Author
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Sharma, Vijay |
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Submitted to: Molecular and Cellular Probes
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 3/1/2006 Publication Date: 10/20/2006 Citation: Sharma, V.K. 2006. 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. 20(5):298-306. Interpretive Summary: Only a 10 - 100 viable cells of E. coli O157:H7 are considered sufficient to cause disease in humans. Although routine culture methods are suitable for detection of live E. coli O157:H7, these methods are less sensitive and can take up to a week to provide specific identification. PCR methods on the other hand can identify E. coli O157:H7 with in 12 hours, but cannot distinguish live from dead bacterial cells. Moreover, PCR targets single copy chromosomal genes that requires longer enrichment period to attain positive results. The objective of the present study was to develop a real-time reverse transcription multiplex polymerase chain reaction (rRT-MPCR) for rapid detection of very low levels of live E. coli O157:H7 cells in cattle feces. By targeting mRNA of two genes that have been shown to be present in only live cells and degraded quickly in dead cells, we were able to detect as few as 1 to 10 cells of E. coli O157:H7 in cattle feces within 12 h period. The meat processing industry, which slaughters more than $50 billion worth of livestock annually, could use this technology to meet current federal regulations that mandate zero tolerance for E. coli O157:H7 contamination of raw meat products. Also, rRT-MPCR assay could be used for understanding the ecology of E. coli O157:H7 in cattle reservoir and for designing of intervention strategies to control the prevalence of E. coli O157:H7 in cattle populations. Technical Abstract: A real-time reverse transcription multiplex polymerase chain reaction (rRT-MPCR) was developed for simultaneous amplification and real-time detection of mRNA encoded by rfbE and eae genes present in enterohemorrhagic Escherichia coli (EHEC) O157:H7. A 129-bp sequence located at the 5’end of gene rfbE and a 106-bp sequence located at the 3’ end of gene eae of EHEC O157:H7 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 rfbEO157- and eaeO157:H7- specific TaqMan probes in rRT-MPCR assay. The ability of two sets of primers and probes to provide specific detection of rfbEO157 and eaeO157:H7 was initially verified by screening RNA prepared from 8 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 using DNA prepared from several E. coli and non-E. coli strains to obtain additional evidence that only rfbEO157- and eaeO157: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 prepared from bovine feces, which were artificially seeded with known amounts of EHEC O157:H7 cells and cultured for a total of five hours in a non-selective broth, was tested in rRT-MPCR as low as 1 cfu per g of feces could be detected. The detection range for each of the two target genes in the seeded fecal cultures was 100 – 105 cfu/g of feces. Thus, the described procedure could be applied to rapid detection and quantification of very low levels of EHEC O157:H7 using total RNA as a template. Since the presence of rfbEO157- and eaeO157:H7-specific mRNA is dependent on actively growing and replicating bacterial cells, rRT-MPCR assay could provide important information about the viability of EHEC O157:H7 in fecal samples. |
