scholarly journals Molecular Detection and Identification of Intimin Alleles in Pathogenic Escherichia coli by Multiplex PCR

1999 ◽  
Vol 37 (8) ◽  
pp. 2719-2722 ◽  
Author(s):  
Sean D. Reid ◽  
David J. Betting ◽  
Thomas S. Whittam
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Molecular Detection ◽  
Pcr Assay ◽  
E Coli ◽  
Detection And Identification ◽  
Pathogenic Escherichia Coli

A multiplex PCR was designed to detect the eae gene and simultaneously identify specific alleles in pathogenicEscherichia coli. The method was tested on 87 strains representing the diarrheagenic E. coli clones. The results show that the PCR assay accurately detects eae and resolves alleles encoding the α, β, and γ intimin variants.

scholarly journals OPTIMIZATION OF MOLECULAR DETECTION FOR Vibrio cholerae and PATHOGENIC Escherichia coli USING MULTIPLEX PCR

2021 ◽  
pp. e299
Author(s):  
Diana Elizabeth Waturangi ◽  
Jason Petrus ◽  
Rico Kosasih ◽  
Felicia Roseline
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Vibrio Cholerae ◽  
False Positive ◽  
Rapid Detection ◽  
Pathogenic Bacteria ◽  
Molecular Detection ◽  
False Negative ◽  
E Coli ◽  
Pathogenic Escherichia Coli

Vibrio cholerae and pathogenic Escherichia coli were considered as main causative agent foodborne diseases especially in many developing countries, such as Indonesia. Thereby, rapid detection of these pathogenic bacteria is necessary to treat food-borne related diseases causing by these bacteria. In this case, multiplex PCR allows multiple genes amplification in one reaction thereby enable to perform rapid detection of these pathogenic bacteria. The objective of this study is to optimize uniplex and multiples PCR of V. cholerae and pathogenic E. coli detection and determine the sensitivity and specificity of this assays. We used various virulence genes for each pathogenic bacterium as markers for uniplex and multiplex PCR detection. Based on this research, the optimum results of V. cholerae and pathogenic E. coli were obtained with a primer concentration of 16 µM for ctxA and ompU, 30 µM for ace, and 50 µM for zot, and toxR; 2 µM for elt and 5 µM for stx, respectively. Finally, based on the standardization method by ISO/TS 20836 these assays had 0% false positive, 0% false negative, 100% specificity, and 100% sensitivity; 0% false positive, 4% false negative, 100% specificity, and 96% sensitivity for V. cholerae and pathogenic E. coli respectively. The optimized method was qualified to be used as a molecular detection for V. cholerae as well as EHEC and ETEC detection according to ISO/TS 20836 (2017)  from drinking water samples.

Usefulness of a Multiplex PCR for Detection of Diarrheagenic Escherichia coli in a Diagnostic Microbiology Laboratory Setting

2016 ◽  
Vol 1 (2) ◽  
pp. 38-42 ◽  
Author(s):  
Khairun Nessa ◽  
Dilruba Ahmed ◽  
Johirul Islam ◽  
FM Lutful Kabir ◽  
M Anowar Hossain
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Clinical Laboratory ◽  
Proteinase K ◽  
Microbiology Laboratory ◽  
Pcr Assay ◽  
E Coli ◽  
Diarrheagenic Escherichia Coli ◽  
Multiplex Pcr Assay ◽  
Diagnostic Microbiology

A multiplex PCR assay was evaluated for diagnosis of diarrheagenic Escherichia coli in stool samples of patients with diarrhoea submitted to a diagnostic microbiology laboratory. Two procedures of DNA template preparationproteinase K buffer method and the boiling method were evaluated to examine isolates of E. coli from 150 selected diarrhoeal cases. By proteinase K buffer method, 119 strains (79.3%) of E. coli were characterized to various categories by their genes that included 55.5% enteroaggregative E. coli (EAEC), 18.5% enterotoxigenic E. coli (ETEC), 1.7% enteropathogenic E. coli (EPEC), and 0.8% Shiga toxin-producing E. coli (STEC). Although boiling method was less time consuming (<24 hrs) and less costly (<8.0 US $/ per test) but was less efficient in typing E. coli compared to proteinase K method (41.3% vs. 79.3% ; p<0.001). The sensitivity and specificity of boiling method compared to proteinase K method was 48.7% and 87.1% while the positive and negative predictive value was 93.5% and 30.7%, respectively. The majority of pathogenic E. coli were detected in children (78.0%) under five years age with 53.3% under one year, and 68.7% of the children were male. Children under 5 years age were frequently infected with EAEC (71.6%) compared to ETEC (24.3%), EPEC (2.7%) and STEC (1.4%). The multiplex PCR assay could be effectively used as a rapid diagnostic tool for characterization of diarrheagenic E. coli using a single reaction tube in the clinical laboratory setting.Bangladesh J Med Microbiol 2007; 01 (02): 38-42

scholarly journals Molecular Detection of Diarrheagenic Escherichia coli from Children with Acute Diarrhea in Tertiary Care Hospitals of Dhaka, Bangladesh.

2013 ◽  
Vol 5 (2) ◽  
pp. 59-66 ◽  
Author(s):  
Sushmita Roy ◽  
SM Shamsuzzaman ◽  
Kazi Z Mamun
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Acute Diarrhea ◽  
Tertiary Care ◽  
Medical Science ◽  
Pcr Assay ◽  
E Coli ◽  
Diarrheagenic Escherichia Coli ◽  
Multiplex Pcr Assay ◽  
Stool Samples

Objective: Multiplex PCR assay was used for diagnosis of diarrheagenic Escherichia coli (DEC) in stool samples of children (under 5 years) with acute diarrhea.  Methods: Samples were collected from January 2011 to December 2011, from Dhaka Medical College Hospital and Dhaka Shishu Hospital. Multiplex PCR with five specific primer pairs to detect enteropathogenic E. coli (eae, bfp), enterotoxigenic E. coli (lt, st) and enteroaggregative E. coli (aat) were used. However, enteroinvasive E. coli, enterohemorrhagicE. coli and diffusely adhererentE. coli were not sought. Result: In total, 135 (67.5%) E. coli were isolated from 200 stool samples. The prevalence of DEC was 68 (34%). Among DEC, most frequently isolated pathotype was EPEC 40 (58.82%), followed by ETEC 24 (35.29%) and EAggEC 18 (26.47%). Among the EPEC, 5 (12.5%) were typical EPEC. Among the 68 DEC positive cases, 22 samples contained more than one pathogenic gene in various combinations. Among the combination of DEC, EPEC+ETEC combination was 6 (27.27%) followed by ETEC+EAggEC 4 (18.18%), EPEC+EAggEC and ETEC+EPEC+EAggEC were both in 3 (13.6%). Conclusion:This study shows that DEC is a common cause of childhood diarrhea in Dhaka city of Bangladesh. By using multiplex PCR assay, DEC can be diagnosed in one PCR reaction that makes a conclusive diagnosis of diarrhea. DOI: http://dx.doi.org/10.3126/ajms.v5i2.8576 Asian Journal of Medical Science, Volume-5(2) 2014: 59-66

scholarly journals Virulence gene profiles of avian pathogenic Escherichia coli isolated from chickens with colibacillosis in Bulawayo, Zimbabwe

2015 ◽  
Vol 82 (1) ◽  
Author(s):  
Joshua Mbanga ◽  
Yvonne O. Nyararai
Keyword(s):  
Escherichia Coli ◽  
Virulence Genes ◽  
Virulence Gene ◽  
Economic Losses ◽  
Avian Pathogenic Escherichia Coli ◽  
Pcr Assay ◽  
E Coli ◽  
Gene Profiles ◽  
Pathogenic Escherichia Coli ◽  
Polymerase Chain

Colibacillosis, a disease caused by avian pathogenic Escherichia coli (APEC), is one of the main causes of economic losses in the poultry industry worldwide. This study was carried out in order to determine the APEC-associated virulence genes contained by E. coli isolates causing colibacillosis in chickens. A total of 45 E. coli isolates were obtained from the diagnostics and research branch of the Central Veterinary Laboratories, Bulawayo, Zimbabwe. These isolates were obtained from chickens with confirmed cases of colibacillosis after postmortem examination. The presence of the iutA, hlyF, ompT, frz, sitD, fimH, kpsM, sitA, sopB, uvrY, pstB and vat genes were investigated by multiplex polymerase chain reaction (PCR) assay. Of the 45 isolates, 93% were positive for the presence of at least one virulence gene. The three most prevalent virulence genes were iutA (80%), fimH (33.3%) and hlyF (24.4%). The kpsM, pstB and ompT genes had the lowest prevalence, having been detected in only 2.2% of the isolates. All 12 virulence genes studied were detected in the 45 APEC isolates. Virulence gene profiles were constructed for each APEC isolate from the multiplex data. The APEC isolates were profiled as 62.2% fitting profile A, 31.1% profile B and 6.7% profile C. None of the isolates had more than seven virulence genes. Virulence profiles of Zimbabwean APEC isolates are different from those previously reported. Zimbabwean APEC isolates appear to be less pathogenic and may rely on environmental factors and stress in hosts to establish infection.

scholarly journals A combined AFLP–multiplex PCR assay for molecular typing of Escherichia coli strains using variable bacterial interspersed mosaic elements

2004 ◽  
Vol 132 (1) ◽  
pp. 61-65 ◽  
Author(s):  
R. HORVÁTH ◽  
M. DENDIS ◽  
J. SCHLEGELOVÁ ◽  
F. RŮŽIČKA ◽  
J. BENEDÍK
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Molecular Typing ◽  
Original Method ◽  
Pcr Assay ◽  
E Coli ◽  
Promising Tool ◽  
Multiplex Pcr Assay ◽  
Chromosomal Sequences

The original method for molecular typing of E. coli strains was developed using the polymorphism in chromosomal sequences of bacterial interspersed mosaic elements (BIMEs) detected by multiplex PCR and analysed by AFLP assay. The applicability of the method in the epidemiology of E. coli was tested on a group of 524 strains of human and veterinary origin. In the studied group 18 different genotypes were detected. Significant differences were found in the frequencies of the genotypes among various groups of strains, suggesting the method could be a promising tool in the epidemiology of E. coli.

IDENTIFICATION OF THE AVIAN PATHOTYPE OF ESCHERICHIA COLI ON LAYER FLOCKS BY MULTIPLEX PCR

2019 ◽  
Vol 7 ◽  
pp. 905-911
Author(s):  
Marilena Burtan ◽  
Virgilia Popa ◽  
Maria Rodica Gurau ◽  
Doina Danes
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Virulence Genes ◽  
Virulence Gene ◽  
Economic Losses ◽  
Ompa Gene ◽  
Avian Pathogenic Escherichia Coli ◽  
E Coli ◽  
Pathogenic Escherichia Coli

Introduction: Colibacillosis in poultry is determined by avian pathogenic Escherichia coli (APEC) and represents an important source of economic losses  in the poultry industry. APEC’s pathogenicity relies on the presence and expression of different virulence factors. The genes ompA , iss  and  fimH, encoding the outer membrane protein, the protein inducing resistance to complement and the synthesis of type 1 fimbria are present in APEC strains. Objective: Escherichia coli strains isolated from layers were analysed to assess the pathotype they belong to. Methods: In order to detect the three genes associated with APEC strains, 16 E. coli isolates were investigated for virulence associated genes ompA, iss and fimH, using multiplex PCR. Results: From the 16 E.coli strains submitted, multiplex PCR assessment revealed that 14 (87.5%) of the E. coli strains isolated contained at least one virulence gene, while 2 (12.5%) strains did not harbour any of the virulence genes tested. The fimH gene was noted in 13 (81.25%) of the strains tested, the ompA gene has been present in 12 (75%) strains and the iss gene was present in 9 (56.25%) strains. Eight (50%) strains were found to present all three investigated genes. Conclusion: Presence of these genes is a strong indicatory to consider those strains as belonging to the APEC pathotype.

Molecular Detection of wzx1 and wzy Genes in Multi Drugs Resistance E. coli Isolates

2018 ◽  
Vol 9 (3) ◽  
Author(s):  
Mona Al-Terehi ◽  
Saba Saadoon Khazaal ◽  
Haidar J Muhammed ◽  
Russul Hikmat Behjet
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Gene Cluster ◽  
Molecular Detection ◽  
E Coli ◽  
Pcr Products ◽  
New Finding ◽  
O Antigens ◽  
Positive Results

Escherichia coli have been genetically changes in Iraqi environments, the present study was carried out to detection E coli isolates sero-group using o-antigens gene cluster, tow genes were amplified used multiplex PCR, the results of present study show that present isolates have more than one plasmid in different size (500-700 bp). PCR results show about 100% of isolates were had a positive results of PCR products of O45 wzx1 gene (451 bp) while 25% of isolates were a positive PCR products of O45 wzy1 gene (255 bp) and about 25% of isolates have tow genes. A new finding in present study that 50% of isolates have other copy of O45 wzx1 gene, these results concluded that it may be important in Iraqi isolates identification and classification however we need more information about genes sequencing.

Development of a Multiplex PCR for Rapid Detection of Verocytotoxin-Producing Escherichia coli O26 in Raw Milk and Ground Beef

2011 ◽  
Vol 74 (1) ◽  
pp. 13-17 ◽  
Author(s):  
V. LORUSSO ◽  
A. DAMBROSIO ◽  
N. C. QUAGLIA ◽  
A. PARISI ◽  
G. LASALANDRA ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Rapid Detection ◽  
Ground Beef ◽  
Raw Milk ◽  
The United States ◽  
Pcr Assay ◽  
E Coli ◽  
Identification And Characterization

Verocytotoxin-producing Escherichia coli (VTEC) O26 is an emergent pathotype that has caused an increasing number of sporadic cases and outbreaks of gastroenteritis, hemorrhagic colitis, and hemolytic uremic syndrome in the United States and Europe. Many cases are associated with the consumption of milk and undercooked or fermented meats. The stx2 strains of VTEC O26 seem to be more likely to cause human infections than isolates expressing only stx1. The isolation and identification of VTEC O26 from foods is labor intensive and time-consuming. We developed a multiplex PCR (M-PCR) assay for the identification and characterization of E. coli O26 VTEC and its detection in raw milk and ground beef. The method is based on the amplification of the wzx, stx1, and stx2 genes for the simultaneous detection of the O26 antigen and verocytotoxin types 1 and 2. This M-PCR assay had a sensitivity of 108 CFU/ml when applied to a bacterial suspension and of 106 CFU/ml or g when applied to both inoculated milk and minced beef samples. This M-PCR assay also was highly specific, and results were consistently negative for negative controls (nonpathogenic E. coli strains, uninoculated milk and beef samples, and samples inoculated with the nontarget microorganisms). This method could be used for the rapid detection of E. coli O26 VTEC from foods and for the rapid identification and characterization of clinical and environmental isolates.

scholarly journals Analysis of the F Antigen-Specific papAAlleles of Extraintestinal Pathogenic Escherichia coli Using a Novel Multiplex PCR-Based Assay

2000 ◽  
Vol 68 (3) ◽  
pp. 1587-1599 ◽  
Author(s):  
James R. Johnson ◽  
Adam L. Stell ◽  
Flemming Scheutz ◽  
Timothy T. O'Bryan ◽  
Thomas A. Russo ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Horizontal Transfer ◽  
Antigenic Determinant ◽  
False Negative ◽  
Phylogenetic Distribution ◽  
Specific Detection ◽  
Pcr Assay ◽  
E Coli ◽  
Pathogenic Escherichia Coli ◽  
Structural Subunit

ABSTRACT Polymorphisms in PapA, the major structural subunit and antigenic determinant of P fimbriae of extraintestinal pathogenicEscherichia coli, are of considerable epidemiological, phylogenetic, and immunotherapeutic importance. However, to date, no method other than DNA sequencing has been generally available for their detection. In the present study, we developed and rigorously validated a novel PCR-based assay for the 11 recognized variants ofpapA and then used the new assay to assess the prevalence, phylogenetic distribution, and bacteriological associations of thepapA alleles among 75 E. coli isolates from patients with urosepsis. In comparison with conventional F serotyping, the assay was extremely sensitive and specific, evidence thatpapA sequences are highly conserved within each of the traditionally recognized F serotypes despite the diversity observed among F types. In certain strains, the assay detected serologically occult copies of papA, of which some were shown to represent false-negative serological results and others were shown to represent the presence of nonfunctional pap fragments. Among the urosepsis isolates, the assay revealed considerable segregation of papA alleles according to O:K:H serotype, consistent with vertical transmission within clones, but with exceptions which strongly suggested horizontal transfer ofpapA alleles between lineages. Sequencing ofpapA from two strains that were papA positive by probe and PCR but F negative in the new PCR assay led to the discovery of two novel papA variants, one of which was actually more prevalent among the urosepsis isolates than were several of the known papA alleles. These findings provide novel insights into the papA alleles of extraintestinal pathogenic E. coli and indicate that the F PCR assay represents a versatile new molecular tool for epidemiological and phylogenetic investigations which should make rapid, specific detection of papA alleles available to any laboratory with PCR capability.

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