Abstract：
:A real-time quantitative polymerase chain reaction (Q-PCR) assay was developed for detecting and quantifying Escherichia coli in water samples from agricultural watersheds. The assay included optimization of DNA extraction and purification from water samples, and Q-PCR amplification conditions using newly designed species-specific oligonucleotide primers derived from conserved flanking regions of the 16S rRNA gene, the internal transcribed spacer region (ITS) and the 23S rRNA gene. The assay was optimized using a pure culture of E. coli with known quantities spiked into autoclaved agricultural water samples. The optimized assay was capable of a minimum quantification limit of 10 cells/ml of E. coli in the spiked agricultural water samples. A total of 121 surface water samples from three agricultural watersheds across Canada were analyzed, and results were compared with conventional culture-based enumerations of E. coli. The Q-PCR assay revealed significantly higher numbers of E. coli in water samples than the culture-based assay in each agricultural watershed. The new Q-PCR assay can facilitate the quantification of E. coli in a single water sample in < 3 h, including melt curve analysis, across a range of agricultural water quality conditions.
journal_name
J Microbiol Methodsjournal_title
Journal of microbiological methodsauthors
Khan IU,Gannon V,Kent R,Koning W,Lapen DR,Miller J,Neumann N,Phillips R,Robertson W,Topp E,van Bochove E,Edge TAdoi
10.1016/j.mimet.2007.02.016subject
Has Abstractpub_date
2007-06-01 00:00:00pages
480-8issue
3eissn
0167-7012issn
1872-8359pii
S0167-7012(07)00069-3journal_volume
69pub_type
杂志文章abstract：:Paper-based nucleic acid testing techniques are increasingly in demand. Hence, we have developed a simple and cheap paper fluidic device to detect multiple gene targets in Leptospira. Fluidic channels of the penta-clover device are drawn using a correction pen on Whatman filter paper 1. The fluid blocks the pores of t...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2020.105962
更新日期：2020-07-01 00:00:00
abstract：:Bacillus thuringiensis is a sporulating bacterium, which produces parasporal inclusions toxic to insects. Widely used methodologies to isolate Bt from soil consist of a thermal shock treatment followed by selective germination of spores. The results presented here suggest that a preliminary 5 h dry-heat treatment larg...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2008.06.008
更新日期：2008-10-01 00:00:00
abstract：:We modified Wayne's pyrazinamidase test against Mycobacterium tuberculosis to indirectly measure pyrazinamidase activity via pyrazinoic acid in liquid medium. The modified pyrazinamidase test was easy to perform and its results were in complete agreement with those of the conventional Wayne's method, highlighting its ...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2018.09.018
更新日期：2018-11-01 00:00:00
abstract：:There is a paucity of knowledge on gene expression in dead bacterial cells. Why would this knowledge be useful? The cells are dead. However, the time duration of gene expression following cell death is often unknown, and possibly in the order of minutes. In addition, it is a challenge to determine if bacterial cells a...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2012.04.004
更新日期：2012-07-01 00:00:00
abstract：:Bacillus anthracis, the causative agent of anthrax, is a potential source of bioterrorism. The existing assays for its identification lack specificity due to the close genetic relationship it exhibits to other members of the B. cereus group. Our comparative analyses of protein sequences from Bacillus species have iden...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2011.08.015
更新日期：2011-12-01 00:00:00
abstract：:Mushrooms are considered as important source of biologically active compounds which include low-molecular-mass protein/peptides (LMMP). In this study, we attempted to profile the LMMP from Lignosus rhinocerus, a wild medicinal mushroom, grown by static cultures (SC) and in stirred tank reactor (STR). Crude water extra...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2011.07.005
更新日期：2011-10-01 00:00:00
abstract：:A rapid and reliable bacterial source tracking (BST) method is essential to counter risks to human health posed by fecal contamination of surface waters. Genetic fingerprinting methods, such as repetitive sequence based-PCR (rep-PCR), have shown promise as BST tools but are time-consuming and labor-intensive. In this ...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2006.10.012
更新日期：2007-03-01 00:00:00
abstract：:The reproducibility and reliability of quantitative microbiological assessments using the DNA-DNA hybridization "checkerboard method" (CKB) were assessed. The data originated from 180 chronic periodontitis patients, who were enrolled in a clinical trial and sampled at baseline, and 3 and 12m post-therapy. The samples ...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2015.01.010
更新日期：2015-03-01 00:00:00
abstract：:We developed an effective and rapid assay to detect both bio-energetic and envelope permeability (BEEP) alterations of Pseudomonas aeruginosa. The assay is based on quantification of extracellular ATP in bacterial cultures using luciferase as a reporter. To demonstrate the validity of our assay we conducted a biased s...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2016.04.009
更新日期：2016-06-01 00:00:00
abstract：:The gas vesicles of the cyanobacterium Microcystis sp. collapse under pressures ranging from 0.65-1.10 MPa, determined from turbidity changes in a pressure nephelometer. In turgid cells, collapse occurs at a lower range of pressures; the difference is equal to the cell turgor pressure. The turgor pressure decreases, h...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2009.02.005
更新日期：2009-05-01 00:00:00
abstract：:An alternative 16S rRNA-targeted oligonucleotide probe specific for Archaea was developed and used for detection of methanogens in anaerobic reactors. The designed probe was checked for its specificity by computer-aided comparative sequence analysis. For in situ application, optimal stringency conditions were adjusted...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2004.11.017
更新日期：2005-04-01 00:00:00
abstract：:Here we present the use of three fluorescent proteins in Staphylococcus aureus, Cerulean, PA-GFP, and mRFPmars. All molecules have an improved codon adaptation for expression in the A + T rich organisms and extend the fluorescent protein portfolio in staphylococcal research. ...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2010.07.022
更新日期：2010-10-01 00:00:00
abstract：:In recent years, the rapid advances of culture-independent methods and new molecular tools have revolutionized our understanding of microbial biodiversity and ecological functions. DNA extraction from microbial communities is a critical step in this process and several methods have been proposed and used, but the infl...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2019.02.005
更新日期：2019-04-01 00:00:00
abstract：:Treatment with 70% ethanol has been proposed as a safe and effective way to inactive bacteria for transport between laboratories prior to identification by MALDI-TOF MS. Ethanol alone does not inactivate spore-forming bacteria and additional chemical or physical treatment is necessary to guarantee inactivation of bact...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2020.105893
更新日期：2020-05-01 00:00:00
abstract：BACKGROUND:In a two-stage exchange protocol for prosthetic joint infection (PJI), bacteria surviving over the antibiotic-loaded cement spacers may cause the persistence of infection with renewed clinical symptoms following the surgery. Culture after sonication of removed prosthesis is more sensitive than conventional p...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2018.12.006
更新日期：2019-01-01 00:00:00
abstract：:Linear B-cell epitopes ((93)AKEFEAAAL(101) and (66)PQLTDVLN(73)) of CfaB were genetically fused to ltb-(gly)5-cfaB(1-25). Sera of rabbits immunized with fusion proteins reacted strongly with solid-phase bound ETEC bacteria bearing CFA/I fimbriae. Sera failed to agglutinate or inhibit hemagglutination promoted by CFA/I...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2014.07.035
更新日期：2014-11-01 00:00:00
abstract：:An optimized Citrobacter braakii phytase gene, appA-c, was chemically synthesized by oligonucleotides synthesis and over-lap PCR method. The appA-c gene encoding 423 amino acids was cloned into expression vector pPIC9 and transformed into methylotropic yeast Pichia pastoris. From about 2000 transformants, 400 transfor...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2009.08.018
更新日期：2009-10-01 00:00:00
abstract：:Flow cytometry was used to characterize bacterioplankton from two tropical environments in Brazil: the eutrophic Guanabara Bay and the oligotrophic southwest Atlantic Ocean. Bacterial abundance was evaluated by flow cytometry, and cells were stained with SYTO 13, allowing demonstration of differences in nucleic acid c...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2003.08.002
更新日期：2003-12-01 00:00:00
abstract：:To establish a simple and rapid high-resolution melting curve (HRM) method, 5 different strains of Salmonella were identified by adding DNA denaturants at different concentrations into the HRM system to change the characteristics of DNA melting and to obtain different Tm (dissolving temperature) values of DNA from dif...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2019.105806
更新日期：2020-05-01 00:00:00
abstract：:Blue native electrophoresis (BNE) has become the most popular method for the global analysis of membrane protein complexes. Although it has been shown to be very useful for that purpose, it can produce the dissociation of complexes with weak interactions and, due to the use of Coomassie Brilliant Blue, does not allow ...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2014.05.003
更新日期：2014-07-01 00:00:00
abstract：:The Carba NP test was developed to detect carbapenemase-producing Enterobacteriaceae, and uses imipenem as the reaction substrate. In Japan, IMP-6 metallo-β-lactamase (MBL) producers, which are usually resistant to meropenem but susceptible to imipenem, and IMP-1 MBL producers, which are usually resistant to both carb...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2016.12.015
更新日期：2017-02-01 00:00:00
abstract：:Virulence is the relative capacity of a pathogenic microorganism to cause damage in susceptible host cells such as those found in airway passages and the gut. In this study, the effect of clinical bacterial isolates on the monolayer integrity of cultured human alveolar basal epithelial cells (A549) was evaluated using...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2020.105833
更新日期：2020-02-01 00:00:00
abstract：:In this study extruded bean was used as a nitrogen source substitute in culture medium formulation. A 3-factor simplex-lattice mixture design was used to establish better growth conditions. Completely substituted medium resulted in 43% of increase in the growth of Saccharomyces cerevisiae. Mixtures containing 1% extru...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2013.01.002
更新日期：2013-03-01 00:00:00
abstract：:Uropathogenic Escherichia coli (UPEC) is the most common causal agent of urinary tract infections (UTIs) in humans. Currently, clinical detection methods take hours (dipsticks) to days (culturing methods), limiting rapid intervention. As an alternative, the use of molecular methods could improve speed and accuracy, bu...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2019.105799
更新日期：2020-02-01 00:00:00
abstract：:Analysis of microbial community structure in complex environmental samples using nucleic acid techniques requires efficient unbiased DNA extraction procedures; however, humic acids and other contaminants complicate the isolation of PCR-amplifiable DNA from compost and other organic-rich samples. In this study, combina...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/s0167-7012(01)00377-3
更新日期：2002-05-01 00:00:00
abstract：:Leishmaniasis, caused by protozoa belonging to the genus Leishmania, is an important public health problem found in >90 countries and with still limited options for treatment. Development of new anti-leishmanial drugs is an urgent need and the identification of new active compounds is a limiting factor that can be acc...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2019.105732
更新日期：2019-11-01 00:00:00
abstract：:Polyhydroxyalkanoates (PHAs) are important for biodegradable plastic production, and prokaryotes play a very important role in PHA production. PHA synthase is a key enzyme for the polymerization of PHAs. There are four classes of PHA synthase. The phaC gene is necessary for the production of all classes of PHA synthas...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2015.06.012
更新日期：2015-09-01 00:00:00
abstract：:To rapidly and simply determine whether or not bacterial colonies growing on agar were Vibrio parahaemolyticus, we developed an immunochromatographic assay (VP-ICA) using two different monoclonal antibodies (designated mAb-VP34 and mAb-VP109) against the delta subunit of V. parahaemolyticus-F0F1 ATP synthase. The epit...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2015.06.009
更新日期：2015-09-01 00:00:00
abstract：:Yersinia pestis is a Department of Health and Human Services select agent as defined in federal regulations. Certain attenuated strains of Y. pestis, such as the pgm(-) strain, are exempt from these regulations. Herein we describe a strategy to verify the absence of the pgm locus in Y. pestis strains being considered ...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2009.02.013
更新日期：2009-06-01 00:00:00
abstract：:Fluorescence in situ hybridization (FISH) can provide information on the morphology, spatial arrangement, and local environment of individual cells enabling the investigation of intact microbial communities. GeneFISH uses polynucleotide probes and enzymatic signal amplification to detect genes that are present in low ...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2021.106147
更新日期：2021-01-22 00:00:00
