[Bacterial DNA alteration by plasma generated atomic nitrogen. Real-time PCR detection contribution].
OBJECTIVE
Bacterial sterilization by the technology of plasma in post-discharge shows a growing interest. The main appeal of this new process resides in its action at dry and low temperature (60 degrees C). This technology would be therefore useful for the complex medical equipment, sensitive to the oxidization, humidity and/or requiring a temperature lower than 60 degrees C. The objective of this survey is to demonstrate the activity of an atomic flux emanating a plasma of pure molecular nitrogen on the bacterial DNA: does the plasma of nitrogen damage the genetic material?


METHODS
The bacteria tested (Bacillus stearothermophilus, Staphylococcus aureus MRSA and Helicobacter pylori) are cultivated on suitable agar, and the bacterial DNA is extracted from every CFU by the technique of the columns (High Pure PCR Template, Roche). Every quantity of DNA extracted is diluted in 1 ml pure water. Then, 50 microl of each of these solutions of DNA are laid down in sterile Nunclon's plates holes, which undergo an advanced emptiness cycle during 60 minutes. The DNA residues will be then introduced during 40 minutes in a plasma sterilization surrounding wall (Plasmalyse), Satelec) where the debit of nitrogen, the pressure and the temperature are adjusted respectively to 1 L x min(-1), 5 Torrs and 60 degrees C. The DNA so ''plasmalysé'', is recovered then by aspiration in 500 mul pure water and processed to undergo an amplification/detection by Real-Time PCR (LightCycler2.0, Roche). The DNA ''plasmalysé'' will be compared to the intact DNA control(1), to the DNA control control(2) having undergone the cycle of emptiness solely, as well as to the DNA control(3) solely heated to 60 degrees C during 40 min.


RESULTS
The amplification curves demonstrated that the only advanced emptiness and the only heat don't have any activity on the bacterial DNA. On the other hand, the DNA ''plasmalysé'' shows a deterioration of the amplified sequences.


CONCLUSIONS
The genomic bacterial DNA, once extracted, is damaged by the gaseous flux of nitrogen plasma. A new sterilization process of the medical material will presumably impose itself in medium-term.
Lakhssassi
N
N
Laboratoire d'écosystème buccal, département d'épidémiologie des maladies infectieuses, faculté de chirurgie dentaire, université Paul-Sabatier, 3 chemin des Maraîchers, 31062 Toulouse cedex 04, France. docteurlakhssassi@yahoo.fr
Cousty
S
S
Villeger
S
S
Diouf
A
A
Ricard
A
A
Sixou
M
M
D001483:Base Sequence; D017931:DNA Primers; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D001411:Geobacillus stearothermophilus / Q000235:genetics*; D016480:Helicobacter pylori / Q000235:genetics*; D016106:Methicillin Resistance; D009584:Nitrogen; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction / Q000379:methods*; D013211:Staphylococcus aureus / Q000235:genetics*
D017931:DNA Primers; D004269:DNA, Bacterial; D009584:Nitrogen