An RNA transcription-based amplification technique (NASBA) for the detection of viable Salmonella enterica.
Possession of mRNA is indicative of cell viability. RTPCR is not appropriate for mRNA detection as it cannot unambiguously detect mRNA in a DNA background. The alternative amplification technique, NASBA, avoids the disadvantages of RTPCR. We have devised a method for detection of viable Salmonella enterica. This involves NASBA amplification of mRNA transcribed from the dnaK gene. Amplification of mRNA extracted from viable and heat-killed cells from the same population produced consistent and highly significant (P > 0.01) differences between the respective signals. The signal obtained from viable cells was completely eradicated by RNase treatment, while PCR amplification of treated and untreated samples was unaffected, indicating that NASBA was unaffected by background DNA.
Simpkins
S A
SA
Central Science Laboratory, Sand Hutton, York, UK.
Chan
A B
AB
Hays
J
J
Pöpping
B
B
Cook
N
N
D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction / Q000379:methods; D012329:RNA, Bacterial / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis / Q000302:isolation & purification; D015203:Reproducibility of Results; D012260:Ribonucleases; D019779:Salmonella enterica / Q000235:genetics / Q000302:isolation & purification*
D017931:DNA Primers; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012333:RNA, Messenger; D012260:Ribonucleases