An improved method for RNA extraction from carcass samples for detection of viable Escherichia coli O157:H7 by reverse-transcriptase polymerase chain reaction
2008
de Wet, S.C. | Denman, S.E. | Sly, L. | McSweeney, C.S.
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. 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. 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. 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.
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