Detection of Salmonella enteritidis by reverse transcription-polymerase chain reaction (PCR)

A reverse transcription-polymerase chain reaction (RT-PCR) method was devel oped for detecting mRNA from the sefA gene of Salmonella enteritidis. Detec tion of target mRNA was examined from cells grown in buffered peptone water at different temperatures (37, 25 and 15 degrees C) and pH (5.5, 7.2 and 8 .5). The results revealed that the levels of transcription of the sefA gene differed depending upon the physiological state of the cells. This affecte d the sensitivity of the RT-PCR assay. When the assay was evaluated for the detection of S. enteritidis PT4 in artificially contaminated minced beef a nd whole egg samples, an enrichment step was used (buffered peptone water, pH 7.2, 37 degrees C, 16 h) to increase the sensitivity of the assay. In th e presence of the normal background flora of each food type, it was possibl e to detect ten cells of S. enteritidis PT4 after a 16-h enrichment using t he RT-PCR assay, with a total testing time of 28 h. Unlike the PCR test for the sefA gene that was tested in parallel, the RT-PCR assay did not detect nonviable (heat-inactivated) S. enteritidis PT4 cells, The results support ed the usefulness of RT-PCR as a method for the detection of viable microor ganisms. (C) 1999 Elsevier Science B.V. All rights reserved.