Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR | Zendy

Baoguang Li | Zendy; Zonglin Hu | Zendy; Christopher A. Elkins | Zendy

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Detection of Live <em>Escherichia coli</em> O157:H7 Cells by PMA-qPCR

Author(s) -

Baoguang Li,

Zonglin Hu,

Christopher A. Elkins

Publication year - 2014

Publication title -

journal of visualized experiments

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 0.596

H-Index - 91

ISSN - 1940-087X

DOI - 10.3791/50967

Subject(s) - propidium monoazide , escherichia coli , open reading frame , biology , microbiology and biotechnology , dna , polymerase chain reaction , genetics , gene , peptide sequence

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

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