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


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.


CONCLUSIONS
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.
de Wet
S C
SC
Queensland Bioscience Precinct, CSIRO Livestock Industries, 306 Carmody Road, St Lucia, Qld 4067, Australia. sharon.dewet@csiro.au
Denman
S E
SE
Sly
L
L
McSweeney
C S
CS
D000818:Animals; D001433:Bacteriolysis; D002238:Carbohydrate Epimerases / Q000235:genetics; D019453:Escherichia coli O157 / Q000235:genetics* / Q000302:isolation & purification*; D008460:Meat / Q000382:microbiology*; D012329:RNA, Bacterial / Q000235:genetics* / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D000637:Transaminases / Q000235:genetics
D012329:RNA, Bacterial; D000637:Transaminases; D002238:Carbohydrate Epimerases; C061272:perosamine synthetase