A reverse transcriptase-polymerase chain reaction assay for detection of viable Escherichia coli O157:H7: investigation of specific target genes.
OBJECTIVE
To determine suitable target genes for detection of the pathogen Escherichia coli O157:H7 by reverse transcriptase-polymerase chain reaction (RT-PCR).


RESULTS
Potential genes used as indicators for viability included rfbE, fliC, stx1, stx2, mobA, eaeA, hly and 16S rRNA. Under normal growth conditions, rfbE, stx1, hly and 16S rRNA amplicons were detected in association with all growth phases. The products of 16S rRNA, mobA, rfbE and stx1 were readily visualized in RNA isolated from viable but non-culturable cells. The 16S rRNA gene was not amplified following heat treatment of cells at 121 degrees C for 15 min and mRNA targets were not amplified after treatment at 60 degrees C for 20 min. In this instance, genes that are not amplified are good targets for determining viability.


CONCLUSIONS
The results of RT-PCR amplification indicate that, under the conditions examined, the rfbE gene is the most appropriate target for detection of viable E. coli O157:H7.


CONCLUSIONS
Prior to detection or identification from an environmental or food sample E. coli O157:H7 may be exposed to many harsh conditions that influence nucleic acid (RNA and DNA) stability. This study gives an insight into the effects of temperature and nutrient deprivation on identification of viable cells using RT-PCR. It also suggests that, if RT-PCR is to be used for detection of live cells in a sample without enrichment, 10(7) cfu of the target organism are required.
Yaron
S
S
Department of Food Science, Rutgers University, New Brunswick, NJ 08901-8520, USA.
Matthews
K R
KR
D015169:Colony Count, Microbial; D003470:Culture Media; D004269:DNA, Bacterial / Q000032:analysis; D004275:DNA, Ribosomal / Q000032:analysis; D019453:Escherichia coli O157 / Q000235:genetics / Q000254:growth & development* / Q000302:isolation & purification; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D005798:Genes, Bacterial; D006801:Humans; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic
D003470:Culture Media; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D029968:Escherichia coli Proteins; D012333:RNA, Messenger; D012336:RNA, Ribosomal, 16S