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Species specific PCR based detection of Escherichia coli from Indian foods.

ABSTRACT: Escherichia coli is a faecal indicator and certain virotypes are known as pathogens. Therefore, detection and prevention of E. coli in food is very important. The existing rapid methods concentrate on detecting the pathogenic E. coli instead of total E. coli population. Present study evaluates the use of two molecular markers (uidA and flanking region of uspA) specific for the E. coli in combination with microbiological method for confirmation. Majority of the isolates (77%) were positive for both the genes tested. However, 22% of the E. coli isolates were positive for any one of the two primer sets [uidA (9%) and flanking region of uspA (13%)]. High levels of E. coli incidences (92% samples) were observed in beef while low occurrence (19% samples) was found in sprouts. Low percentage (7.3%) of E. coli isolates was positive for virulence genes tested (lt, ipaH, aggR, eaeA, stx1 and stx2). Two isolates were positive for stx genes. However, none of the isolates including stx positive isolates were E. coli 0157:H7. Maximum number of the E. coli (44%) isolates was characterized under phylogenetic group B2. This phylogenetic group comprises of extra intestinal and virulent E. coli strains.

SUBMITTER: Godambe LP

PROVIDER: S-EPMC5453913 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Species specific PCR based detection of Escherichia coli from Indian foods.

Godambe Lipika Pansare LP Bandekar Jayant J Shashidhar Ravindranath R

3 Biotech 20170601 2

Escherichia coli is a faecal indicator and certain virotypes are known as pathogens. Therefore, detection and prevention of E. coli in food is very important. The existing rapid methods concentrate on detecting the pathogenic E. coli instead of total E. coli population. Present study evaluates the use of two molecular markers (uidA and flanking region of uspA) specific for the E. coli in combination with microbiological method for confirmation. Majority of the isolates (77%) were positive for bo ...[more]

PMID: 28573400

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Project description: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.ImportanceDetection 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.

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Species specific PCR based detection of Escherichia coli from Indian foods.

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Species specific PCR based detection of Escherichia coli from Indian foods.

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