Novel multiplex-PCR test for Escherichia coli detection

Abstract

Escherichia coli is a diverse and ubiquitous strain of both commensal and pathogenic bacteria. In this study, we propose the use of multiplex polymerase chain reaction (PCR), using amplification of three genes (cydA, lacY, and ydiV), as a method for determining the affiliation of the tested strains to the E. coli species. The novelty of the method lies in the small number of steps needed to perform the diagnosis and, consequently, in the small amount of time needed to obtain it. This method, like any other, has some limitations, but its advantage is fast, cheap, and reliable identification of the presence of E. coli. Sequences of the indicated genes from 1,171 complete E. coli genomes in the NCBI database were used to prepare the primers. The developed multiplex PCR was tested on 47,370 different Enterobacteriaceae genomes using in silico PCR. The sensitivity and specificity of the developed test were 95.76% and 99.49%, respectively. Wet laboratory analyses confirmed the high specificity, repeatability, reproducibility, and reliability of the proposed test. Because of the detection of three genes, this method is very cost and labor-effective, yet still highly accurate, specific, and sensitive in comparison to similar methods.

Importance: Detection of E. coli from environmental or clinical samples is important due to the common occurrence of this species of bacteria in all human and animal environments. As commonly known, these bacteria strains can be commensal and pathogenic, causing numerous infections of clinical importance, including infections of the digestive system, urinary, respiratory, and even meninges, particularly dangerous for newborns. The developed multiplex polymerase chain reaction test, confirming the presence of E. coli in samples, can be used in many laboratories. The test provides new opportunities for quick and cheap analyses, detecting E. coli using only three pairs of primers (analysis of the presence of three genes) responsible for metabolism and distinguishing E. coli from other pathogens from the Enterobacteriaceae family. Compared to other tests previously described in the literature, our method is characterized by high specificity and sensitivity.

Keywords: Escherichia coli; PCR; detection; diagnostics; multiplex PCR.

Fig 1

Pie charts presenting the composition of the Enterobacteriaceae genomes of varying completeness-level data sets used to perform isPCR. (A) Pie chart presenting the contribution of different bacterial genera to 43,730 data set used for isPCR, with 336 genomes denoted as “others” for clarity. (B) Proportion of distinct genera in the “others” data set. Taxa whose percent occurrence could not be clearly denoted on the graph have the value shown on the right side of the legend.

Fig 2

Confusion matrix presenting proportion of correctly and improperly classified E. coli and non-E. coli strain (denoted as “Other”). The bar on the right serves as a color scale indicating the number of bacterial genomes—the darker the color the higher the number of genomes.

Fig 3

Scatter plot where x-axis indicates sensitivity/recall (proportion of E. coli detected), while the y-axis shows specificity (proportion of correct detections). The closer the dot is to the top rightmost corner the greater the accuracy. The parameters are calculated based on the isPCR performed on the Enterobacteriaceae data set, which composition is presented in Fig. 1. The color of the dot fill indicates the detection method tested and corresponds to the legend found in the box. The closer the dot is to the top rightmost corner the greater the accuracy.

Fig 4

Bar chart revealing predicted accuracy of different E. coli detection methods. The accuracy was calculated based on the isPCR performed on the Enterobacteriaceae data set, whose composition is presented in Fig. 1.

Fig 5

Bar chart revealing what percentage of Shigella genus members remained undetected after subjecting them to primer pairs found in different E. coli assays. The results were obtained from isPCR analysis.

Fig 6

Column plot indicating what percentage of each of the Shigella species remained undetected after subjecting them to different E. coli detection assays. The results were obtained from isPCR analysis.

Fig 7

The PCR products after amplification of cydA, lacY, and ydiV genes as a multiplex on genomic DNA of bacterial strains (electrophoresis in 2% agarose gel). NTC—no template control, PC—positive control. M—marker; the sizes of the molecular size markers are shown in bp on the left side of the figure.

Fig 8

The PCR products after amplification of cydA, lacY, and ydiV genes as a multiplex on genomic DNA of bacterial strains (electrophoresis in 2% agarose gel). NTC—no template control, PC—positive control. M—marker; the sizes of the molecular size markers are shown in bp on the left side of the figure.

Fig 9

The PCR products after amplification of cydA, lacY, and ydiV genes as a multiplex on different quantities of genomic DNA of E. coli (electrophoresis in 2% agarose gel). NTC—no template control, PC—positive control. M—marker; the sizes of the molecular size markers are shown in bp on the left side of the figure.