[Microbial diagnosis with PCR will become clinically beneficial with a faster analysis].
PCR was introduced in 1985 by Mullis and was immediately recognized as a valuable tool in biomedical research and was awarded the Nobel Prize in 1993. Two culture-negative meningitis cases are described where Haemophilus influenzae and Neisseria meningitidis were found by 16SRNA-PCR. The modern real time PCR technology using fluorescent probes (hybridization probes, lightup probes, molecular beacons etc) for detection of the PCR-product or on DNA microarray chips, is under development for routine use. Multiplex technology can be used to simultaneously detect multiple microorganisms as well as resistance genes. Using super-convection with ultracentrifugation high-speed PCR, results can be obtained in 10 minutes and the amplificate can also be analyzed by DNA-sequencing to achieve species identification as well as detection of resistance gene mutations. The technique has mainly been applied to viruses, but is now slowly adapted to bacteria, fungi, protozoa and helminths. PCR is especially well suited for slow growing bacteria like Mycobacteria, fastidious organisms like Bartonella and contagious agents like tularemia, but also for malaria and fungi, where the advantages in sensitivity and speed can be exploited. The limit for application to routine analysis will depend on the development of simple and fast procedures for nucleic acid extraction, as well as interpretation of the PCR analysis per se, since highly efficient thermocyclers now are on the markets.
Fohlman
Jan
J
Institutionen för medicinska vetenskaper, sektionen för infektion, Akademiska sjukhuset, Uppsala. jan.fohlman@ltkronoberg.se
Blomberg
Jonas
J
Fröman
Gunnar
G
Engstrand
Lars
L
Johansson
Anders
A
Friman
Göran
G
D000328:Adult; D001424:Bacterial Infections / Q000175:diagnosis* / Q000382:microbiology; D005260:Female; D006801:Humans; D008297:Male; D016920:Meningitis, Bacterial / Q000134:cerebrospinal fluid / Q000175:diagnosis* / Q000382:microbiology; D008583:Meningitis, Haemophilus / Q000134:cerebrospinal fluid / Q000175:diagnosis / Q000382:microbiology; D008585:Meningitis, Meningococcal / Q000134:cerebrospinal fluid / Q000175:diagnosis / Q000382:microbiology; D009345:Neisseria meningitidis / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D013997:Time Factors