Rapid Cycle Real-Time PCR - Methods and Applications [antikvár]

Introduction for Microbiology Volume of Rapid Cycle Real-Time PCR Several commercial testing platforms have become available or soon will be available which combine rapid cycle polymerase chain reaction with fluorescent probe detection of amplified target nucleic acid in the same closed vessel. This technology is referred to as rapid cycle real-time PCR. One testing platform which exploits this technology, the LightCycler instrument (Roche Diagnostics Corporation), has become particularly popular. The LightCycler system was the first system available that permitted the operator to evaluate target amplification after each PCR cycle. Recently an automated nucleic acid extraction system, the MagNA Pure LC (Roche Diagnostics Corporation), has been developed to complement the LightCycler system. The combination of the MagNA Pure LC and LightCycler system now permits complete automation of extraction, purification, concentration, amplification and detection of target nucleic acid from patient samples. Although the LightCycler system was initially intended for research purposes, it became quickly obvious to many microbiologists, that the system had immense potential for diagnostic microbiology testing. The LightCycler-MagNA Pure LC system could not only replace cumbersome and work intense procedures required for conventional, "home-brewed" PCR assays, but as well could replace standard antigen or culture-based assays. As an example, the accepted diagnostic approach for detecting Group A Streptococcus (Streptococcus pyogenes) from throat swabs is to screen for group A streptococcal antigens using a commercial direct immunoassay. If the result for this "rapid antigen" assay is negative, a culture is performed. This two-step procedure follows guidelines provided by the Infectious Diseases Society of America and other professional organizations and is required because current rapid antigen assays lack sensitivity. We have developed a LightCycler assay which is -10% more sensitive and approximately half the cost ($l-$2 vs $2-$3) and requires much less time to perform (<1 hr vs up to 48 h) than the combined antigen-culture assay. In addition to accuracy, speed and cost issues, the LightCycler system and other rapid cycle real-time PCR platforms have other advantages over conventional PCR methods. These include decreased risk for contamination (specimen or amplicon) and adaptability for a wide range of testing. The containment of the PCR reaction and amplicon detection in the same closed vessel, as is possible with the LightCycler system, lessens the possibility of amplicon contamination including amplicon "carryover" from previous assay runs. As well, the possibility of specimen to specimen contamination is essentially eliminated. The versatility for testing is exemplified by applications