Comparison of direct PCR and PCR amplification after DNA extraction for the detection of viable enterotoxigenic Escherichia coli in laboratory microcosms

1996 ◽  
Vol 26 (1-2) ◽  
pp. 21-26 ◽  
Author(s):  
Zohreh Tamanai-Shacoori ◽  
Anne Jolivet-Gougeon ◽  
Michel Cormier
Keyword(s):  
Escherichia Coli ◽  
Dna Extraction ◽  
Pcr Amplification ◽  
Enterotoxigenic Escherichia Coli ◽  
Direct Pcr

Simple ‘universal’ DNA extraction procedure compatible with direct PCR amplification

1996 ◽  
Vol 52 (4) ◽  
pp. 295-295 ◽  
Author(s):  
D. Goldenberger ◽  
I. Perschil ◽  
M. Ritzler ◽  
M. Altwegg
Keyword(s):  
Dna Extraction ◽  
Extraction Procedure ◽  
Pcr Amplification ◽  
Direct Pcr

Detection of enterotoxigenic Escherichia coli in water by polymerase chain reaction amplification and hybridization

1994 ◽  
Vol 40 (4) ◽  
pp. 243-249 ◽  
Author(s):  
Z. Tamanai-Shacoori ◽  
A. Jolivet-Gougeon ◽  
M. Pommepuy ◽  
M. Cormier ◽  
R. R. Colwell
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Polymerase Chain Reaction Amplification ◽  
Pcr Amplification ◽  
Environmental Water ◽  
Latex Agglutination ◽  
Enterotoxigenic Escherichia Coli ◽  
Enzyme Linked Immunosorbent Assay ◽  
Chain Reaction ◽  
Polymerase Chain

Enterotoxigenic Escherichia coli was studied in waste water, river water, and seawater from six locations along the west coast of Normandy by using the polymerase chain reaction (PCR) to amplify the heat labile (LT) gene. Cellular DNA was extracted from centrifugation pellets and amplified using PCR. The PCR products were detected by gel electrophoresis and confirmed by hybridization assay, using an 850 base pair HindIII DNA fragment probe from pEWD299 conjugated to digoxigenin and specific for the LT gene. Results of the PCR amplification were compared with those of GM1 enzyme-linked immunosorbent assay, latex agglutination, and colony hybridization. The PCR method was found to be more precise and less time consuming, especially when compared with methods requiring culture of isolates for enumeration of enterotoxigenic E. coli in water.Key words: enterotoxigenic Escherichia coli, PCR, environmental water, digoxigenin.

scholarly journals Comparison and Validation of Ichthyoplankton DNA Extraction Methods

2021 ◽  
Vol 4 (4) ◽  
pp. 87
Author(s):  
Diouri Lamia ◽  
Uwiringiyeyezu Théophile ◽  
Abdelouahab Hinde ◽  
Malki Mohamed ◽  
Baibai Tarik ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Dna Extraction ◽  
Classical Method ◽  
Pcr Amplification ◽  
Temporal Distribution ◽  
Extraction Methods ◽  
Direct Pcr ◽  
Fisheries Resources ◽  
Morphological Criteria

Ichthyoplankton is the cluster of planktonic organisms that consists of fish eggs and larvae. These planktonic stages belong to the temporary zooplankton, representing future exploitable stocks. The study of the early ontogenesis of fish plays a key role in the understanding and evaluation of these populations through the study of their abundance and their spatio-temporal distribution. To better understand and protect these fisheries resources, it is essential to identify the different stages of fish embryonic development. This identification is usually performed using the classical method, based on morphological criteria under a binocular magnifying glass; however, this methodology is not always sufficient and is time consuming and, therefore, it is necessary to rely increasingly on molecular tools. The major problem with these tools is the yield and quality of the nucleic acids extracted from ichthyoplankton, especially in the case of eggs, which are small. Several methods have been used for DNA extraction from ichthyoplankton, either automated or manual, but very often from larvae or adults. In the present work, five fish egg DNA extraction protocols were compared based on their DNA yield and extraction quality, verified by agarose gel electrophoresis and quantitative PCR amplification. The results showed that extraction by our heat-protocol for direct PCR (Hp-dPCR) presents the simplest and cheapest protocol of all the kits used in this study, providing a sufficient quantity and quality of nucleic acids to be used for PCR amplification, and being within the reach of third world laboratories that often do not have sufficiently large budgets to obtain automated kits.

scholarly journals Improved direct lysis PCR amplification method of microalgal culture for sequencing and species identification

2021 ◽  
Vol 948 (1) ◽  
pp. 012013
Author(s):  
F Fitriyah ◽  
Y Faramitha ◽  
D A Sari ◽  
I Kresnawaty ◽  
T Panji ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Species Identification ◽  
Dna Barcode ◽  
Pcr Amplification ◽  
Extraction Methods ◽  
Rbcl Gene ◽  
Direct Pcr ◽  
Nannochloropsis Gaditana ◽  
Amplification Method ◽  
Microalgal Culture

Abstract Molecular approach plays important role in species identification for microalgae which involves sequencing of specific DNA barcode present in the genome. This approach involved preparation of template DNA for polymerase chain reaction (PCR) which is time consuming and requires large amounts of algal cells. Microalgal direct PCR have been used frequently for species identification, which simplified the DNA isolation procedure. However, the recent attempts to amplify the rbcL gene of microalga using the previously reported protocol led to poor repeatability. In this study, Nannochloropsis gaditana NIES-2587 was cultured in f/2 liquid medium. The culture growth was estimated on optical density value and the lysis process was improved using gradual temperature procedure during the PCR process. The same culture was extracted using manual DNA extraction method for comparison. The DNA obtained from both methods were amplified using RbclN primer pair to amplify 1486 bp partial sequence of Nannochloropsis rbcL gene, followed by the sequencing of the PCR product. Molecular identification based on the sequence result and BLAST analysis indicated that direct PCR and manual DNA extraction methods successfully produced high sequences result and confirmed the identity of microalgae species into N. gaditana strain CCMP527 with a genetic similarity of >99%.

scholarly journals DMSO Improves the Ski-Slope Effect in Direct PCR

2021 ◽  
Vol 11 (4) ◽  
pp. 1943
Author(s):  
Joo-Young Kim ◽  
Ju Yeon Jung ◽  
Da-Hye Kim ◽  
Seohyun Moon ◽  
Won-Hae Lee ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Pcr Amplification ◽  
Analytical Techniques ◽  
Direct Pcr ◽  
Dna Profile ◽  
Novel Technologies ◽  
Specific Amplification ◽  
Polymerase Chain ◽  
Dna Profiles

Analytical techniques such as DNA profiling are widely used in various fields, including forensic science, and novel technologies such as direct polymerase chain reaction (PCR) amplification are continuously being developed in order to acquire DNA profiles efficiently. However, non-specific amplification may occur depending on the quality of the crime scene evidence and amplification methods employed. In particular, the ski-slope effect observed in direct PCR amplification has led to inaccurate interpretations of the DNA profile results. In this study, we aimed to reduce the ski-slope effect by using dimethyl sulfoxide (DMSO) in direct PCR. We confirmed that DMSO (3.75%, v/v) increased the amplification yield of large-sized DNA sequences more than that of small-sized ones. Using 50 Korean buccal samples, we further demonstrated that DMSO reduced the ski-slope effect in direct PCR. These results suggest that the experimental method developed in this study is suitable for direct PCR and may help to successfully obtain DNA profiles from various types of evidence at crime scenes.

scholarly journals Structure of the master regulator Rns reveals an inhibitor of enterotoxigenic Escherichia coli virulence regulons

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Charles R. Midgett ◽  
Kacey Marie Talbot ◽  
Jessica L. Day ◽  
George P. Munson ◽  
F. Jon Kull
Keyword(s):  
Escherichia Coli ◽  
Small Molecules ◽  
Family Member ◽  
Shigella Flexneri ◽  
Decanoic Acid ◽  
Enterotoxigenic Escherichia Coli ◽  
Gram Negative Bacteria ◽  
Enteric Infections ◽  
First Time ◽  
Arac Family

AbstractEnteric infections caused by the gram-negative bacteria enterotoxigenic Escherichia coli (ETEC), Vibrio cholerae, Shigella flexneri, and Salmonella enterica are among the most common and affect billions of people each year. These bacteria control expression of virulence factors using a network of transcriptional regulators, some of which are modulated by small molecules as has been shown for ToxT, an AraC family member from V. cholerae. In ETEC the expression of many types of adhesive pili is dependent upon the AraC family member Rns. We present here the 3 Å crystal structure of Rns and show it closely resembles ToxT. Rns crystallized as a dimer via an interface similar to that observed in other dimeric AraC’s. Furthermore, the structure of Rns revealed the presence of a ligand, decanoic acid, that inhibits its activity in a manner similar to the fatty acid mediated inhibition observed for ToxT and the S. enterica homologue HilD. Together, these results support our hypothesis that fatty acids regulate virulence controlling AraC family members in a common manner across a number of enteric pathogens. Furthermore, for the first time this work identifies a small molecule capable of inhibiting the ETEC Rns regulon, providing a basis for development of therapeutics against this deadly human pathogen.

scholarly journals Deletion of FaeG alleviated Enterotoxigenic Escherichia coli F4ac-induced apoptosis in the intestine

AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Pengpeng Xia ◽  
Yunping Wu ◽  
Siqi Lian ◽  
Guomei Quan ◽  
Yiting Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Clinical Symptoms ◽  
Mucosal Damage ◽  
Enterotoxigenic Escherichia Coli ◽  
Intestinal Epithelial ◽  
Antibody Microarray ◽  
E Coli ◽  
Fimbrial Subunit ◽  
Induced Apoptosis ◽  
Weaning Piglets

AbstractEnterotoxigenic Escherichia coli (ETEC) F4ac is a major constraint to the development of the pig industry, which is causing newborn and post-weaning piglets diarrhea. Previous studies proved that FaeG is the major fimbrial subunit of F4ac E. coli and efficient for bacterial adherence and receptor recognition. Here we show that the faeG deletion attenuates both the clinical symptoms of F4ac infection and the F4ac-induced intestinal mucosal damage in piglets. Antibody microarray analysis and the detection of mRNA expression using porcine neonatal jejunal IPEC-J2 cells also determined that the absence of FaeG subunit alleviated the F4ac promoted apoptosis in the intestinal epithelial cells. Thus, targeted depletion of FaeG is still beneficial for the prevention or treatment of F4ac infection.

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