Absolute and relative real-time PCR in the quantification of tst gene expression among methicillin-resistant Staphylococcus aureus: evaluation by two mathematical models.
OBJECTIVE
Absolute and relative quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) by the use of two mathematical models were applied in order to study the expression of tst gene encoding the toxic shock syndrome toxin-1 (TSST-1), among methicillin-resistant Staphylococcus aureus (MRSA).


RESULTS
Thirteen epidemic MRSA belonging to different clones and carrying a variety of toxin genes were selected. tst gene expression was achieved by using absolute and relative quantitative real-time RT-PCR and the SYBR Green I. Absolute RT-PCR showed a statistically significant higher level of tst expression among strains isolated from soft tissue infections. Relative quantification was performed in relation to 23S rRNA expression by the application of two mathematical models, the 2(-DeltaDeltaCt) and the Pfaffl analysis methods.


CONCLUSIONS
tst gene expression was best calculated by the relative real-time RT-PCR analysis applying the Pfaffl analysis method, taking into account the reactions' efficiencies. Level of tst expression was related to patients' infection and did not depend on the MRSA genetic profile.


CONCLUSIONS
The results indicate that the application of the Pfaffl analysis method in the evaluation of relative real-time RT-PCR is more adequate.
Chini
V
V
Department of Microbiology, School of Medicine, University of Patras, Patras, Greece.
Foka
A
A
Dimitracopoulos
G
G
Spiliopoulou
I
I
D001427:Bacterial Toxins / Q000235:genetics*; D004768:Enterotoxins / Q000235:genetics*; D015870:Gene Expression; D005838:Genotype; D006801:Humans; D016106:Methicillin Resistance / Q000235:genetics*; D008962:Models, Theoretical; D012329:RNA, Bacterial / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013203:Staphylococcal Infections / Q000175:diagnosis* / Q000382:microbiology; D013211:Staphylococcus aureus / Q000235:genetics / Q000302:isolation & purification*; D018089:Superantigens / Q000235:genetics*
D001427:Bacterial Toxins; D004768:Enterotoxins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D018089:Superantigens; C030107:enterotoxin F, Staphylococcal