[Present status and future on automated PCR testing for microorganisms].
PCR is a continually evolving technology. In the years since in inception, there have been many improvements made to the assay, and also to the automation. Some of the early developmental highlights in PCR are listed as follows; 1) The amplification process was automated with the introduction of thermocyclers. 2) False-positive results from carryover of amplification products have been largely eliminated by the incorporation of dUTP into amplification reactions and the subsequent restriction of dU-containing amplicons with UNG (AmpErase). 3) A combined RT-PCR assay for the amplification of RNA that utilizes a single thermoactive enzyme (Tth DNA polymerase) was recently developed. This assay eliminates the need to change buffer/enzymes between the RT and PCR steps. 4) The methods for detecting amplicons has been made simpler and faster. The use of radioactivity and electrophoresis have been eliminated. The development of microwell plate assays provides users with a familiar format (similar to EIAs) and makes use of reagents that are readily available. Based on the above developments and clinical requirements, the developments of the automated PCR testing are progressed. The recent introduction of the COBAS AMPLICOR represents a major advance in PCR instrumentation. With the COBAS AMPLICORE, both the amplification and detection process are automated. This greatly reduces the hand-on time required to carry out PCR assays and further improves assay reproducibility. Further developments for full automation are on going, i.e., kinetic PCR, 5' exonuclease assay, etc.
Hirose
T
T
Diagnostic Division, Nippon Roche K.K., Tokyo.
D001331:Automation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012329:RNA, Bacterial / Q000032:analysis; D015203:Reproducibility of Results
D012329:RNA, Bacterial