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PCR and the detection of uncultured pathogens
ELSEVTER
Journal
of Microbiological
Methods
30 (1997) 235-253
Journal “Microbiological Methods
Abstracts of the Second European Meeting on Diagnostic PCR, Kurhaus Hotel, The Hague, The Netherlands, 16- 17 October 1997
2 PCR and the detection of uncultured pathogens Martin Ahwegg Depurtrnent of Medical Microbiology, University Gloriustrusse .?O, 8028 Ziirich, Switzerland
of Ziirich,
For more than a century, cultures in liquid and on solid media have been essential tools for micobiologists allowing the recovery of many different kinds of bacteria. Despite considerable efforts to improve the sensitivity and the selectivity of culture media, we have become increasingly aware of the fact that cultures may be of limited value for various reasons: long generation times; loss of viability due to previous antibiotic therapy or during transport of the specimens to the laboratory; no discrimination between virulent and avirulent strains within a given species; or organisms that so far have not been cultured in vitro. In recent years, the polymerase chain reaction and other amplification techniques have provided a means for the extremely sensitive and specific detection of microorganisms also in clinical specimens. In contrast to cultures, however, specific amplification assays can only be used if one or a few particular organisms need to be looked for. The concept of broad-spectrum PCR amplifying part of the 16s rRNA genes of all (most) bacteria by using primers directed against conserved regions of these molecules followed by direct sequencing (i.e. without subcloning) of the amplified fragment or by using specific probes for identification provides a promising approach for the detection of any bacteria present in a particular clinical material including as yet unknown organisms. We have successfully used this approach for detecting bacteria in culture-negative specimens (Trophe~ma whip&ii in a case of spondylodiscitis, Borrelia burgdorferi in synovial specimens of patients with reactive arthritis, T. whippelii and other organisms in heart valves of patients with endocarditis, etc.). This technique, however. is limited to monobacterial infections and normally sterile specimens and it is prone to reagent contamination (bacterial DNA in water. primers, enzymes, etc.). In addition, it is usually less sensitive than species-specific assays because relatively large fragments need to be amplified to provide sufficient sequence information for identification.
0167.7012/97/$17.00 0 1997 Elsevier Science PII SO167-7012(97)00069-9
B.V. All rights reserved.
3 PCR in routine clinical diagnosis Beverly Dale Director qf Scient$c Affairs, Roche Molecular Atlantic Avenue. Alameda, CA, 94501, USA
Systems,
1145
The conception of the principle of polymerase chain reaction by Kary Mullis in 1983 revolutionized the field of molecular biology and resulted in the awarding of the Nobel Prize for the invention of PCR in 1993. Although early applications of the technology confirmed that PCR had the potential to impact significantly in the field of infectious disease diagnosis, a number of technical advances were required before the introduction of commercial PCR kits in 1992, nine years after the visualization of PCR. These improvements included, among others: the availability of simple, rapid, and reliable sample preparation methods; the optimization of a sensitive non-radioactive detection method already familiar to the clinical laboratorian, the calorimetric microwell format; the introduction of a sterilization system that minimizes the impact of amplicon contamination; the extension of single tube PCR technology to RNA as well as DNA with the exploitation of single enzyme reverse transcription and amplification; and, the clear demonstration that PCR could be configured into a reliable quantitative format with the introduction of the MONITORTM quantitative assays. The introduction of these commercial kits, the AMPLICORB product line, represented the first phase in the broad introduction of PCR technology into the routine clinical diagnostic laboratory. Complete integration of PCR into the clinical lab will depend upon continuing development of the technology in two specific areas: simplification and demonstration of clinical utility. Simplijication: In clinical diagnosis, simplification equates to ease of use and economy of time, two issues best addressed with automation of all processes of testing. The AMPLICORB test system introduced in 1992 requires hands-on manipulation in all three steps of the PCR process, sample preparation, amplification, and detection. In 1995, the COBAS AMPLICORrM test system was introduced, bringing to the clinical laboratory automation of the amplification and detection PCR processes. The first COBAS
Journal
of Microbiological
Methods
30 (1997) 235-253
Journal “Microbiological Methods
Abstracts of the Second European Meeting on Diagnostic PCR, Kurhaus Hotel, The Hague, The Netherlands, 16- 17 October 1997
2 PCR and the detection of uncultured pathogens Martin Ahwegg Depurtrnent of Medical Microbiology, University Gloriustrusse .?O, 8028 Ziirich, Switzerland
of Ziirich,
For more than a century, cultures in liquid and on solid media have been essential tools for micobiologists allowing the recovery of many different kinds of bacteria. Despite considerable efforts to improve the sensitivity and the selectivity of culture media, we have become increasingly aware of the fact that cultures may be of limited value for various reasons: long generation times; loss of viability due to previous antibiotic therapy or during transport of the specimens to the laboratory; no discrimination between virulent and avirulent strains within a given species; or organisms that so far have not been cultured in vitro. In recent years, the polymerase chain reaction and other amplification techniques have provided a means for the extremely sensitive and specific detection of microorganisms also in clinical specimens. In contrast to cultures, however, specific amplification assays can only be used if one or a few particular organisms need to be looked for. The concept of broad-spectrum PCR amplifying part of the 16s rRNA genes of all (most) bacteria by using primers directed against conserved regions of these molecules followed by direct sequencing (i.e. without subcloning) of the amplified fragment or by using specific probes for identification provides a promising approach for the detection of any bacteria present in a particular clinical material including as yet unknown organisms. We have successfully used this approach for detecting bacteria in culture-negative specimens (Trophe~ma whip&ii in a case of spondylodiscitis, Borrelia burgdorferi in synovial specimens of patients with reactive arthritis, T. whippelii and other organisms in heart valves of patients with endocarditis, etc.). This technique, however. is limited to monobacterial infections and normally sterile specimens and it is prone to reagent contamination (bacterial DNA in water. primers, enzymes, etc.). In addition, it is usually less sensitive than species-specific assays because relatively large fragments need to be amplified to provide sufficient sequence information for identification.
0167.7012/97/$17.00 0 1997 Elsevier Science PII SO167-7012(97)00069-9
B.V. All rights reserved.
3 PCR in routine clinical diagnosis Beverly Dale Director qf Scient$c Affairs, Roche Molecular Atlantic Avenue. Alameda, CA, 94501, USA
Systems,
1145
The conception of the principle of polymerase chain reaction by Kary Mullis in 1983 revolutionized the field of molecular biology and resulted in the awarding of the Nobel Prize for the invention of PCR in 1993. Although early applications of the technology confirmed that PCR had the potential to impact significantly in the field of infectious disease diagnosis, a number of technical advances were required before the introduction of commercial PCR kits in 1992, nine years after the visualization of PCR. These improvements included, among others: the availability of simple, rapid, and reliable sample preparation methods; the optimization of a sensitive non-radioactive detection method already familiar to the clinical laboratorian, the calorimetric microwell format; the introduction of a sterilization system that minimizes the impact of amplicon contamination; the extension of single tube PCR technology to RNA as well as DNA with the exploitation of single enzyme reverse transcription and amplification; and, the clear demonstration that PCR could be configured into a reliable quantitative format with the introduction of the MONITORTM quantitative assays. The introduction of these commercial kits, the AMPLICORB product line, represented the first phase in the broad introduction of PCR technology into the routine clinical diagnostic laboratory. Complete integration of PCR into the clinical lab will depend upon continuing development of the technology in two specific areas: simplification and demonstration of clinical utility. Simplijication: In clinical diagnosis, simplification equates to ease of use and economy of time, two issues best addressed with automation of all processes of testing. The AMPLICORB test system introduced in 1992 requires hands-on manipulation in all three steps of the PCR process, sample preparation, amplification, and detection. In 1995, the COBAS AMPLICORrM test system was introduced, bringing to the clinical laboratory automation of the amplification and detection PCR processes. The first COBAS