Quantification of bacterial transcripts during infection using competitive reverse transcription-PCR (RT-PCR) and LightCycler RT-PCR.
Bacteria have evolved sophisticated regulatory circuits to modulate their gene expression in response to disparate environments. In order to monitor bacterial gene expression and regulation in the host, methods for direct transcript analysis from clinical specimens are needed. For most bacterial infections, amplification of the mRNAs of interest is necessary due to the low numbers of cells present and the low levels of specific transcripts. Here we compare two methods of quantitative reverse transcription-PCR (RT-PCR)-competitive RT-PCR using a one-tube system followed by standard gel analysis and the real-time detection of PCR product formation by fluorescence resonance energy transfer technology using the LightCycler unit. We isolated Staphylococcus aureus RNA directly from clinical specimens obtained from cystic fibrosis patients with chronic S. aureus lung infection and from an animal model of foreign-body infection with no further cultivation of the bacteria. Competitive RT-PCR and LightCycler RT-PCR were tested for their ability to quantify the transcription of a constitutively expressed gyrase gene (gyr) and a highly regulated alpha-toxin gene (hla) of S. aureus. Reproducible results were obtained with both methods. A sensitivity of 10(4) (gyr) and 10(3) (hla) copies, respectively, was reached, which was sufficient for the quantification of transcripts during bacterial infection. Overall, the competitive RT-PCR is a robust technique which does not need special RNA purification. On the negative side, it is labor intensive and time consuming, thus limiting the numbers of samples which can be analyzed at a given time. LightCycler RT-PCR is very susceptible to even traces of inhibitors, but it allows high-throughput processing of samples.
Institut für Allgemeine Hygiene und Umwelthygiene, Universität Tübingen, Wilhelmstrasse 31, 72024 Tübingen, Germany.
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Goerke
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Bayer
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Wolz
D000818:Animals; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D009693:Nucleic Acid Hybridization / Q000379:methods; D012313:RNA / Q000138:chemical synthesis; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation* / Q000592:standards; D012680:Sensitivity and Specificity; D013203:Staphylococcal Infections / Q000382:microbiology*; D013211:Staphylococcus aureus / Q000235:genetics*; D014158:Transcription, Genetic / Q000235:genetics*
D017931:DNA Primers; D004269:DNA, Bacterial; D012313:RNA