Real-time reverse transcription-multiplex PCR for simultaneous and specific detection of rfbE and eae genes of Escherichia coli O157:H7

Molecular and Cellular Probes
Vijay K Sharma

Abstract

A real-time reverse transcription multiplex polymerase chain reaction (rRT-MPCR) was developed for detection of mRNA encoded by rfbE and eae genes of enterohemorrhagic Escherichia coli (EHEC) O157:H7. A 129-bp and a 106-bp sequence specific to rfbE and eae, respectively, were targeted for reverse transcription, amplification, and real-time detection. A single-step RT-PCR kit containing a mixture of reverse transcriptases converted mRNA into cDNA, which was subsequently amplified by Taq polymerase included in the same kit. The real-time detection of amplification products was achieved by incorporating rfbE(O157)- and eae(O157:H7)-specific TaqMan probes in rRT-MPCR. The ability of two sets of primers and probes for specific detection of rfbE(O157) and eae(O157:H7) was initially verified by screening RNA of eight E. coli serotypes possessing different O antigens and eae alleles. These two sets of primers and probes were also tested in a standard real-time PCR (rPCR) using DNA prepared from several E. coli and non-E. coli strains to verify that only rfbE(O157)- and eae(O157:H7)-specific sequences were amplified and detected. The rRT-MPCR was then evaluated for detecting low-level contamination of EHEC O157:H7 in feces. When RNA pre...Continue Reading

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Citations

Mar 16, 2007·Analytical and Bioanalytical Chemistry·Tsuyoshi IshiiKeizo Yamashita
Jun 29, 2010·Applied Microbiology and Biotechnology·Hari P DwivediLee-Ann Jaykus
Mar 19, 2013·Biosensors & Bioelectronics·M Barreiros dos SantosJ Samitier
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Apr 14, 2009·Diagnostic Microbiology and Infectious Disease·Laurent ChassagneJulien Delmas
Jun 9, 2015·Diagnostic Microbiology and Infectious Disease·Tanushree SaxenaMedicherla Krishna Mohan
Apr 2, 2015·Critical Reviews in Food Science and Nutrition·Manisha MangalRam K Gupta
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Nov 26, 2019·Journal of Environmental Science and Health. Part. B, Pesticides, Food Contaminants, and Agricultural Wastes·Mahmoud ElafifyAdel Abdelkhalek
Jul 1, 2014·Comprehensive Reviews in Food Science and Food Safety·Vincenzina Fusco, Grazia Marina Quero

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