scholarly journals Comparison of multiplex PCR with serogrouping and PCR-RFLP of fliC gene for the detection of enteropathogenic Escherichia coli (EPEC)

2011 ◽  
Vol 15 (4) ◽  
pp. 365-369
Author(s):  
Saeid Bouzari ◽  
Mohammad M Aslani ◽  
Mana Oloomi ◽  
Anis Jafari ◽  
Amir Dashti
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Enteropathogenic Escherichia Coli ◽  
Flic Gene ◽  
Pcr Rflp

Molecular Strategies for the Detection, Identification, and Differentiation between Enteroinvasive Escherichia coli and Shigella spp.

2005 ◽  
Vol 68 (2) ◽  
pp. 239-245 ◽  
Author(s):  
CESAR I. BIN KINGOMBE ◽  
MARIA-LUCIA CERQUEIRA-CAMPOS ◽  
JEFFREY M. FARBER
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Shigella Flexneri ◽  
Shigella Dysenteriae ◽  
Shigella Boydii ◽  
Epidemiological Tool ◽  
Pcr Rflp ◽  
Shigella Spp

A strategy for the detection, identification, and differentiation of enteroinvasive Escherichia coli (EIEC) and Shigella spp. has been developed. The strategy includes (i) a multiplex PCR for the amplification of two virulence genes, i.e., iuc (222 bp) and ipaH (629 bp); (ii) amplification of the ial gene (a 1,038-bp amplicon) located within a large plasmid; and (iii) restriction fragment length polymorphism (RFLP) of the ial gene amplicon. The multiplex PCR provided three patterns. Pattern 1 (iuc−/ipaH+) was found in 10 (67%) of 15 EIEC strains tested, pattern 2 (iuc+/ipaH−) in only 2 (4.4%) of 46 non-EIEC isolates, whereas pattern 3 (iuc+/ipaH+) was observed in all Shigella spp. and also in 5 (33%) of 15 EIEC strains tested. The pattern 3 EIEC strains were all positive for the ial gene. The PCR-RFLP of the ial gene amplicon using the endonuclease AclI was used to differentiate Shigella spp. from the EIEC strains that belonged to pattern 3. The ial gene was present in 21 (38%) of 56 and 6 (40%) of 15 Shigella spp. and EIEC strains tested, respectively. The PCR-RFLP of the ial gene amplicon divided the strains in two types. Type 1 did not contain the restriction enzyme site and was found in 6 (100%) of 6 EIEC strains, 4 (80%) of 5 Shigella boydii, and 4 (100%) of 4 Shigella dysenteriae strains tested. Type 2, which gave two fragments of 286 and 752 bp, was observed in 5 (83%) of 6 Shigella flexneri strains and 6 (100%) of 6 Shigella sonnei strains. Detection, identification, and differentiation of Shigella spp. and EIEC were achieved by analyses of the PCR patterns and RFLP types. To our knowledge, this is the first study to demonstrate a simple and rapid method for detecting, identifying, and differentiating, at the molecular level, Shigella spp. and EIEC strains. This method will have tremendous utility as an epidemiological tool and in helping to develop policies, risk assessments, and national and international methods for Shigella spp.

Determination of flagellar types by PCR-RFLP analysis of enteropathogenic Escherichia coli (EPEC) and Shiga toxin-producing E. coli (STEC) strains isolated from animals in São Paulo, Brazil

2012 ◽  
Vol 92 (1) ◽  
pp. 18-23 ◽  
Author(s):  
Claudia de Oliveira Ayala ◽  
Ana Carolina Ramos Moreno ◽  
Marina Baquerizo Martinez ◽  
Ylanna Kelner Burgos ◽  
Antonio Fernando Pestana de Castro ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Rflp Analysis ◽  
Sao Paulo ◽  
São Paulo ◽  
Enteropathogenic Escherichia Coli ◽  
E Coli ◽  
Pcr Rflp

scholarly journals Identification of new flagellin-encoding fliC genes in Escherichia coli isolated from domestic animals using RFLP-PCR and sequencing methods

2013 ◽  
Vol 33 (4) ◽  
pp. 417-422 ◽  
Author(s):  
Cláudia de Moura ◽  
Monique Ribeiro Tiba ◽  
Marcio José da Silva ◽  
Domingos da Silva Leite
Keyword(s):  
Escherichia Coli ◽  
Fragment Length Polymorphism ◽  
Domestic Animals ◽  
Flic Gene ◽  
E Coli ◽  
New Genes ◽  
Pcr Rflp ◽  
Specific Antisera ◽  
Flagellar Antigen ◽  
Restriction Fragment Length

Identification of Escherichia coli requires knowledge regarding the prevalent serotypes and virulence factors profiles allows the classification in pathogenic/non-pathogenic. However, some of these bacteria do not express flagellar antigen invitro. In this case the PCR-restriction fragment length polymorphism (RFLP-PCR) and sequencing of the fliC may be suitable for the identification of antigens by replacing the traditional serology. We studied 17 samples of E. coli isolated from animals and presenting antigen H nontypeable (HNT). The H antigens were characterized by PCR-RFLP and sequencing of fliC gene. Three new flagellin genes were identified, for which specific antisera were obtained. The PCR-RFLP was shown to be faster than the serotyping H antigen in E. coli, provided information on some characteristics of these antigens and indicated the presence of new genes fliC.

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

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