A multiplex PCR assay for the identification of human diarrheagenic Escherichia coli was developed. The targets selected for each category were eae for enteropathogenic E. coli, stx for Shiga toxin-producing E. coli, elt and est for enterotoxigenic E. coli, ipaH for enteroinvasive E. coli, and aggR for enteroaggregative E. coli. This assay allowed the categorization of a diarrheagenic E. coli strain in a single reaction tube.

Multiplex PCR assay for identification of human diarrheagenic Escherichia coli.

Pathotypes of diarrheagenic Escherichia coli in children attending a tertiary care hospital in South India

Detection of the enteroaggregative Escherichia coli heat-stable enterotoxin 1 (EAST1) gene and its relationship with fimbrial and enterotoxin markers in E. coli isolates from pigs with diarrhoea.

A PCR-based assay that can simultaneously detect and differentiate five different types of nosocomial bacterial pathogens was developed. Six pairs of selected primers targeting femA (132 bp) and mecA (310 bp) of methicillin-resistant Staphylococcus aureus, gltA (722 bp) of Acinetobacter baumannii, phoA (903 bp) of Escherichia coli, mdh (364 bp) of Klebsiella pneumoniae and oprL (504 bp) of Pseudomonas aeruginosa were used in this study. The conditions were optimized for the multiplex PCR to ensure specific amplification of the selected targets. Sensitivity and specificity tests were also carried out using a blind test approach on 50 bacterial cultures and resulted in 100% for both positive and negative predictive values.

Simultaneous detection of methicillin-resistant Staphylococcus aureus, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa by multiplex PCR.

Since the initial discovery of universal stress protein A (UspA) 25 years ago, remarkable advances in molecular and biochemical technologies have revolutionized our understanding of biology. Many studies using these technologies have focused on characterization of the uspA gene and Usp-type proteins. These studies have identified the conservation of Usp-like proteins across bacteria, archaea, plants, and even some invertebrate animals. Regulation of these proteins under diverse stresses has been associated with different stress-response genes including spoT and relA in the stringent response and the dosR two-component signaling pathways. These and other foundational studies suggest Usps serve regulatory and protective roles to enable adaptation and survival under external stresses. Despite these foundational studies, many bacterial species have multiple paralogs of genes encoding these proteins and ablation of the genes does not provide a distinct phenotype. This outcome has limited our understanding of the biochemical functions of these proteins. Here, we summarize the current knowledge of Usps in general and UspA in particular across different genera as well as conclusions about their functions from seminal studies in diverse organisms. Our objective has been to organize the foundational studies in this field to identify the significant impediments to further understanding of Usp functions at the molecular level. We propose ideas and experimental approaches that may overcome these impediments and drive future development of molecular approaches to understand and target Usps as central regulators of stress adaptation and survival. Despite the fact that the full functions of Usps are still not known, creative many applications have already been proposed, tested, and used. The complementary approaches of basic research and applications, along with new technology and analytic tools, may yield the elusive yet critical functions of universal stress proteins in diverse systems.

Twenty-Five Years of Investigating the Universal Stress Protein: Function, Structure, and Applications.

A multiplex polymerase chain reaction (PCR) system was developed for identification of enterotoxigenic Escherichia coli (ETEC) strains and to differentiate them from other gram negative enteric bacteria. This test simultaneously amplifies heat-labile (LTI) and heat-stable (STI and STII) toxin sequences and the E. coli-specific universal stress protein (uspA). The specificity of the method was validated by single PCR tests performed with the reference E. coli and non-E. coli strains and with bacteria isolated from pig feces. The multiplex PCR allowed the rapid and specific identification of enterotoxin-positive E. coli and may be used as a method for direct determination of ETEC and to differentiate them from other E. coli and gram-negative enteric isolates.

https://journals.sagepub.com/doi/pdf/10.1177/104063870101300405

Jacek Osek

Papers

Multiplex Polymerase Chain Reaction Assay for Identification of Enterotoxigenic Escherichia Coli Strains