Structural comparison of O-antigen gene clusters of Legionella pneumophila and its application of a serogroup-specific multiplex PCR assay

2015 ◽  
Vol 108 (6) ◽  
pp. 1405-1423 ◽  
Author(s):  
Boyang Cao ◽  
Zhenyang Tian ◽  
Suwei Wang ◽  
Zhiyan Zhu ◽  
Yamin Sun ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Legionella Pneumophila ◽  
Gene Clusters ◽  
Structural Comparison ◽  
Pcr Assay ◽  
Antigen Gene ◽  
O Antigen ◽  
Multiplex Pcr Assay

scholarly journals Genetic Analysis of the Cronobacter sakazakii O4 to O7 O-Antigen Gene Clusters and Development of a PCR Assay for Identification of All C. sakazakii O Serotypes

2012 ◽  
Vol 78 (11) ◽  
pp. 3966-3974 ◽  
Author(s):  
Yamin Sun ◽  
Min Wang ◽  
Quan Wang ◽  
Boyang Cao ◽  
Xin He ◽  
...  
Keyword(s):  
Gene Clusters ◽  
Cronobacter Sakazakii ◽  
Pcr Assay ◽  
Gram Negative ◽  
Content Type ◽  
Antigen Gene ◽  
O Antigen ◽  
Multiplex Pcr Assay ◽  
Food Borne ◽  
Invasive Infections

ABSTRACTThe Gram-negative bacteriumCronobacter sakazakiiis an emerging food-borne pathogen that causes severe invasive infections in neonates. Variation in the O-antigen lipopolysaccharide in the outer membrane provides the basis for Gram-negative bacteria serotyping. The O-antigen serotyping scheme forC. sakazakii, which includes seven serotypes (O1 to O7), has been recently established, and the O-antigen gene clusters and specific primers for threeC. sakazakiiserotypes (O1, O2, and O3) have been characterized. In this study, theC. sakazakiiO4, O5, O6, and O7 O-antigen gene clusters were sequenced, and gene functions were predicted on the basis of homology.C. sakazakiiO4 shared a similar O-antigen gene cluster withEscherichia coliO103. The general features and anomalies of all sevenC. sakazakiiO-antigen gene clusters were evaluated and the relationship between O-antigen structures and their gene clusters were investigated. Serotype-specific genes for O4 to O7 were identified, and a molecular serotyping method for allC. sakazakiiO serotypes, a multiplex PCR assay, was developed by screening against 136 strains ofC. sakazakiiand closely related species. The sensitivity of PCR-based serotyping method was determined to be 0.01 ng of genomic DNA and 103CFU of each strain/ml. This study completes the elucidation ofC. sakazakiiO-antigen genetics and provides a molecular method suitable for the identification ofC. sakazakiiO1 to O7 strains.

scholarly journals Molecular analysis ofShigella boydiiO1 O-antigen gene cluster and its PCR typing

2005 ◽  
Vol 51 (5) ◽  
pp. 387-392 ◽  
Author(s):  
Jiang Tao ◽  
Lei Wang ◽  
Dan Liu ◽  
Yue Li ◽  
David A Bastin ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Gene Clusters ◽  
Pcr Assay ◽  
Pcr Screening ◽  
E Coli ◽  
Antigen Gene ◽  
O Antigen ◽  
Genes Encoding ◽  
Type Strains ◽  
Overnight Culture

Shigella is an important human pathogen and is closely related to Escherichia coli. O-antigen is the most variable part of the lipopolysaccharide on the cell surface of Gram-negative bacteria and plays an important role in pathogenicity. The O-antigen gene cluster of S. boydii O1 was sequenced. The putative genes encoding enzymes for rhamnose synthesis, transferases, O-unit flippase, and O-unit polymerase were identified on the basis of homology. The O-antigen gene clusters of S. boydii O1 and E. coli O149, which share the same O-antigen form, were found to have the same genes and organization by adjacent gene PCR assay. Two genes specific for S. boydii O1 and E. coli O149 were identified by PCR screening against E. coli- and Shigella-type strains of the 186 known O-antigen forms and 39 E. coli clinical isolates. A PCR sensitivity of 103to 104CFU/mL overnight culture of S. boydii O1 and E. coli O149 was obtained. S. boydii O1 and E. coli O149 were differentiated by PCR using lacZ- and cadA-based primers.Key words: O-antigen gene cluster, S. boydii O1, E. coli O149, molecular typing.

Escherichia coliserogroup O2 and O28ac O-antigen gene cluster sequences and detection of pathogenicE. coliO2 and O28ac by PCR

2010 ◽  
Vol 56 (4) ◽  
pp. 308-316 ◽  
Author(s):  
Pina M. Fratamico ◽  
Xianghe Yan ◽  
Yanhong Liu ◽  
Chitrita DebRoy ◽  
Brian Byrne ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Gene Cluster ◽  
Gene Clusters ◽  
Rapid Identification ◽  
Open Reading Frames ◽  
E Coli ◽  
Antigen Gene ◽  
O Antigen ◽  
Pcr Assays

The O-antigen gene clusters of Escherichia coli serogroups O2 and O28ac were sequenced, and PCR assays were developed to identify strains belonging to these 2 serogroups. Sixteen and 8 open reading frames were mapped to these loci in E. coli O2:H4 U 9-41 and E. coli O28ac:H25 96-3286, respectively. The wzx (O-antigen flippase) and wzy (O-antigen polymerase) genes in the E. coli O2 and O28ac O-antigen gene clusters were selected as targets for PCR assays for their identification. PCR assays targeting the wzx and wzy genes were specific for these serogroups, with one exception. Escherichia coli serogroup O42 strains gave positive results with wzx and wzy PCR assays targeting E. coli O28ac, and antiserum raised against O42 cross-reacted with serogroup O28ac strains. The O-antigen gene cluster of a strain of E. coli serogroup O42 was sequenced, and there were only 3 nt differences between the O-antigen gene clusters of the O28ac and O42 strains. Multiplex PCR assays targeting the O2 wzx gene, the stx1, stx2, hly, eae, and saa genes, and the O28ac wzx, ial, ipaC, and ipaH genes were developed for detecting Shiga toxin-producing E. coli O2 strains and enteroinvasive E. coli O28ac strains, respectively. The O2 and O28ac wzx and wzy genes can be used as diagnostic markers in PCR assays for rapid identification of these serogroups as an alternative to serotyping, and the multiplex PCR assays targeting serogroup-specific genes in combination with virulence genes can be used to identify and to detect pathogenic serogroup O2 and O28ac strains.

scholarly journals Development of PCR Assays Targeting Genes in O-Antigen Gene Clusters for Detection and Identification of Escherichia coli O45 and O55 Serogroups

2005 ◽  
Vol 71 (8) ◽  
pp. 4919-4924 ◽  
Author(s):  
Chitrita DebRoy ◽  
Pina M. Fratamico ◽  
Elisabeth Roberts ◽  
Michael A. Davis ◽  
Yanhong Liu
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Gene Cluster ◽  
Real Time Pcr ◽  
Gene Clusters ◽  
E Coli ◽  
Antigen Gene ◽  
O Antigen ◽  
Detection And Identification ◽  
Pcr Assays

ABSTRACT The Escherichia coli O45 O-antigen gene cluster of strain O45:H2 96-3285 was sequenced, and conventional (singleplex), multiplex, and real-time PCR assays were designed to amplify regions in the wzx (O-antigen flippase) and wzy (O-antigen polymerase) genes. In addition, PCR assays targeting the E. coli O55 wzx and wzy genes were designed based on previously published sequences. PCR assays targeting E. coli O45 showed 100% specificity for this serogroup, whereas by PCR assays specific for E. coli O55, 97/102 strains serotyped as E. coli O55 were positive for wzx and 98/102 for wzy. Multiplex PCR assays targeting the E. coli O45 and the E. coli O55 wzx and wzy genes were used to detect the organisms in fecal samples spiked at levels of 106 and 108 CFU/0.2 g feces. Thus, the PCR assays can be used to detect and identify E. coli serogroups O45 and O55.

scholarly journals Development of a Multiplex PCR Assay Targeting O-Antigen Modification Genes for Molecular Serotyping of Shigella flexneri

2011 ◽  
Vol 49 (11) ◽  
pp. 3766-3770 ◽  
Author(s):  
Qiangzheng Sun ◽  
Ruiting Lan ◽  
Yiting Wang ◽  
Ailan Zhao ◽  
Shaomin Zhang ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Shigella Flexneri ◽  
Pcr Assay ◽  
Molecular Serotyping ◽  
O Antigen ◽  
Multiplex Pcr Assay

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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