An improved method for RNA extraction from carcass samples for detection of viable Escherichia coli 0157:H7 by reverse-transcriptase polymerase chain reaction

de Wet, S. C., Denman, S. E., Sly, L. and McSweeney, C. S. (2008) An improved method for RNA extraction from carcass samples for detection of viable Escherichia coli 0157:H7 by reverse-transcriptase polymerase chain reaction. Letters in Applied Microbiology, 47 5: 399-404.


Author de Wet, S. C.
Denman, S. E.
Sly, L.
McSweeney, C. S.
Title An improved method for RNA extraction from carcass samples for detection of viable Escherichia coli 0157:H7 by reverse-transcriptase polymerase chain reaction
Formatted title An improved method for RNA extraction from carcass samples for detection of viable Escherichia coli 0157:H7 by reverse-transcriptase polymerase chain reaction
Journal name Letters in Applied Microbiology   Check publisher's open access policy
ISSN 0266-8254
Publication date 2008-11
Sub-type Article (original research)
DOI 10.1111/j.1472-765X.2008.02462.x
Volume 47
Issue 5
Start page 399
End page 404
Total pages 6
Place of publication Oxford, England
Publisher Wiley- Blackwell Publishing
Collection year 2009
Language eng
Subject C1
060501 Bacteriology
920109 Infectious Diseases
Formatted abstract Aims: To develop a rapid RNA extraction procedure for maximizing bacterial RNA yield from carcass samples with low abundance of Escherichia coli O157:H7 without pre-enrichment.

Methods and Results: Nontarget bacterial cells were added to the sample prior to RNA extraction, facilitating the co-precipitation of target RNA along with nontarget RNA and thus enhancing the recovery. This method was developed using a serial dilution of log phase target cells (E. coli O157:H7), combined with a high number of nontarget cells (E. coli K12). Cells were lysed by a bead beating method followed by RNA purification using a commercial kit. A reverse-transcriptase PCR assay for the detection of rfbE gene in E. coli O157:H7 was used to demonstrate that the procedure increased the recovery of amplifiable RNA target with a detection limit of approximately 63 CFU ml−1 in cultures and 27·5 CFU ml−1 in carcass liquor.

Conclusions: An RNA extraction procedure was developed to detect low numbers (<30 viable cells ml−1) of E. coli O157:H7 in carcass liquor without pre-enrichment.

Significance and Impact of the Study: This method could be applied for the detection of E. coli O157:H7 in low abundance on carcasses where rapid detection and early intervention is essential for safety in the livestock industry.
Keyword Carass
E. coli
Reverse-transcriptase PCR
RNA extraction
viable cells
Q-Index Code C1
Q-Index Status Confirmed Code
Additional Notes Published Online: 16 Oct 2008

Document type: Journal Article
Sub-type: Article (original research)
Collections: 2009 Higher Education Research Data Collection
School of Chemistry and Molecular Biosciences
 
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Created: Mon, 30 Mar 2009, 10:42:57 EST by Glenda Chown on behalf of School of Chemistry & Molecular Biosciences