Multiplex real time PCR panels to identify fourteen colonization factors of enterotoxigenic <i>Escherichia coli</i> (ETEC)

<div><p>Enterotoxigenic <i>Escherichia coli</i> (ETEC) is a leading cause of childhood diarrhea in low income countries and in travelers to those areas. Inactivated enterotoxins and colonization factors (CFs) are leading vaccine candidates, therefore it is important to determine the prevailing CF types in different geographic locations and populations. Here we developed real time PCR (qPCR) assays for 14 colonization factors, including the common vaccine targets. These assays, along with three enterotoxin targets (STh, STp, and LT) were formulated into three 5-plex qPCR panels, and validated on 120 ETEC isolates and 74 <i>E</i>. <i>coli</i> colony pools. The overall sensitivity and specificity was 99% (199/202) and 99% (2497/2514), respectively, compared to the CF results obtained with conventional PCR. Amplicon sequencing of discrepant samples revealed that the qPCR was 100% accurate. qPCR panels were also performed on nucleic acid extracted from stool and compared to the results of the ETEC isolates or E. coli colony pools cultured from them. 95% (105/110) of the CF detections in the cultures were confirmed in the stool. Additionally, direct testing of stool yielded 30 more CF detections. Among 74 randomly selected <i>E</i>. <i>coli</i> colony pools with paired stool, at least one CF was detected in 63% (32/51) of the colony pools while at least one CF was detected in 78% (47/60) of the stool samples (P = NS). We conclude that these ETEC CF assays can be used on both cultures and stool samples to facilitate better understanding of CF distribution for ETEC epidemiology and vaccine development.</p></div

Comparison of enteropathogen detection on 129 stool and swab sample pairs collected on the same day.

<p>Sensitivity and specificity of detection on swabs was calculated using the results from the corresponding stool as the reference.</p

Comparison of total nucleic acid extract versus 1:1 mixed DNA and RNA extracts.

<p>For the RNA derived targets below, we compared Cqs between nucleic acid extracted with QIAamp Stool DNA mini kit versus a 1:1 mixture of QIAamp stool DNA mini kit extract and FUJIfilm QuickGene RNA tissue kit or QIAamp viral RNA mini kit extract.</p

Validation of qPCR assays on clinical samples, using confirmatory PCR assays and amplicon sequencing.

<p>The assays are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158199#pone.0158199.s001" target="_blank">S1 Table</a>.</p

Demonstration of quantification on spiked analytical samples.

<p>Five lots of stool with different amount of inhibitors were prepared, then the mixture of target nucleic acid was spiked. Extraction and testing with TAC were performed, target copy numbers were calculated based on standard curves and normalized to extrinsic controls. Target copy numbers (circles) were log2 transformed in order to be on the same scale as Cq.</p