ZASTOSOWANIE MULTIPLEKS PCR DO IDENTYFIKACJI SZCZEPÓW MRSA I MRCNS W PODŁOŻU STOSOWANYM W AUTOMATYCZNYM SYSTEMIE DO POSIEWU KRWI
[APPLICATION OF MULTIPLEX PCR FOR IDENTIFICATION OF MRSA AND MRCNS STRAINS IN MEDIUM OF AUTOMATIC BLOOD CULTURE SYSTEM]

Streszczenie.

Wykrywanie techniką PCR patogenów obecnych w dodatnich posiewach krwi systemu BACTEC lub BacT/Alert sprawia wiele trudności ze względu na obecność licznych inhibitorów amplifikacji. Celem niniejszej pracy było opracowanie skutecznej metody umożliwiającej szybkie wykrycie gronkowców w tego rodzaju próbkach i jednoczesne określenie ich oporności na meticylinę.

Abstract.

Rapid detection and identification of methicillin-resistant staphylococci in patient’s blood is essential for the prompt antimicrobial therapy of infection. Molecular-based diagnosis, in comparison to conventional methods, allows to increase sensitivity and to reduce time necessary to achieve positive results and also enable to test susceptibility. The aim of the study was the use of multiplex PCR to detect region of the 16S rRNA that is unique to staphylococci, the S. aureus-specific clfA gene and the mecA gene which is a determinant of methicillin resistance, in a medium of the BACTEC blood
culture system. Three different extraction methods were examined in order to ascertain the most suitable
method to isolate staphylococcal DNA. The only method which removed PCR inhibitors contained in BACTEC blood culture material use Triton X-100 and lysostaphyin to lysis bacterial cells. The use of High Template Preparation Kit failed to yield a positive result in all investigated probes. Third of the methods, based on alkali lysis, resulted in DNA which could be amplified by PCR only in case of
S. aureus blood culture. The findings of our study suggest that not all DNA extraction methods are appropriate because some of them may not remove potent amplification inhibitors found in blood and medium of system BACTEC.