  
Pig Farms ◽  
Cattle Farms

ABSTRACT The microbiological quality of coastal or river water can be affected by fecal contamination from human or animal sources. To discriminate pig fecal pollution from other pollution, a library-independent microbial source tracking method targeting Bacteroidales host-specific 16S rRNA gene markers by real-time PCR was designed. Two pig-specific Bacteroidales markers (Pig-1-Bac and Pig-2-Bac) were designed using 16S rRNA gene Bacteroidales clone libraries from pig feces and slurry. For these two pig markers, 98 to 100% sensitivity and 100% specificity were obtained when tested by TaqMan real-time PCR. A decrease in the concentrations of Pig-1-Bac and Pig-2-Bac markers was observed throughout the slurry treatment chain. The two newly designed pig-specific Bacteroidales markers, plus the human-specific (HF183) and ruminant-specific (BacR) Bacteroidales markers, were then applied to river water samples (n = 24) representing 14 different sites from the French Daoulas River catchment (Brittany, France). Pig-1-Bac and Pig-2-Bac were quantified in 25% and 62.5%, respectively, of samples collected around pig farms, with concentrations ranging from 3.6 to 4.1 log10 copies per 100 ml of water. They were detected in water samples collected downstream from pig farms but never detected near cattle farms. HF183 was quantified in 90% of water samples collected downstream near Daoulas town, with concentrations ranging between 3.6 and 4.4 log10 copies per 100 ml of water, and BacR in all water samples collected around cattle farms, with concentrations ranging between 4.6 and 6.0 log10 copies per 100 ml of water. The results of this study highlight that pig fecal contamination was not as frequent as human or bovine fecal contamination and that fecal pollution generally came from multiple origins. The two pig-specific Bacteroidales markers can be applied to environmental water samples to detect pig fecal pollution.

scholarly journals Molecular detection of Toxoplasma gondii in water samples from Scotland and a comparison between the 529bp real-time PCR and ITS1 nested PCR

2015 ◽  
Vol 87 ◽  
pp. 175-181 ◽  
Author(s):  
Beth Wells ◽  
Hannah Shaw ◽  
Giles Innocent ◽  
Stefano Guido ◽  
Emily Hotchkiss ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Real Time ◽  
Water Samples ◽  
Real Time Pcr ◽  
Nested Pcr ◽  
Molecular Detection

scholarly journals Quantitative detection of E. coli, E. coli O157 and other shiga toxin producing E. coli in water samples using a culture method combined with real-time PCR

2006 ◽  
Vol 4 (4) ◽  
pp. 487-498 ◽  
Author(s):  
Leo Heijnen ◽  
Gertjan Medema
Keyword(s):  
Surface Water ◽  
Real Time ◽  
Shiga Toxin ◽  
Water Samples ◽  
Real Time Pcr ◽  
Matrix Effects ◽  
Quantitative Detection ◽  
Attractive Alternative ◽  
E Coli ◽  
Wastewater Samples

Recent water related outbreaks of shiga toxin producing E. coli O157 have resulted in increased attention of the water industry to this potentially deadly pathogen. Current methods to detect E. coli O157 and its virulence genes are laborious and time-consuming. Specificity, sensitivity and simple use of a real-time PCR method makes it an attractive alternative for the detection of STEC E. coli O157. This study describes the development and application of real-time PCR methods for the detection of E. coli O157, shiga toxin genes (Stx1 and Stx2) and E. coli. The specificity of the methods was confirmed by performing colony-PCR assays on characterized bacterial isolates, demonstrating the applicability of these assays as rapid tests to confirm the presence of E. coli or E. coli O157 colonies on culture plates. Sensitive culture-PCR methods were developed by combining culture enrichment with real-time PCR detection. This rapid method allowed detection of low concentrations of E. coli O157 in the presence of high concentrations of non-O157-E. coli (1:104). Culture-PCR methods were applied to 27 surface water and 4 wastewater samples. E. coli O157 and both Stx genes were detected in two wastewater samples, whereas only E. coli O157 was detected in two surface water samples. Culture-PCR methods were not influenced by matrix effects and also enabled quantitative (MPN) detection of E. coli in these samples.

scholarly journals Real-Time PCR for Quantification of Giardia and Cryptosporidium in Environmental Water Samples and Sewage

2003 ◽  
Vol 69 (9) ◽  
pp. 5178-5185 ◽  
Author(s):  
Rebecca A. Guy ◽  
Pierre Payment ◽  
Ulrich J. Krull ◽  
Paul A. Horgen
Keyword(s):  
Real Time ◽  
Water Samples ◽  
Real Time Pcr ◽  
Simultaneous Detection ◽  
Environmental Water ◽  
Qpcr Assay ◽  
Detection Methods ◽  
Environmental Water Samples ◽  
Dna Yield ◽  
Chelex 100

ABSTRACT The protozoan pathogens Giardia lamblia and Cryptosporidium parvum are major causes of waterborne enteric disease throughout the world. Improved detection methods that are very sensitive and rapid are urgently needed. This is especially the case for analysis of environmental water samples in which the densities of Giardia and Cryptosporidium are very low. Primers and TaqMan probes based on the β-giardin gene of G. lamblia and the COWP gene of C. parvum were developed and used to detect DNA concentrations over a range of 7 orders of magnitude. It was possible to detect DNA to the equivalent of a single cyst of G. lamblia and one oocyst of C. parvum. A multiplex real-time PCR (qPCR) assay for simultaneous detection of G. lamblia and C. parvum resulted in comparable levels of detection. Comparison of DNA extraction methodologies to maximize DNA yield from cysts and oocysts determined that a combination of freeze-thaw, sonication, and purification using the DNeasy kit (Qiagen) provided a highly efficient method. Sampling of four environmental water bodies revealed variation in qPCR inhibitors in 2-liter concentrates. A methodology for dealing with qPCR inhibitors that involved the use of Chelex 100 and PVP 360 was developed. It was possible to detect and quantify G. lamblia in sewage using qPCR when applying the procedure for extraction of DNA from 1-liter sewage samples. Numbers obtained from the qPCR assay were comparable to those obtained with immunofluorescence microscopy. The qPCR analysis revealed both assemblage A and assemblage B genotypes of G. lamblia in the sewage. No Cryptosporidium was detected in these samples by either method.

scholarly journals Rapid And Sensitive Detection Of Lawsonia Intracellularis In Pigs By Real-Time Loop-Mediated Isothermal Amplification

2015 ◽  
Vol 65 (1) ◽  
pp. 20-29 ◽  
Author(s):  
PARK Byung-Yong ◽  
SHIM Kwan-Seob ◽  
KIM Won-Il ◽  
HOSSAIN Md Mukter ◽  
KIM Bumseok ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Sensitive Detection ◽  
Detection Methods ◽  
Conventional Pcr ◽  
Fecal Samples ◽  
Loop Mediated Isothermal Amplification ◽  
Time Loop

Abstract A simple and rapid real-time loop-mediated isothermal amplification (LAMP) assay designed to detect Lawsonia (L.) intracellularis, an important bacteria causing proliferative enteropathy in pigs. A set of four primers targeting the ubiquinone/menaquinone biosynthesis methylase (ubiE) gene was designed for the LAMP reaction. Additionally, serial 10-fold dilutions of cultured L. intracellularis and spiked feces were also used for the optimization of real-time LAMP. The lower limit of the linear range of the assay in L. intracellularis was 1.0 × 100 L. intracellularis. Real-time LAMP was 10 and 100 times more sensitive than real-time PCR and conventional PCR detection methods, respectively. Based on testing of 213 porcine fecal samples using real-time LAMP, realtime PCR and PCR, the agreement quotients of real-time LAMP with conventional PCR and with real-time PCR were 0.77 and 0.95, respectively. This study demonstrated that real-time LAMP was a powerful tool for the rapid and sensitive detection of L. intracellularis in porcine fecal samples.

Real-time PCR detection of Toxoplasma gondii in surface water samples in São Paulo, Brazil

2019 ◽  
Vol 118 (2) ◽  
pp. 631-640 ◽  
Author(s):  
Ana Tereza Galvani ◽  
Ana Paula Guarnieri Christ ◽  
José Antonio Padula ◽  
Mikaela Renata Funada Barbosa ◽  
Ronalda Silva de Araújo ◽  
...  
Keyword(s):  
Surface Water ◽  
Toxoplasma Gondii ◽  
Real Time ◽  
Water Samples ◽  
Real Time Pcr ◽  
Sao Paulo ◽  
Pcr Detection ◽  
São Paulo ◽  
Surface Water Samples

scholarly journals  Recovery of Cryptosporidium from spiked water and stool samples measured by PCR and real time PCR

2012 ◽  
Vol 57 (No. 5) ◽  
pp. 224-232 ◽  
Author(s):  
M. Adamska ◽  
A. Leonska-Duniec ◽  
M. Sawczuk ◽  
A. Maciejewska ◽  
B. Skotarczak
Keyword(s):  
Real Time ◽  
Water Samples ◽  
Real Time Pcr ◽  
18S Rrna ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
High Concentration ◽  
Intestinal Protozoan ◽  
Wide Range ◽  
Stool Samples

Cryptosporidium parvum is a common intestinal protozoan parasite infecting humans and a wide range of animals, whose diagnostics present considerable difficulties. These arise from the exceptionally robust nature of the oocyst’s walls, which necessitates more stringent treatments for disruption and recovery of DNA for analysis using molecular methods. In the case of water, which is the major source of Cryptosporidium oocysts, investigations concern the detection of the presence of the oocysts. Their concentration in water is very low, and moreover, many substances that may have significance as inhibitors of DNA amplification, are present in environmental water and stool. We have carried out trials in order to assess the effectiveness of recovery of C. parvum oocysts, from spiked environmental and distilled water samples, filtrated and concentrated with the use of special laboratory equipment. Inactivation of inhibitors was carried out with use of bovine serum albumin (BSA) in PCR mixes at ten different concentrations. DNA extraction was carried out from stool samples spiked with C. parvum oocysts, concentrated using two methods, and unconcentrated. Nested PCR and a TaqMan nested real time PCR assay, targeting the 18S rRNA gene, was used to detect C. parvum DNA in spiked water and additionally in spiked stool samples. The obtained results showed that losses of C. parvum oocysts occur during the filtration and concentration of spiked water samples. The addition of small amounts of BSA (5–20 ng/µl) to PCR and TaqMan PCR mixes increases the sensitivity of both methods, but a high concentration of BSA (100 ng/µl and above) has an inhibiting effect on the polymerase reaction. The extraction of DNA from C. parvum oocysts from spiked stool samples preceded by concentration with PBS, ether and Percoll resulted in a higher copy number of the 18S rRNA gene.  

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