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An improved method for RNA extraction from carcass samples for detection of viable Escherichia coli O157:H7 by reverse‐transcriptase polymerase chain reaction
Author(s) -
De Wet S.C.,
Denman S.E.,
Sly L.,
McSweeney C.S.
Publication year - 2008
Publication title -
letters in applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.698
H-Index - 110
eISSN - 1472-765X
pISSN - 0266-8254
DOI - 10.1111/j.1472-765x.2008.02462.x
Subject(s) - rna extraction , rna , escherichia coli , reverse transcriptase , biology , microbiology and biotechnology , lysis , polymerase chain reaction , lysis buffer , reverse transcription polymerase chain reaction , messenger rna , chromatography , gene , chemistry , biochemistry
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.