improved method for RNA extraction from carcass samples for detection of viable Escherichia coli O157:H7 by reverse-transcriptase polymerase chain reaction

• Published in: Letters in applied microbiology Vol. 47; no. 5; pp. 399 - 404
• Main Authors: de Wet, S.C, Denman, S.E, Sly, L, McSweeney, C.S
• Format: Journal Article
• Language: English
• Published: Oxford, UK Oxford, UK : Blackwell Publishing Ltd 01.11.2008; Blackwell Publishing Ltd
• Subjects: Animals; Bacteriolysis; Carass; Carbohydrate Epimerases - genetics; E. coli O157:H7; Escherichia coli; Escherichia coli O157 - genetics; Escherichia coli O157 - isolation & purification; Escherichia coli O157:H7; Meat - microbiology; Reverse Transcriptase Polymerase Chain Reaction - methods; reverse-transcriptase PCR; RNA extraction; RNA, Bacterial - genetics; RNA, Bacterial - isolation & purification; Sensitivity and Specificity; Transaminases - genetics; viable cells
• ISSN: 0266-8254; 1472-765X
• DOI: 10.1111/j.1472-765X.2008.02462.x

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.