Comparison of culture and qPCR methods in detection of mycobacteria from drinking waters

2013 ◽  
Vol 59 (4) ◽  
pp. 280-286 ◽  
Author(s):  
Noora H.J. Räsänen ◽  
Helena Rintala ◽  
Ilkka T. Miettinen ◽  
Eila Torvinen
Keyword(s):  
Surface Water ◽  
Quantitative Polymerase Chain Reaction ◽  
Water Source ◽  
Assimilable Organic Carbon ◽  
Environmental Mycobacteria ◽  
Drinking Waters ◽  
Qpcr Method ◽  
Sensitive Tool ◽  
Different Types ◽  
Polymerase Chain

Environmental mycobacteria are common bacteria in man-made water systems and may cause infections and hypersensitivity pneumonitis via exposure to water. We compared a generally used cultivation method and a quantitative polymerase chain reaction (qPCR) method to detect mycobacteria in 3 types of drinking waters: surface water, ozone-treated surface water, and groundwater. There was a correlation between the numbers of mycobacteria obtained by cultivation and qPCR methods, but the ratio of the counts obtained by the 2 methods varied among the types of water. The qPCR counts in the drinking waters produced from surface or groundwater were 5 to 34 times higher than culturable counts. In ozone-treated surface waters, both methods gave similar counts. The ozone-treated drinking waters had the highest concentration of assimilable organic carbon, which may explain the good culturability. In warm tap waters, qPCR gave 43 times higher counts than cultivation, but both qPCR counts and culturable counts were lower than those in the drinking waters collected from the same sites. The TaqMan qPCR method is a rapid and sensitive tool for total quantitation of mycobacteria in different types of clean waters. The raw water source and treatments affect both culturability and total numbers of mycobacteria in drinking waters.

A proteomics analysis of adventitious root formation after leaf removal in lotus (Nelumbo nucifera Gaertn.)

2018 ◽  
Vol 73 (9-10) ◽  
pp. 375-389 ◽  
Author(s):  
Libao Cheng ◽  
Huiying Liu ◽  
Runzhi Jiang ◽  
Shuyan Li
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Adventitious Root Formation ◽  
Nelumbo Nucifera ◽  
Leaf Removal ◽  
Proteomics Analysis ◽  
Citrate Cycle ◽  
Different Types ◽  
Genes Encoding ◽  
Polymerase Chain ◽  
Isobaric Tags

AbstractThe formation of adventitious roots (ARs) is an important process for lotus (Nelumbo nucifera), which does not have a well-formed main root. In lotus, the removal of leaves above the waterline significantly promoted AR formation, while the removal of leaves below the waterline inhibited AR formation. Proteins were identified using isobaric tags for relative and absolute quantization technique. The number of proteins decreased with increasing sequencing coverage, and most of the identified proteins had fewer than 10 peptides. In the A1/A0 and A2/A1 stages, 661 and 154 proteins showed increased abundance, respectively, and 498 and 111 proteins showed decreased abundance, respectively. In the B1/B0 and B2/B1 stages, 498 and 436 proteins showed increased abundance, respectively, and 358 and 348 proteins showed decreased abundance, respectively. Among the proteins showing large differences in abundance, 17 were identified as being related to AR formation. Proteins involved in the glycolytic pathway and the citrate cycle showed differences in abundance between the two types of leaf removal. The transcriptional levels of nine genes encoding relevant proteins were assessed by quantitative polymerase chain reaction. The results of this study illustrate the changes in metabolism after different types of leaf removal during AR formation in lotus.

scholarly journals The Distribution and Detection of Grapevine red blotch virus in its Host Depend on Time of Sampling and Tissue Type

Plant Disease ◽  
2018 ◽  
Vol 102 (11) ◽  
pp. 2187-2193 ◽  
Author(s):  
Felicia J. Setiono ◽  
Debotri Chatterjee ◽  
Marc Fuchs ◽  
Keith L. Perry ◽  
Jeremy R. Thompson
Keyword(s):  
Polymerase Chain Reaction ◽  
Seasonal Variations ◽  
Quantitative Polymerase Chain Reaction ◽  
Virus Titer ◽  
Tissue Type ◽  
Chain Reaction ◽  
Variable Distribution ◽  
Qpcr Method ◽  
Polymerase Chain ◽  
Virus Distribution

Grapevine red blotch virus (GRBV) is the causal agent of grapevine red blotch, an emerging disease that affects cultivated grapevine such as Vitis vinifera. The ability to detect viruses in grapevine is often hindered by low virus titers compounded by a variable distribution in the plant and seasonal variations. In order to examine these two variables in relation to GRBV, we developed a quantitative polymerase chain reaction (qPCR) method that incorporates both internal and external references to enhance assay robustness. In greenhouse-grown vines infected with GRBV, qPCR identified highest virus titers in the petioles of fully expanded leaves and significantly reduced levels of virus in the shoot extremities. In vineyard-grown vines infected with GRBV, the virus titer in July and October 2016 followed a pattern similar to that found for the greenhouse-grown plants but, most strikingly, close to half (44%) of the samples analyzed in June 2015 tested negative for infection. The technique presented and results obtained highlight the variability of virus distribution in its host and provide a useful guide for selecting the best tissues for optimal GRBV diagnosis.

Surveillance of human viral contamination and physicochemical profiles in a surface water lagoon

2012 ◽  
Vol 66 (12) ◽  
pp. 2682-2687 ◽  
Author(s):  
G. Fongaro ◽  
M. A Nascimento ◽  
A. Viancelli ◽  
D. Tonetta ◽  
M. M. Petrucio ◽  
...  
Keyword(s):  
Surface Water ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Quantitative Polymerase Chain Reaction ◽  
Human Adenovirus ◽  
Human Consumption ◽  
Viral Contamination ◽  
Human Waste ◽  
Viral Particles ◽  
Polymerase Chain

The present study evaluated the contamination of a surface water lagoon (Peri Lagoon) in Florianópolis, Santa Catarina, Brazil, by human adenovirus (HAdV), polyomavirus JC (JCPyV), hepatitis A virus (HAV) and rotavirus species A (RVA). Efforts were driven to determine the correlation between viral presence and the physicochemical parameters of the lagoon and measure the distribution of these viruses throughout the year (June 2010 to May 2011). A total of 48 samples were collected, concentrated and analyzed by qPCR (quantitative polymerase chain reaction). Approximately 96% of the samples were positive for HAdV (46/48), 65% were positive for RVA (31/48), 21% were positive for JCPyV (10/48) and 12% were positive for HAV (6/48). The presence of JCPyV was positively correlated with that of NO2−N, and also there was a positive correlation between the presence of each one of the viruses (HAdV, HAV and RVA) in winter. Samples from water dedicated for human consumption and recreation tested positive for HAdV by qPCR. These samples were also subjected to viral integrity and viability assays: 83% (10/12) contained intact viral particles and 66% (8/12) contained infectious particles. Our results demonstrate the release of human waste into water sources, justifying the urgent need to add viral parameters to water quality surveillance.

scholarly journals Enumeration of viable and non-viable larvated Ascaris eggs with quantitative PCR

2012 ◽  
Vol 10 (4) ◽  
pp. 594-604 ◽  
Author(s):  
Maria Raynal ◽  
Eric N. Villegas ◽  
Kara L. Nelson
Keyword(s):  
Polymerase Chain Reaction ◽  
Detection Limit ◽  
Quantitative Polymerase Chain Reaction ◽  
High Heat ◽  
Chain Reaction ◽  
Direct Microscopy ◽  
Development Stages ◽  
Target Dna ◽  
Qpcr Method ◽  
Polymerase Chain

The goal of this study was to further develop an incubation-quantitative polymerase chain reaction (qPCR) method for quantifying viable Ascaris eggs by characterizing the detection limit and number of template copies per egg, determining the specificity of the method, and testing the method with viable and inactivated larvated eggs. The number of template copies per cell was determined by amplifying DNA from known numbers of eggs at different development stages; the value was estimated to be 32 copies. The specificity of the method was tested against a panel of bacteria, fungi, protozoa and helminths, and no amplification was found with non-target DNA. Finally, fully larvated eggs were inactivated by four different treatments: 254 nm ultraviolet light, 2,000 ppm NH3-N at pH 9, moderate heat (48 °C) and high heat (70 °C). Concentrations of treated eggs were measured by direct microscopy and incubation-qPCR. The qPCR signal decreased following all four treatments, and was in general agreement with the decrease in viable eggs determined by microscopy. The incubation-qPCR method for enumerating viable Ascaris eggs is a promising approach that can produce results faster than direct microscopy, and may have benefits for applications such as assessing biosolids.

scholarly journals A survey of assimilable organic carbon, biodegradable organic carbon and coliform growth response in US drinking waters

2005 ◽  
Vol 5 ◽  
pp. 207-224 ◽  
Author(s):  
L. A. Kaplan ◽  
D. J. Reasoner ◽  
E. W. Rice ◽  
T. L. Bott
Keyword(s):  
Surface Water ◽  
Organic Carbon ◽  
Growth Response ◽  
Water Sources ◽  
Plate Count ◽  
Assimilable Organic Carbon ◽  
Test Water ◽  
Drinking Waters ◽  
Colony Forming Units ◽  
Growth Promoting

The primary objectives of this study were : 1) to document concentrations of Assimilable Organic Carbon (AOC) in a survey of a broad range of drinking waters and treatment processes; 2) compare the Colilorm Growth Response (CGR) to AOC concentrations; and 3) compare Biodegradable Dissolved Organic Carbon (BDOC) concentrations to AOC concentrations. AOC was measured with mixed cultures of Pseudomonas fluorescens strain P-17 and Spirillum sp. strain NOX. Test waters were transferred to 40-ml vials, pasteurized, inoculated, and incubated at 15°C. Colony forming units was the test parameter. CGR was performed with pasteurized test water inoculated with Enterobacter cloacae. Samples were incubated at 20°C for 5 days and response determined from colony forming units. The BDOC assay was performed in 40-ml vials, with glass fiber filtered, pasteurized test water, inoculated with the indigenous microflore from a stream, and incubated for 28 days in the dark et room temperature. The survey involved 109 samples from 79 drinking water supplies located throughout the United States and Canada, including 26 groundwater and 53 surface water sources. Utility personnel were supplied with sample bottles and instructions for sampling and pasteurtzing the test waters. Pasteurized water was sent to the Stroud Water Research Center for AOC, BDOC, and DOC analyses, and to the Risk Reduction Engineering Laboratory for the CGR assay. Densities of coliforms and heterotrophic plate count bacteria (HPC) were measured in the test waters by utility personnel. DOC concentrations ranged from 203 to 4943 µg/L. AOC concentrations ranged from 18 to 322 µg/L, or 2.4 % to 44.0 % of the DOC. High pH values in 5 test waters inhibited the growth of both AOC bioassay organisms. BDOC concentrations ranged from 1 to 1521 µg/L, or 0.4% to 52.8 % of DOC. The CGR assay indicated that 79 % of the test waters did not promote coliform growth, 7 % were strongly growth promoting, all from surface water sources, and 14 % were moderately growth promoting. No coliforms and only low densities at HPC organisms were reported by utilities for treatment plant effluents. The correlation of AOC and BDOC was significant (P≪0.01), with a correlation coefficient of r=0.594. Significant correlations were also found for AOC and DOC, and BDOC and DOC. Correlations of CGR and either AOC or BDOC were not statistically significant.

scholarly journals Effect of D-Mannose on Gene Expression of Neuraminidase Produced from Different Clinical Isolates of Pseudomonas aeruginosa

2019 ◽  
Vol 16 (2) ◽  
pp. 0291
Author(s):  
Shehab Et al.
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Antibacterial Agents ◽  
Quantitative Polymerase Chain Reaction ◽  
Clinical Isolates ◽  
Chain Reaction ◽  
Gene Copies ◽  
Different Types ◽  
Polymerase Chain

The present work aimed to investigate the neuraminidase (nan1) gene expression in 32 different clinical isolates of Pseudomonas aeruginosa to explore the role of the enzyme in different types of infection and might give a better understanding of host cell-pathogens interaction. In addition, the effect of monosaccharide D-mannose on neuraminidase gene expression in eight isolates was studied by utilizing a reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The results demonstrated that the highest expression of nan1 gene was in otitis samples (208,913.81) which were significantly higher than that from other infections (P < 0.01). While, the concentrations of gene copies obtained from urine, sputum and burns samples were 93,535.34, 92,254.64 and 74,029.63respectively. While the least expression in wound samples (32,017.06). This suggests that neuraminidase in ear samples might be more virulent and invasive followed by that from urine, sputum, burns and wounds samples. The considerable interest of addition D-mannose significantly reduced the rate of neuraminidase activity reached fivefold in some isolates. This indicates that D-mannose down regulates nan1 gene expression. Hence, this sugar could be used in the development of potential new antibacterial agents where it acts as a competitive neuraminidase inhibitors.

scholarly journals Effect of D-Mannose on Gene Expression of Neuraminidase Produced from Different Clinical Isolates of Pseudomonas aeruginosa

2019 ◽  
Vol 16 (2) ◽  
pp. 0291
Author(s):  
Shehab Et al.
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Antibacterial Agents ◽  
Quantitative Polymerase Chain Reaction ◽  
Clinical Isolates ◽  
Chain Reaction ◽  
Gene Copies ◽  
Different Types ◽  
Polymerase Chain

The present work aimed to investigate the neuraminidase (nan1) gene expression in 32 different clinical isolates of Pseudomonas aeruginosa to explore the role of the enzyme in different types of infection and might give a better understanding of host cell-pathogens interaction. In addition, the effect of monosaccharide D-mannose on neuraminidase gene expression in eight isolates was studied by utilizing a reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The results demonstrated that the highest expression of nan1 gene was in otitis samples (208,913.81) which were significantly higher than that from other infections (P < 0.01). While, the concentrations of gene copies obtained from urine, sputum and burns samples were 93,535.34, 92,254.64 and 74,029.63respectively. While the least expression in wound samples (32,017.06). This suggests that neuraminidase in ear samples might be more virulent and invasive followed by that from urine, sputum, burns and wounds samples. The considerable interest of addition D-mannose significantly reduced the rate of neuraminidase activity reached fivefold in some isolates. This indicates that D-mannose down regulates nan1 gene expression. Hence, this sugar could be used in the development of potential new antibacterial agents where it acts as a competitive neuraminidase inhibitors.

scholarly journals Q fever: A neglected disease of camels in Giza and Cairo Provinces, Egypt

2019 ◽  
Vol 12 (12) ◽  
pp. 1945-1950 ◽  
Author(s):  
Hend H. A. M. Abdullah ◽  
Hany A. Hussein ◽  
Khaled A. Abd El-Razik ◽  
Ashraf M. A. Barakat ◽  
Yousef A. Soliman
Keyword(s):  
Q Fever ◽  
Quantitative Polymerase Chain Reaction ◽  
Epidemiological Data ◽  
Acute Stage ◽  
Enzyme Linked Immunosorbent Assay ◽  
Igg Antibodies ◽  
Molecular Tools ◽  
Blood Samples ◽  
Sensitive Tool ◽  
Polymerase Chain

Background and Aim: Q fever is a zoonotic disease caused by Coxiella burnetii. Cattle, sheep, and goat are the main reservoir of C. burnetii. In Egypt, the epidemiological data about C. burnetii in camels are limited. Therefore, the current study was conducted to identify C. burnetii infection in camels by different molecular tools and to estimate its seropositivity through the detection of anti-C. burnetii antibodies in camel sera. Materials and Methods: Blood samples were collected 112 from camels in Giza and Cairo Provinces, Egypt. All blood samples were screened by trans-quantitative polymerase chain reaction (trans-qPCR) for C. burnetii and positive samples subjected to standard PCR using the superoxide dismutase enzyme coding gene of C. burnetii. Sera of studied camels were examined for the presence of antibodies against C. burnetii using enzyme-linked immunosorbent assay. Results: Out of 112 camels, 19 were positive for C. burnetii by qPCR with an overall prevalence of 16.9% (18.6% in Giza and 15.1% in Cairo Provinces, respectively). The seroprevalence of anti-C. burnetii IgG antibodies in the examined camels was 4.5% (5/112). Conclusion: Trans-qPCR assay is a rapid and sensitive tool for the detection of C. burnetii in acute stage. Camels should be considered one of the major reservoirs for C. burnetii in Egypt.

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