Development and assessment of real-time PCR assays to detect diffusely adherent Escherichia coli Open Access

Abstract

The most newly recognized and one of the least studied pathotypes of diarrheagenic E. coli bacteria is Diffusely Adherent E. coli (DAEC). While molecular assays have been developed for detection of DAEC, no quantitative real-time PCR (qPCR) assay exists. The aim of this project was to create a rapid and specific real-time PCR assay able to exclusively detect organisms that define the DAEC classification of E. coli. We aligned 85 DAEC whole genome sequences in order to find DNA regions exclusive to this pathotype with which to design and evaluate candidate primers and probes for a DAEC-specific qPCR assay. A thorough comparison and assessment of each experimental design was performed. By using a known DAEC strain, as well as E. coli strains from other pathotypes, several parameters of the assay were assessed, including: specificity, sensitivity, efficiency, and the ability to discern closely related, yet genetically distinct pathotypes. In the future, this assay may provide a useful and more convenient way to identify DAEC in clinical cases and outbreak investigations. This rapid test would also greatly enhance the understanding and knowledge base of a lesser-known pathotype that is suspected of being an important cause of diarrhea in children, especially in resource-poor areas of the world. The value of this test may be further increased if incorporated into a multi-pathogen panel of diarrheagenic microbes, particularly when used in global settings that are most affected by diarrheal illness.

Table of Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

E. coli strains and nucleic acid extraction . . . . . . . . . . . . . . . . . . . . . 3

PCR and amplicon analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

DAEC genome sequences and analysis . . . . . . . . . . . . . . . . . . . . . . . 5

Assay design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Assay validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

PCR and amplicon visualization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

DAEC genome sequences and analysis . . . . . . . . . . . . . . . . . . . . . . . . 8

Assay design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Analytical validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Index of Figures

Figure 1: PCR amplified product visualization . . . . . . . . . . . . . . . . . . 8

Table 1: Real-time PCR assay designs . . . . . . . . . . . . . . . . . . . . . . . . 9

Figure 2: Locations of primers and probes . . . . . . . . . . . . . . . . . . . . . 9

Table 2: Limit of detection analysis . . . . . . . . . . . . . . . . . . . . . . . . . . 10

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, General Biology, Microbiology
Keyword	real-time PCR Escherichia coli quantitative PCR
Committee Chair / Thesis Advisor	Levy, Karen, Emory University Winchell, Jonas, Centers for Disease Control and Prevention
Committee Members	O'Toole, Kate, Emory University Koelle, Katia, Emory University

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