Development and validation of a resistance and virulence gene microarray targeting Escherichia coli and Salmonella enterica

Abstract

A microarray was developed to simultaneously screen Escherichia coli and Salmonella enterica for multiple genetic traits. The final array included 203 60-mer oligonucleotide probes, including 117 for resistance genes, 16 for virulence genes, 25 for replicon markers, and 45 other markers. Validity of the array was tested by assessing inter-laboratory agreement among four collaborating groups using a blinded study design. Internal validation indicated that the assay was reliable (area under the receiver-operator characteristic curve=0.97). Inter-laboratory agreement, however, was poor when estimated using the intraclass correlation coefficient, which ranged from 0.27 (95% confidence interval 0.24, 0.29) to 0.29 (0.23, 0.34). These findings suggest that extensive testing and procedure standardization will be needed before bacterial genotyping arrays can be readily shared between laboratories.

Figure 1

Receiver-operator characteristic (ROC) curve using sensitivity and specificity values based on thresholds for a positive result at increments of 0.05 normalized fluorescence intensity. The area under the curve = 0.97. Numbers inside the graph indicate thresholds associated with data points.

Figure 2

Sensitivity and specificity for normalized fluorescence intensity threshold values at increments of 0.05.

photo 1

BSA Immersion

photo 2

Slide Rinse

photo 3

Slide Spin

photo 4

Sample Application

photo 5

Hybridization Chamber

photo 6

Immersion in 55 °C Water Bath

photo 7

Slide Aspiration on Chamber/Tip Box

photo 8

Coplin Jar

photo 9

Horizontal Staining Jar

Figure 1. The two wells furthest from the frosted end of the slide do not contain printed arrays

The letters “B” indicate the location of the biotin positive control spots. Well diameter ∼ 7.9mm, between wells a) ∼1.1 mm, b) ∼0.95 mm.

Figure 2

Locations of gene probes on array.