Details

Autor(en) / Beteiligte

• Yaron, S
• Matthews, K.R

Titel

reverse transcriptase-polymerase chain reaction assay for detection of viable Escherichia coli O157:H7: investigation of specific target genes

Ist Teil von

• Journal of applied microbiology, 2002-01, Vol.92 (4), p.633-640

Ort / Verlag

Oxford UK: Blackwell Science Ltd

Erscheinungsjahr

2002

Link zum Volltext

Quelle

MEDLINE

Beschreibungen/Notizen

• Aims: To determine suitable target genes for detection of the pathogen Escherichia coli O157:H7 by reverse transcriptase-polymerase chain reaction (RT-PCR). Methods and 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. Significance and Impact of the Study: 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.