scholarly journals DNA-based real-time detection and quantification of aeromonads from fresh water beaches on Lake Ontario

2009 ◽  
Vol 7 (2) ◽  
pp. 312-323 ◽  
Author(s):  
Izhar U. H. Khan ◽  
Alyssa Loughborough ◽  
Thomas A. Edge
Keyword(s):  
Real Time ◽  
Fresh Water ◽  
Water Samples ◽  
Quantitative Polymerase Chain Reaction ◽  
Lake Ontario ◽  
Enteric Bacteria ◽  
Recreational Water ◽  
Recreational Waters ◽  
Standard Curve ◽  
High Concentrations

The present study was designed to develop a novel, rapid, direct DNA-based protocol to enumerate aeromonads in recreational waters. An Aeromonas genus-specific real-time quantitative polymerase chain reaction (Q-PCR) protocol was developed and optimized using newly designed genus-specific oligonucleotide primers derived from the gyrase B subunit (GyrB) gene. A standard curve was developed based on the PCR protocol with a minimum quantification limit of 10 cell equivalents ml−1 achieved using an autoclaved water sample from recreational water spiked with known quantities of an Aeromonas ATCC strain. The Q-PCR protocol was validated and applied to detect and quantify the total number of aeromonads in water samples collected from two fresh water beaches on Lake Ontario. The Q-PCR protocol revealed significantly higher numbers of aeromonads in all water samples than a culture-based assay at both beaches. Foreshore sand was found to serve as a reservoir of high concentrations of Aeromonas similar to this phenomenon noted for enteric bacteria like Eschershia coli. The new real-time Q-PCR protocol facilitated the rapid quantification of total numbers of Aeromonas cells present in recreational water samples in <3 hours without culturing.

scholarly journals Testing for viral material in water of public bathing areas of the Danube during summer, Vojvodina, Serbia, 2014

2016 ◽  
Vol 21 (15) ◽  
Author(s):  
Aleksandra Jovanović Galović ◽  
Sanja Bijelović ◽  
Vesna Milošević ◽  
Ivana Hrnjaković Cvjetkovic ◽  
Milka Popović ◽  
...  
Keyword(s):  
Surface Waters ◽  
Water Samples ◽  
Genetic Material ◽  
Recreational Water ◽  
Recreational Waters ◽  
Quality Of Water ◽  
Viral Indicators ◽  
Urban Wastewater Treatment ◽  
Recreational Use

From August to September 2014 a water quality study was conducted on five popular public Danube beaches in Vojvodina, Serbia. To assess the safety of Danube water for bathing, physical, chemical, bacteriological tests were performed. While many parameters for monitoring the quality of water are regulated by law, there are neither national nor international legislations addressing the presence of viruses in recreational waters. In this study, we performed analyses that surpassed national requirements, and investigated if adenovirus, enterovirus or rotavirus genetic material was present in samples of recreational water collected for quality monitoring. Of 90 water samples obtained during the study, enterovirus material was not found in any sample, but adenovirus and rotavirus genetic materials were respectively detected in 60 and 31 samples. Statistical analyses showed a significant correlation between adenovirus DNA and total coliforms in the water. Even when water samples were adequate for recreational use, adenoviruses were detected in 75% (57/76) of such samples. Our results indicate that implementation of viral indicators in recreational water might be helpful to better assess public health safety. This might be particularly relevant in areas where urban wastewater treatment is insufficient and surface waters affected by wastewater are used for recreation.

scholarly journals Relationships between the occurrence of Giardia and Cryptosporidium and physicochemical properties of marine waters of the Pacific Coast of Mexico

2010 ◽  
Vol 8 (4) ◽  
pp. 797-802 ◽  
Author(s):  
Dalia Magana-Ordorica ◽  
Kristina Mena ◽  
Jose B. Valdez-Torres ◽  
Marcela Soto-Beltran ◽  
Josefina Leon-Felix ◽  
...  
Keyword(s):  
Water Samples ◽  
Physicochemical Parameters ◽  
Pacific Coast ◽  
Microbial Quality ◽  
Recreational Water ◽  
Regression Analyses ◽  
Recreational Waters ◽  
Untreated Sewage ◽  
The Pacific

Untreated sewage has adversely affected the quality of marine recreational waters worldwide. Exposure to marine recreational water with poor microbial quality may pose a threat to bathers. The objectives of this study were to assess the effect of physicochemical parameters on Cryptosporidium and Giardia presence in marine recreational water of Sinaloa, Mexico, by Logistic Regression Analyses. Thirty-two 10-litre water samples were collected from two tourist beaches, Altata and Mazatlan, between November 2006 and May 2007. Water samples were processed by the EPA 1623 method and pH, temperature, salinity and turbidity were also determined. Cryptosporidium and Giardia were present in 71 and 57% of the samples collected from Altata, respectively. In Mazatlan, Cryptosporidium and Giardia were found in 83 and 72% of the samples, respectively. The overall concentration of Cryptosporidium ranged from 150 to 2,050 oocysts/10 L with an average of 581 oocysts/10 L and Giardia ranged from 10 to 300 cysts/10 L with an average of 73 cysts/10 L. The occurrence of both parasites increased in water with decreasing temperatures and increasing turbidity of the water.

Real-time quantitative PCR detection ofEscherichia coliO157:H7

2007 ◽  
Vol 4 (1) ◽  
pp. 15-19 ◽  
Author(s):  
Chen Si ◽  
Huang Kun-Lun ◽  
Xu Wen-Tao ◽  
Li Yuan ◽  
Luo Yun-Bo
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Polymerase Chain Reaction ◽  
Amplification Product ◽  
Chain Reaction ◽  
Standard Curve ◽  
Real Time Quantitative Pcr ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Dissociation Curves

AbstractA rapid and accurate real-time quantitative polymerase chain reaction (real-time PCR) method with SYBR Green I was established for detectingEscherichia coliO157:H7. A pair of primers were designed to amplify theeaegene. The dissociation curves showed that the amplification product was very specific. The optimal conditions and standard curve were established. The result indicated that real-time PCR was 1000 times more sensitive than ordinary PCR.

scholarly journals Optimized Quantification of Unculturable Candidatus Liberibacter Spp. in Host Plants Using Real-Time PCR

Plant Disease ◽  
2008 ◽  
Vol 92 (6) ◽  
pp. 854-861 ◽  
Author(s):  
Wenbin Li ◽  
Dayan Li ◽  
Elizabeth Twieg ◽  
John S. Hartung ◽  
Laurene Levy
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Polymerase Chain Reaction ◽  
Disease Diagnosis ◽  
Amplification Efficiency ◽  
Citrus Species ◽  
Geographic Locations ◽  
Standard Curve ◽  
Candidatus Liberibacter ◽  
Universal Standard

Citrus huanglongbing (HLB) is caused by the phloem-limited and psyllid-vectored Candidatus Liberibacter spp. and is a destructive disease of citrus that is rapidly increasing in importance. The disease was reported recently in the principle citrus-producing areas of São Paulo, Brazil in 2004 and in Florida in 2005. A variety of laboratory methods have been developed to confirm a symptom-based disease diagnosis or for the detection or identification of the pathogen; however, no quantitative information has been available on the pathogen titer in either host or vector interactions because the pathogen remains unculturable in artificial media. We previously developed a quantitative polymerase chain reaction (PCR)-based assay for detection of Ca. Liberibacter spp. and, in this study, we evaluated the effects of sample composition on quantification of the pathogen in citrus plants by TaqMan real-time PCR. Standard curves were established using cloned plasmids containing target DNA from the pathogen and with total DNA samples from field-grown HLB-infected citrus plants. Regression analysis showed that a standard curve established with DNA extracted from naturally infected field-grown plants was more accurate than the standard curve constructed from plasmids containing the amplification targets as cloned inserts. Nontarget DNA and putative PCR inhibitors from citrus plants decreased the sensitivity and the amplification efficiency of real-time PCR when plasmids provided the template target in “spiked” healthy citrus DNA extracts. This effect varied among plant tissue types, citrus species, and geographic locations. Based on these sample effects, a universal standard curve has been established for quantification of the pathogen in various citrus tissues of different citrus species planted in different geographic locations. Sample storage at 4°C for 2 months prior to PCR assay did not affect subsequent quantification of the pathogen. The validated quantitative real-time PCR method and the universal standard curve will be very useful for studies of host–pathogen interactions and epidemiology, and in the development of control strategies for the disease.

scholarly journals Comparison of enterovirus and adenovirus concentration and enumeration methods in seawater from Southern California, USA and Baja Malibu, Mexico

2012 ◽  
Vol 10 (3) ◽  
pp. 419-430 ◽  
Author(s):  
Lauren M. Sassoubre ◽  
David C. Love ◽  
Andrea I. Silverman ◽  
Kara L. Nelson ◽  
Alexandria B. Boehm
Keyword(s):  
Cell Culture ◽  
Water Samples ◽  
Membrane Filtration ◽  
Baja California ◽  
Quantitative Polymerase Chain Reaction ◽  
Skim Milk ◽  
Water Quality Monitoring ◽  
Recreational Waters ◽  
California Water ◽  
Etiological Agents

Despite being important etiological agents of waterborne illness, the sources, transport and decay of human viruses in recreational waters are not well understood. This study examines enterovirus and adenovirus concentrations in coastal water samples collected from four beaches impacted by microbial pollution: (1) Malibu Lagoon, Malibu; (2) Tijuana River, Imperial Beach; (3) Baja Malibu, Baja California; and (4) Punta Bandera, Baja California. Water samples were concentrated using a flocculation-based skim milk method and dead-end membrane filtration (MF). Viruses were enumerated using cell culture infectivity assays and reverse transcription quantitative polymerase chain reaction (RT-QPCR). Across concentration and quantification methods, enteroviruses were detected more often than adenoviruses. For both viruses, MF followed by (RT)QPCR yielded higher concentrations than skim milk flocculation followed by (RT)QPCR or cell culture assays. Samples concentrated by skim milk flocculation and enumerated by (RT)QPCR agreed more closely with concentrations enumerated by cell culture assays than MF followed by (RT)QPCR. The detection of viruses by MF and (RT)QPCR was positively correlated with the presence of infectious viruses. Further research is needed to determine if detection of viruses by rapid methods such as (RT)QPCR can be a useful water quality monitoring tool to assess health risks in recreational waters.

scholarly journals Developing a Real-Time PCR Assay for Detection and Quantification of Pratylenchus neglectus in Soil

Plant Disease ◽  
2013 ◽  
Vol 97 (6) ◽  
pp. 757-764 ◽  
Author(s):  
Guiping Yan ◽  
Richard W. Smiley ◽  
Patricia A. Okubara ◽  
Andrea M. Skantar ◽  
Catherine L. Reardon
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Management Practices ◽  
Quantitative Polymerase Chain Reaction ◽  
Internal Transcribed Spacer Region ◽  
Pratylenchus Neglectus ◽  
Standard Curve ◽  
Field Soils ◽  
Plant Parasitic ◽  
Detection And Quantification

Pratylenchus neglectus is one of the most widespread and economically important nematodes that invades plant roots and restricts wheat productivity in the Pacific Northwest. It is challenging to quantify P. neglectus using microscopic methods for studies that require large-scale sampling, such as assessment of rotation crops, wheat cultivars, and other management practices. A real-time quantitative polymerase chain reaction (qPCR) assay was developed to detect and quantify P. neglectus from DNA extracts of soil. The primers, designed from the internal transcribed spacer region of rDNA, showed high specificity with a single melt curve peak to DNA from eight isolates of P. neglectus but did not amplify DNA from 28 isolates of other plant-parasitic and non-plant-parasitic nematodes. A standard curve (R2 = 0.96; P < 0.001) was generated by amplifying DNA extracted from soil to which nematodes were added. The soil standard curve was validated using sterilized soil inoculated with lower numbers of P. neglectus. A significant positive relationship (R2 = 0.66; P < 0.001) was observed for nematode numbers quantified from 15 field soils using qPCR and the Whitehead tray and microscopic method but the qPCR generally tended to provide higher estimates. Real-time PCR potentially provides a useful platform for efficient detection and quantification of P. neglectus directly from field soils.

scholarly journals A novel genetic marker for the rapid detection of Bacteroides fragilis in recreational water as a human-specific faecal indicator

2011 ◽  
Vol 9 (2) ◽  
pp. 253-264 ◽  
Author(s):  
Chang Soo Lee ◽  
Jason W. Marion ◽  
Jiyoung Lee
Keyword(s):  
16S Rrna ◽  
Real Time ◽  
Real Time Pcr ◽  
Detection System ◽  
Bacteroides Fragilis ◽  
Human Colon ◽  
Recreational Water ◽  
Recreational Waters ◽  
Pcr Products ◽  
Us Epa

Bacteroides spp. has gained substantial interest among the suggested potential candidates for alternative faecal indicators for untreated recreational waters by the US EPA. Interest in Bacteroides as a faecal indicator is based upon the relative abundance of selected members of the Bacteroides genus in the human colon and human faeces. In this study, we developed a real-time PCR detection system based on gyrase B subunit genes (gyrB) specific to Bacteroides fragilis. The gryB-based method was compared with previously described 16S rRNA-based real-time qPCR methods and evaluated for specificity, sensitivity and robustness in detecting B. fragilis from untreated recreational water impacted by human and non-human faecal sources. The new gyrB-based system only detected B. fragilis, whereas the 16S rRNA-based methods generated cross-amplifications with other Bacteroides and Prevotella species. We used a procedure of prefiltration, filtration, sonication and DNA concentration in order to improve the DNA extraction efficiency and the sensitivity of the real-time PCR while removing interference. The amplification and sequencing of PCR products generated by the gyrB-based method confirmed that gyrB-amplified sequences only contained B. fragilis. This rapid method is useful for quantifying faecal contamination and may assist beach and watershed managers in elucidating possible contamination sources.

scholarly journals Real Time PCR Based Quantification of Banana Bunchy Top Virus (BBTV) Titre in Banana cv. Grand Naine (Musa acuminata)

2019 ◽  
pp. 1-7
Author(s):  
N. Tanuja ◽  
A. Ramanathan ◽  
S. Varanavasiappan ◽  
E. Kokila Devi ◽  
L. Arul ◽  
...  
Keyword(s):  
Coat Protein ◽  
Real Time ◽  
Coat Protein Gene ◽  
Real Time Pcr ◽  
Protein Gene ◽  
Quantitative Polymerase Chain Reaction ◽  
Sybr Green ◽  
Insect Vector ◽  
Banana Bunchy Top Virus ◽  
Standard Curve

Banana Bunchy Top Disease (BBTD) is one of the most severe viral diseases affecting major banana growing belts in India. Banana Bunchy Top Virus (BBTV) is transmitted by a black banana aphid (Pentalonia nigronervosa L Coquerel) in a persistent manner. BBTV virions are limited to the phloem tissue of banana resulting in low titre in banana. A reliable method to quantify the BBTV in the banana will be useful for monitoring the insect vector mediated BBTV transmission in banana, an essential requirement for characterizing the transgenic banana transformed for BBTV resistance. A protocol for real time PCR based absolute quantification of BBTV is reported in the present study. The partial BBTV coat protein gene (459 bp) was isolated, cloned into a plasmid vector and used to construct a standard curve using an SYBR green-based assay with known copies of BBTV coat protein gene. Using the standard curve, BBTV viral load was estimated in BBTV infected symptomatic and asymptomatic leaf samples of banana cultivar Grand Naine through SYBR green-based quantitative Polymerase Chain Reaction (qPCR). The study demonstrated that a higher viral tire was associated with BBTV disease symptoms appearance, whereas the low titre resulted in asymptomatic plants.

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