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J. Med. Microbiol. Vol. 46 (1W7), 188-194 1997 The Pathological Society of Great Britain and lrcland ( ~ REVIEW ARTICLE The Possibilities and limitations of nucleic acid amphfication technology in diagnostic microbiology M. VANEECHOUTTE and J. VAN ELDERE* Department of Clinical Chemistry, Microbiology and Immunology, Blok A, University Hospital, B 9000 Ghent and *Laboratory of Microbiology, University Hospital St Rafael, B 3000 Leuven, Belgium Nucleic acid amplification technology is examined from the critical viewpoint of a clinical microbiologist working in a routine diagnostic bacteriology laboratory. Widely recognised limitations of amplification technology include those of false-positive and false-negative results, the difficulty of obtaining quantitative results, the problem of using this technology for susceptibility testing, and the difficulty of detecting routinely the wide range of possible pathogens contained in a clinical sample. On the positive side, amplification technology brings welcome new possibilities for rapid detection of specific pathogens in a sample, including viruses, slowly growing bacteria, fastidious o r uncultivable bacteria, fungi and protozoa. Other possible applications include screening normally sterile clinical samples for non-specific bacterial contamination and the use of amplification-based DNA fingerprinting methods for identification and typing of microorganisms. Nevertheless, it is predicted that-in contrast to research and reference facilities-routine bacteriology laboratories will continue to rely on culture as the preferred ‘amplification method’ for most diagnostic applications. Introduction This review presents a critical examination of the use of amplification technology in microbiological diagnosis rather than a simple enumeration of its possibilities. The technology is examined from the viewpoint of a clinical microbiologist working in a routine clinical laboratory to assess whether and where amplification technology could replace existing techniques. At the same time this is an attempt to fuel a discussion on the possibilities and limitations of DNA technology in clinical microbiology, with a particular einphasi s on diagnostic bacteriology. In clinical microbiology, the possibility of detecting the presence of organisms in clinical samples directly is the most obvious and most thoroughly studied application of enzymic amplification of nucleic acids [ 1 , 21. For many microbiologists, this application holds the promise that culture will no longer be necessary in the future. Although, at first sight, amplification-based specific detection is a very appealing approach in efforts to enhance diagnostic abilities, several drawbacks limit its application in routine clinical diagnosis. Received 16 July 1996; accepted 18 Aug. 1996. Corresponding author: Dr M. Vaneechoutte. It remains to be established whether, and to what extent, nucleic acid amplification (i.e., enzymic amplification of part(s) of the genome) will be able to replace culture (i.e., biological amplification of the complete genome). Other possible applications of nucleic acid amplification, apart from species-specific amplification for detection purposes, will also be considered. Limitations of nucleic acid amplification technology for detection purposes Practical limitations Some technical problems of nucleic acid amplification technology for detection purposes are recognised widely. Flilse-positive results. These were the most important problem in a study in which seven laboratories were asked to amplify Mycobacteriunz bovis strain BCG from blinded spiked samples [3]. False-positive results may be caused by the fact that DNA (and RNA) amplification is highly sensitive and thus prone to contamination. The most likely sources of contamination are other samples and products from previous amplifications. Processing of negative samples along Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 04 May 2017 11:13:56 NUCLEIC ACID AMPLIFICATION TECHNOLOGY with each extraction batch can control for contamination by sample carryover, and a convenient solution to sample carryover during extraction procedures might be the application of automated nucleic acid extraction techniques ( e g , GenePure 34 1; Applied Biosystems). So far as contamination with amplification products from previous amplifications is concerned, the need for extensive precautions is already well-known [4, 51. Further precautions can be taken by using uracil-Nglycosylase (UNG) to prevent contamination [4, 61. However, both approaches to contamination control should be employed, as illustrated by the fact that the laboratory with the highest number of false-positive results in the study of Noordhoek et al. [3] used UNG decontamination. Although, theoretically, DNA stability may be another possible source of false-positive results as it may lead to amplification of DNA from dead organi sms-e. g ., after successful antibiotic treatmentthis may be a minor problem in practice, as indicated by a study which showed that the frequency of positive results obtained in a PCR test declined rapidly after appropriate therapy for M. tuberculosis [7]. Amplification of more labile RNA targets [8-101 does not suffer from this problem, but requires RNAase-free reagents and equipment to avoid false-negative results. Finally, low specificity of the amplification reaction can also be a source of false-positive results [3]. Therefore, amplification specificity should be controlled by a second amplification with internal primers [ 111 or by hybridisation with a specific probe [8, 9, 11-13]. Generally, commercial systems provide sufficient specificity controls. False-rzegative results. These may result from an intrinsically low sensitivity of the amplification reaction. Besides optimal amplification design and thorough optimisation of the amplification conditions, several approaches to enhance intrinsic sensitivity are possible, such as re-amplification (nested PCR) [ 1 11, the use of repeated sequences as the target DNA ( e g , IS6110 of M. tuberculosis [3, 14]), and several ingenious hybridisation techniques [9, 10, 12, 151. Theoretically, amplification of mRNA or rRNA [8101-which takes advantage of the fact that each actively replicating organism has c. 2000 rRNA molecules compared to only one or a few rRNA cistrons [9]-should also lead to greater sensitivity. Another cause of false negative results can be the presence of polymerase inhibitors in the clinical sample. Addition to each amplification reaction of an artificial target-with an amplification product that can be differentiated readily from that of the real target [16, 171-can be used as a control. Extensive DNA purification [18, 191 is required to remove inhibitors, although this procedure may increase the risk of contamination as it usually comprises multiple steps. Numerous DNA extraction and purification procedures have been published and several systems are now available commercially. Comparative evaluation of 189 these DNA extraction and purification techniques may be of more interest than comparison of the amplification methods. The maximum sample volume of 10-50 pl for enzymic amplification-compared to 0.5- 10 ml for culture-is another important reason for false negative results. For DNA amplification reactions that start from a maximum sample volume of 50 p l (in a 100pl total reaction volume), it can be calculated that 20 DNA target moleculks (or organisms)/ml of sample is the detection limit with ideal DNA extraction and amplification conditions if no concentration of the organisms or the DNA in the total sample has been performed. For detection of pathogens in samples with very low initial inocula-e.g., blood and food-the achievable sensitivity of amplification technology is inadequate and well below that of culture. Thus, semiautomated blood culture can detect one or a few organisms/lO ml of blood-which is often the concentration of bacteria present during septicaemia-and culture can detect a few organisms in contaminated food. Concentration can be achieved by centrifugation or precipitation, but for many clinical samples this means that DNA from the host ( e g , leukocyte DNA from blood samples) will also be concentrated and may inhibit the amplification reaction. Other concentration techniques have been described that use either immunomagnetic procedures to separate target organisms and sequences [20-241 or filtration [25]. This last method has been shown to detect 1 cfu of Listeria mnnocytogenes in 300 ml of milk. RNA amplification may be a possible solution for the problem of small sample volume, as the multiple RNA molecules originating from a single organism may seed the complete sample after cell lysis, increasing the chance that the selected sample contains an amplifiable target. Despite these efforts, amplification of targets from clinical samples in general may have rather limited sensitivity, in contrast to that which is often reported from research studies. For example, with M. tuberculosis many studies report a low sensitivity for paucibacillary specimens (i.e., specimens with <lo4 cfu/ml). Besides the small sample volume, the known tendency for cell clumping [26] of this microorganism provides another explanation of false negative results or low sensitivity following DNA amplification [26]. Although efforts to enhance sensitivity differ markedly from one report to another, the overall levels of sensitivity reached tend to be in agreement. For example, in two studies that amplified a repetitive sequence from M. tuberculosis in sputum samples, a PCR sensitivity of 100% was reported in one study for 29 culture-positive samples when extracting DNA by a simple heating protocol, performing amplification by a single PCR and visualising the product by ethidium bromide staining [7], while the other study [13] reported a PCR sensitivity of 75% for 24 culturepositive samples following DNA purification with a Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 04 May 2017 11:13:56 190 M . VANEECHOUTTE A N D J. VAN ELDERE chaotropic agent and silica, a nested PCR and hybridisation. Similarly, limited sensitivity was reported in a comparison of two commercially available M. tuberculosis tests; indeed, smear-negative, culturepositive samples were detected poorly [27], although it is specifically for this kind of specimen that mycobacteriologists seek more sensitive and reliable diagnosis [26]. Perhaps DNA amplification sensitivity per se-i.e., without confounding factors such as the presence of amplification. inhibitors-can best be evaluated by studies such as that in which the performance of ‘home-made’ PCR tests for detection of M. tuberculosis was compared between different laboratories [3]. The laboratories were sent samples (water, saliva or sputum) spiked with different inocula (0-lo7) of M. hovis strain BCG. The IS6110 insertion element was the amplification target for all laboratories. The data showed that only two laboratories with no falsepositive results detected 80% of the samples containing 500 cells/ml of water (most published studies report a sensitivity of 1-2 cells/25- 100 pl reaction, or 10-40 cells/ml). Furthermore, a sensitivity only 10-fold higher than that of microscopy-i.e., 5 X lo3 cells/ml of sputum-was achieved reproducibly by only one laboratory without false-positive results. From these results-and from the fact that so many completely different techniques are reported to yield good results-it is clear that more comparative studies are needed to enable a thorough comparison of the value of the different methodologies. QuantlJication problems. A further problem of nucleic acid amplification technology is the difficulty in obtaining quantitative results. Quantification is often important for diagnostic work, particularly for samples where the mere presence of an organism is not an indication of infection, as the organism can be present in low numbers as a commensal, or for samples (e.g., urine) where the number of organisms present is used as an indication of infection. Several possible approaches for quantification, each with its own problems, have been reviewed by Reischl and Kochanowski [28] but, in summary, a reproducible quantitative amplification assay is very difficult to construct and remains laborious and cumbersome. The commercially available 5 ’nuclease assay (TaqmanTM LS-SOB PCR detection system; ABI Prism 7700 Sequence Detection System; Applied BioSystems) [29] may be a valuable alternative. However, for bacteriological diagnosis, the capital investment is enormous compared to the cost of culture techniques available currently. The current situation As indicated by the preceding summary of some of the possible solutions to the different technical problems of amplification technology, scientists have been very inventive in building in amplification controls and preventing contamination, in rendering amplification semi-quantitative and in enhancing sensitivity and specificity. However, these measures often involve additional labour, costs and complications that make the technology even less applicable for routine diagnostic work. Automation of amplification technology therefore becomes necessary. Perhaps of most importance is the fact that the sensitivity of nucleic acid amplification technology in a routine clinical laboratory situation remains to be established by comparative studies. It is rather surprising that even for M. tuberculosis-which is probably the bacterium studied most intensively with regard to nucleic acid amplification detection methods - the current situation is that ‘reliable detection of M. tuberculosis is still dependent on conventional laboratory methods until the usefulness of PCR methods has been established’ [3, 301. Fundamental limitations of ampllJication-based detection Even if the preceding limitations of DNA technology are accepted as teething troubles of a new technology which will be resolved sooner rather than later, there are some theoretical constraints that limit the application of this technology in the clinical microbiology laboratory, and especially in the field of bacteriology. Susceptibility testing. This requirement poses a fundamental problem for the introduction of current amplification-based detection methods into diagnostic bacteriology. Information on the susceptibility of a pathogen is often considered to be of more clinical importance than its correct identification. There is a basic problem in that amplification-based detection of all pathogens, even if this was possible (see below), would simply mean extra work unless it was also possible to perform culture-free susceptibility testing. However, for the following reasons, culture seems to be essential for susceptibility testing. First, different primer sets are needed to cover all possible resistance mechanisms, as the encoding genes are scattered all over the genome. Moreover, the genetic basis of resistance to a single class of antibiotics may be very diverse. Thus, p-lactam resistance can be a consequence of P-lactamase production, altered penicillinbinding proteins, porin alterations resulting in reduced membrane permeability, or other mechanisms. Multiplex PCRs - in which up to three or four sets of primers are used simultaneously-provide only a very partial and tedious solution. On the other hand, the Kirby-Bauer diffusion antibiogram-despite its many shortcomings [3 11-enables rapid screening on a single plate of genetically unrelated resistance mechanisms towards antibiotics of different classes. Second, defective or constitutively suppressed resistance genes may be amplified. Finally, the simultaneous demonstration of the presence of a particular resistance mechanism Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 04 May 2017 11:13:56 NUCLEIC ACID AMPLIFICATION TECHNOLOGY and pathogen in a clinical sample is not definite proof of the presence of a resistant pathogen, as similar resistance mechanisms can be found in commensals. Thus, even genotypic susceptibility testing should start ideally from isolated, cultured organisms. Even if a DNA-based non-culture method could be developed for the detection of all known resistance mechanisms, this would allow detection of only the known (i.e., sequenced) mechanisms, while newly evolving resistance mechanisms-which are recognised readily on a diffusion antibiogram by an experienced microbiologist-would escape attention. In general, it can be said that nucleic acid amplification technology is very useful for gaining an insight of molecular mechanisms of resistance, and that it can be used by specialised reference laboratories for large-scale monitoring of certain resistance mechanisms, but that it has little future in the daily work of a routine clinical bacteriology laboratory. Spec$c detection. This is the application of DNA amplification thought currently to be of most use in microbiological diagnosis. Species-specific primers are used for amplification (or species-specific probes for hybridisation). Thus the method is limited to detection of a single species or genus. However, most clinical samples may contain a wide range of possible pathogens. Specific detection of all these pathogens would require the use of different primer sets (and probes), which would be highly impracticable. Again, multiplex PCRs [32] or amplification with a single pair of primers that yields products with different lengths according to the species [33] are only partial solutions. Furthermore, in contrast to virology and parasitology, the current culture alternative used in routine bacteriology is relatively fast and less labour-intensive, as most pathogens can be grown easily and quickly on a limited number of growth media, leading in some instances to an immediate preliminary identification. It is also relevant that culture technology itself is continuing to improve. Thus, for M. tuberculosis, the recent major advances in reduced detection time and enhanced sensitivity have been achieved by combining non-radioactive detection methods with automated liquid culture techniques (Bactec, Becton Dickinson; BacT/Alert, Organon Teknika). The last few years have also seen the development of semi-automated liquid culture systems that permit identification and susceptibility testing of several pathogens in <24 h (VITEK, bioMeri eux; Micr oScan, Baxter) . 19 1 gical agent(s) directly from clinical samples; (b) identification of cultured organism(s) to species level; (c) assessment of the antibiotic susceptibility of isolated organisms; and-to a lesser extent and mainly in hospital laboratories-(d) differentiation between strains of the same species in order to trace nosocomial outbreaks (i.e., strain identification or typing). Detection of micro-organisms Specific detection. As discussed previously, although amplification for specific detection purposes has limited practical applicability for covering the whole array of possible pathogens, it brings welcome new possibilities for detection of viruses, slowly growing bacteria (e.g., Mycobacterium spp.), fastidious or noncultivable bacterial pathogens (e.g., Borrelia, Chlamydia, Ehrlichia, Mycoplasma, Tropheryma [34], certain fungi [4] and protozoa [35]. These are all organisms for which the current alternatives-culture or serology-are slower than DNA amplification technology, but just as labour intensive, or for which no alternatives are available. Consequently several companies (GenProbe, Roche, Abbott, Organon Teknika, Becton Dickinson, Genetrak, Chiron, etc.) have either marketed or are about to market amplificationbased kits for specific detection of some viruses (HIV, HBV, HCV, HSV, etc.) and selected bacterial species ( M . tuberculosis, Chlamydia, etc.). Amplification for specific detection purposes can also be applied routinely in reference laboratories that process a large number of samples while searching for only one or a few species (e.g., screening for Salmonella or Listeria spp. in food quality control or for M, tuberculosis in clinical samples). Universal detection. This application involves the detection of any micro-organism in normally sterile samples. The presence of highly conserved genomic regions throughout the eubacterial kingdom [36] or within some eukaryote groups (e.g., fungi [37]) enables the design of ‘universal’ primers that enable amplification of all members of a particular group. Such primers can be used to screen for the presence of ( e g ) bacteria in normally sterile clinical samples such as blood, cerebrospinal fluid and tissue samples. This approach has been used to detect non-cultivable species [34]. However, limited sensitivity may be a problem (see above). Culture-based applications of ampllJication Possible applications of amplification technology in diagnostic microbiology Given the limitations of nucleic acid-based technology, it is worth considering how this versatile technology could be applied usefully to the different tasks of a clinical microbiology laboratory. The main potential applications include: (a) detection of possible aetiolo- Susceptibility testing. The limitations outlined previously mean that amplification-based susceptibility testing may be applicable only for slowly growing bacteria. Susceptibility testing of potential multipledrug-resistant isolates of M. tuberculosis is the most obvious application [38, 391. DNA technology (hybridisation or amplification) may also indicate latent resistance by detection of non-expressed resistance Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 04 May 2017 11:13:56 192 M. VANEECHOUTTE AND J. VAN ELDERE genes (e.g., some mecA gene positive strains of Staphylococcus aweus which are methicillin-sensitive until challenged in vivo [40]). This is an example of how culture-based susceptibility testing can sometimes yield false-negative results that are resolved by DNA technology. However, it should be noted that both of these applications of amplification technology for susceptibility testing still start from cultured organisms. Identification. Since there still seems to be a need for viable organisms in pure culture (see above), the many problems of phenotypic identification could be solved by the use of amplification-based techniques for determining species identity of cultured organisms. Several PCR-based DNA fingerprinting methods have been claimed to produce species-specific fingerprints, including rDNA RFLP analysis [41, 421, rDNA SSCP analysis [43], tRNA inter-repeat length polymorphism analysis [44] and rDNA spacer length polymorphism analysis [45]. These PCR-based DNA fingerprinting techniques have several advantages over the phenotypic identification methods used currently. They may be faster (automatable) and more reliable, they may enable finer discrimination (allowing differentiation between genomic species), and they may replace the different biochemical techniques and identification schemes by a single universally applicable approach. They also have several advantages compared to approaches based on specific amplification and hybridisation as: (a) they are independent of sequence information so that new species can be added to a databank as new patterns, obviating the need to develop new primers or probes; (b) they are universally applicable, which means that all species can be amplified with the same set of primers; and (c) they are fast, requiring only simple DNA extraction, amplification, restriction and electrophoresis techniques, but no hybridisation. These techniques are currently being evaluated and developed, and fingerprint databanks have yet to be constructed. All of these fingerprinting techniques are indirect means for sequence polymorphism analysis. The reason why the most direct and informative approach of DNA sequencing-although strongly advocated by some groups [8, 461-is not applicable currently as a routine technique is, of course, the inherent high workload. However, several improvements and novel approaches are being developed. First, biochemical sequencing, based on strand termination by dideoxynucleotide incorporation, has developed rapidly since the availability of thermostable polymerases because sufficient product for direct sequencing, together with isolation of the target DNA, can be obtained without the need of cloning and because starting from much lower amounts of target (e.g., by means of cycle sequencing) becomes possible. Analysis of the sequence reaction products has been automated by the use of fluorescent DNA fragment electrophoresis equipment (e.g., ABI 377 DNA sequencer, Applied BioSystems; ALF Express DNA sequencer, Pharmacia Biotech; ABI Prism 3 10 capillary electrophoresis apparatus, Applied BioSystems), sometimes referred to by the somewhat misleading term of ‘automated sequencer’. This equipment has also been shown to be useful for DNA fingerprint analysis [47, 481. Other breakthroughs may come from unexpected directions. The possibility of developing microchips coated with an array of oligonucleotides of known sequence at well-determined locations [49, 501 may allow sequence determination of a target based on the combination of the hybridisation events of the target with different probes. Other applications of bioelectronics [5 I] include coupling oligonucleotides to a fibreoptic biosensor [52] to enable rapid analysis of hybridisation events. Developments in microscopy might even allow a DNA sequence to be literally ‘read’ through direct recognition of the morphology of the nucleotides [53]. As amplification usually yields a homogeneous mixture of molecules, this approach might not be too difficult. However, although these developments might turn sequence determination into a rapid technique that may supplant the indirect sequence analysis techniques of DNA fingerprinting and classical hybridisation, it should be emphasised that the possible availability of these rapid sequencing techniques does not alleviate the limitations of specific DNA amplification technology outlined above. Tvping. The techniques of arbitrarily-primed PCR or RAPD [54, 551, rRNA spacer length polymorphism analysis (‘PCR-ribotyping’) [56, 571, inter-repeat spacer length polymorphism analysis [5 81, tandem repeat number polymorphism analysis [59, 601 and gene restriction analysis (PCR-RFLP) [6 I , 621 are examples of the numerous DNA amplification-based possibilities for differentiating between strains of a single species [63]. The major advantage of these simple and rapid PCR-based techniques is that they bring genotypic epidemiological analysis within the reach of most routine diagnostic laboratories, so that typing becomes an effective tool for dealing with nosocomial outbreaks. Currently, most phenotypic typing methods (e.g., serotyping, phage typing, etc.) and other genotypic typing methods (e.g., chromosomal DNA restriction analysis with low frequency restriction enzymes, ribotyping) are slow or are carried out in reference laboratories because they are complicated or need special reagents. As a consequence, too much time is often lost for the typing results to be used effectively in tracing the source of nosocomial outbreaks. Rapid sequencing techniques might also enable strain differentiation and could eventually replace these indirect means of sequence polymorphism analysis. Conclusions In summary, from the critical viewpoint of this review, the application of amplification methods for species- Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 04 May 2017 11:13:56 NUCLEIC ACID AMPLIFICATION TECHNOLOGY specific detection is usefully limited to viruses, to poorly or non-cultivable bacteria, or to reference laboratories that concentrate on detecting a single pathogen. This is because of the many technical difficulties of this technology and the continuing need for cultured bacteria to perform susceptibility testing, and because the non-universality and lack of standardisation of amplification-based detection make it impracticable for routine laboratories. Susceptibility testing based on DNA technology is applicable for large-scale studies of the epidemiology of particular resistance mechanisms and for obtaining a molecular insight into resistance mechanisms, but will probably remain limited in the clinical bacteriology laboratory to organisms for which culture is slow or impossible. Therefore, it can be predicted that routine bacteriology will also continue to rely on culture for detection of most pathogens. Several culture-dependent, but broadly applicable, nucleic acid-based amplification techniques are being developed for discrimination of different strains within a species and for differentiation between species, although rapid sequence determination might conceivably replace the indirect approaches of hybridisation and DNA fingerprinting in the future. Thus, the overall conclusion of this review is that provided culture is not unduly slow-as is the case for most known pathogenic bacteria-it remains the preferred ‘nucleic acid amplification method’ for diagnostic microbiology, in part because it yields a complete organism and genome instead of one or a few DNA fragments. Therefore, it seems unlikely that the achievements of Watson, Crick, Mullis and Faloona will replace those of Pasteur in our routine bacteriology laboratories in the immediate future. Rather, microbiologists will continue to take the best of both approaches. The authors thank Anne-Mie Vandamme for critical reading of the manuscript. References 1. s.‘ilk1 RK, Scharf S, Faloona F et al. Enzymatic amplification of P-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 198.5; 230: 13501354. 2. Mullis KB, Faloona FA. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enz-ymol 1987; 155: 335-350. 3. Noordhoek GT, Kolk AHJ, Bjune G et ul. Sensitivity and specificity of PCR for detection of Mycobucterium tuberculosis: a blind comparison study among seven laboratories. J Clin Microbiol 1994; 32: 277-284. 4. E m s RK, Bromley SE, Day SP et ul. Molecular Diagnostic Methods for Infectious Diseases, Approved Guideline. NCCLS MM3-A. December 1995. 5 . Kwok S, Higuchi R. Avoiding false positives with PCR. Nuture 1989; 339: 237-238. 6. Loewy ZG, Mecca J, Diaco R. Enhancement of Borrelia burgdoi$eri PCR by uracil N-glycosylase. J Clin Micvohiol 1994; 32: 135-138. 7. Kocagoz T, Yilmaz E, Ozkara S et al. Detection of Mycobucterium t u b e i d o s i s in sputum samples by polymerase chain reaction using a simplified procedure. J Clin Microbiol ‘ ’ 193 1993; 31: 1435-1438. 8. Boddinghaus B, Rogall T, Flohr, Blocker H, Bottger EC. Detection and identification of Mycobacteria by amplification of rRNA. J Clin Microbiol 1990; 28: 175 1- 1759. 9. Jonas V, Alden MJ, Curry JI et al. Detection and identification of Mycobacterium tuberculosis directly from sputum sediments by amplification of rRNA. J Clin Microbiol 1993; 31: 2410-2416. 10. van der Vliet GME, Schukkink RAF, van Gemen B, Schepers P, Klatser PR. Nucleic acid sequence-based amplification (NASBA) for the identification of mycobacteria. J Gen Microbiol 1993; 139: 2423-2429. 11. Pierre C, Lecossier D, Boussougant Y et al. Use of a reamplification protocol improves sensitivity of detection of Mjmbacterium tuberculosis in clinical samples by amplification of DNA. J Clin Microbiol 1991; 29: 712-717. 12. Fiss EH, Chehab FF, Brooks GF. DNA amplification and reverse dot blot hybridization for detection and identification of Mycobacteria to the species level in the clinical laboratory. J Clin Microbiol 1992; 30: 1220-1224. 13. Wilson SM, McNerney R, Nye PM, Godfrey-Faussett PD, Stoker NG, Voller A. Progress toward a simplified polymerase chain reaction and its application to diagnosis of tuberculosis. J Clin Microbiol 1993; 31: 776-782. 14. Zwadyk P, Down JA, Myers N, Dey MS. Rendering of mycobacteria safe for molecular diagnostic studies and development of a lysis method for strand displacement amplification and PCR. J Clin Microbiol 1994; 32: 21402146. 15. Stuyver L, Rossau R, Wyseur A et al. Typing of hepatitis C virus isolates and characterization of new subtypes using a line probe assay. J Gen Virol 1993; 74: 1093-1 102. 16. Pallen MJ, Puckey LH, Wren BW. A rapid, simple method for detecting PCR failure. PCR Methods Appli 1992; 2: 91-92. 17. Ursi J-P, Ursi D, Ieven M, Pattyn SR. Utility of an internal control for the polymerase chain reaction. Application to detection of Mycoplusma pneiimoniae in clinical specimens. APMIS 1992; 100: 635-639. 18. Boom R, Sol CJA, Salimans MMM, Jansen CL, Wertheimvan Dillen PME, van der Noordaa J. Rapid and simple method for purification of nucleic acids. J Clin Microbiol 1990; 28: 495-503. 19. Cheung RC, Matsui SM, Greenberg HB. Rapid and sensitive method for detection of hepatitis C virus RNA by using silica particles. J Clin Microbiol 1994; 32: 2593-2597. 20. Mazurek GH, Reddy V, Murphy D, Ansari T. Detection of Mycobacterium tuberculosis in cerebrospinal fluid following immunomagnetic enrichment. J Clin Microbiol 1996; 34: 450-453. 21. Olsvik 0, Popovic T, Skjerve E et al. Magnetic separation techniques in diagnostic microbiology. Clin Microbiol Rev 1994; 7: 43-54. 22. Fluit AC, Torensma R, Visser MJ et ul. Detection of Listeriu monocytogenes in cheese with the magnetic immuno-polymerase chain reaction assay. Appl Environ Microbiol 1993; 59: 1289-1293. 23. Mangiapan G, Vokurka M, Schouls L et al. Sequence capture-PCR improves detection of mycobacterial DNA in clinical specimens. J Clin Microbiol 1996; 34: 1209- 1215. 24. Niederhauser C, Luthy J, Candrian U. Direct detection of Listeria monocytogenes using paramagnetic bead DNA extraction and enzymatic DNA amplification. Mol Cell Probes 1994; 8: 223-228. 25. Starbuck MA, Hill PJ, Stewart GS. Ultra sensitive detection of Listeria monocytogenes in milk by the polymerase chain reaction (PCR). Lett Appl Microbiol 1992; 15: 248-252. 26. Ieven M, Goossens H. Molecular diagnostics in the clinical laboratory: a plea for prudence! Acta Clin Belg 1995; 50: 255-259. 27. Vuorinen P, Miettinen A, Vuento R, Hallstrom 0. Direct detection of Mvcobacterium tuberculosis complex in respiratory specimens by Gen-Probe Amplified Mycobucterium Tuberculosis Direct Test and Roche Amplicor Mycobacterium tuberculosis Test. J Clin Microbiol 1995; 33: 1856- 1859. 28. Reischl U, Kochanowski B. Quantitative PCR. A survey of the present technology. Mol Biotechnol 1995; 3: 55-71. 29. Bassler HA, Flood SJ, Livak KJ, Marmaro J, Knorr R, Batt CA. Use of a fluorogenic probe in a PCR-based assay for the detection of Listeriu monocytogenes. Appl Environ Microbiol Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 04 May 2017 11:13:56 194 M. VANEECHOUTTE AND J. VAN ELDERE 1995; 61: 3724-3728. 30. Centers for Disease Control and Prevention. Diagnosis of tuberculosis by nucleic acid amplification methods applied to clinical specimens. Morbid Mortal Weekly Rep 1993; 42: 686. 3 1. Barry AL, Thornsberry C. Susceptibility tests: diffusion test procedures. In: Balows A, Hausler WJ, Herrmann KL, Isenberg HD, Shadomy HJ (eds) Manual of clinical microbiology, 5th edn. Washington DC, American Society of Microbiology 1991: 1 1 17- 1 125. 32. Wilton S, Cousins D. Detection and identification of multiple mycobacterial pathogens by DNA amplification in a single tube. PCR Methods Appl 1992; 1: 269-273. 33. Jordan JA. PCR identification of four medically important Candida species by using a single primer pair. J Clin Microbiol 1994; 32: 2962-2967. 34. Relman DA, Falkow S. Identification of uncultured microorganisms: expanding the spectrum of characterized microbial pathogens, Infect Agents Dis 1992; 1: 245-253. 35. Weiss JB. DNA probes and PCR for diagnosis of parasitic infections. Clin Microbiol Rev 1995; 8: 113-130. 36. Woese CR. Bacterial evolution. Microbiol Rev 1987; 51: 221 -271. 37. Makimura K, Murayama SY, Yamaguchi H. Detection of a wide range of medically important fungi by the polymerase chain reaction. J Med Microbiol 1994; 40: 358-364. 38. Telenti A, Imboden P, Marchesi F et al. Detection of rifampicin-resistance mutations in Mycobacterium tuberculosis. Lancet 1993; 341: 647-650. 39. De Beenhouwer H, Lhiang Z, Jannes G et al. Rapid detection of rifampicin resistance in sputum and biopsy specimens from tuberculosis patients by PCR and line probe assay. Tuber Lung Dis 1995; 76: 425-430. 40. Unal S, Hoskins J, Flokowitsch JE, Wu CYE, Preston DA, Skatrud PL. Detection of methicillin-resistant Staphylococci by using the polymerase chain reaction. J Clin hficrobiol 1992; 30: 1685-1691. 41. Vilgalys R, Hester M. Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. J Bacteriol 1990; 172: 42384246. 42. Vaneechoutte M, Dijkshoorn L, Tjernberg 1 et al. Identification of Acinetobacter genomic species by amplified ribosomal DNA restriction analysis. J Clin Microbiol 1995; 33: 11-15. 43. Widjojoadmotjo MN, Fluit AC, Verhoef J. Rapid identification of bacteria by PCR-single-strand conformation polymorphism. J Clin Microbiol 1994; 32: 3002-3007. 44. Welsh J, McClelland M. Genomic fingerprints produced by PCR with consensus tRNA gene primers. Nucleic Acids Res 1991; 19: 861-866. 45. Jensen MA, Webster JA, Straus N. Rapid identification of bacteria on the basis of polymerase chain reaction-amplified ribosomal DNA spacer polymorphisms. Appl Environ Microbiol 1993; 59: 945-952. 46. Kirschner P, Springer B, Vogel U et ai. Genotypic identification of Mycobacteria by nucleic acid sequence determination: report of a 2-year experience in a clinical laboratory. J Clin Microbiol 1993; 31: 2882-2889. 47. Martin F, Vairelles D, Henrion B. Automated ribosomal DNA fingerprinting by capillary electrophoresis of PCR products. Anal Biochem 1993; 214: 182-189. 48. Wiedmann-Al-Ahmad M, Tichy H-V, Schon G. Characterization of Acinetobacter type strains and isolates obtained from wastewater treatment plants by PCR fingerprinting. Appl Environ Microbiol 1994; 60: 4066-407 1. 49. Smith LM. The future of DNA sequencing. Science 1993; 262: 530-532. 50. Lipshutz RJ, Morris D, Chee M et al. Using oligonucleotide probe arrays to access genetic diversity. Biotechniques 1995; 19: 442-447. 5 1. Eggers M, Ehrlich D. A review of microfabricated devices for gene-based diagnostics. Hematol Pathoi 1995; 9: 1 - 15. 52. Strachan NJ, Gray DI. A rapid general method for the identification of PCR products using a fibre-optic biosensor and its application to the detection of Listeria. Lett Appl Microbiol 1995; 21: 5-9. 53. Kopelman R, Tan W. Near-field optics: imaging single molecules. Science 1993; 262: 1382-1384. 54. Welsh J, McClelland M. Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res 1990; 18: 72137218. 55. Williams JGK, Kubelik AR, Livak KJ, Rafalski JA, Tingey SY DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res 1990; 18: 653 1-6535. 56. Gurtler Y Typing of Clostridium diJJicile strains by PCRamplification of variable length 16S-23s rDNA spacer regions. J Gen Microbiol 1993; 139: 3089-3097. 57. Kostman JR, Edlind TD, LiPuma JJ, Stull TL. Molecular epidemiology of Pseudomonas cepacia determined by polymerase chain reaction ribotyping. J Clin Microbiol 1992; 30: 2084-2087. 58. Versalovic J, Koeuth T, Lupski JR. Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Res 1991; 19: 6823683 1 . 59. Goh S-H, Byrne SK, Zhang JL, Chow AW. Molecular typing of Staphylococcus aureus on the basis of coagulase gene polymorphisms. J Clin Microbiol 1992; 30: 1642- 1645. 60. Frenay HME, Theelen JPG, Schouls LM et al. Discrimination of epidemic and nonepidemic methicillin-resistant Staphylococcus aureus strains on the basis of protein A gene polymorphism. J Clin Microbiol 1994; 32: 846-847. 61. Gardiner D, Hartas J, Currie B, Mathews JD, Kemp DJ, Sriprakash KS. Vir typing: a long-PCR typing method for group A streptococci. PCR Methods Appl 1995; 4: 288-293. 62. King V, Clayton CL. Genomic investigation of phenotypic variation in Campylobacter jejuni flagellin. FEMS Microbiol Lett 1991; 84: 107-111. 63. Vaneechoutte M. Review of DNA fingerprinting techniques for micro-organisms: a proposal for classification and nomenclature. Mol Biotechnol 1996; 6 (in press). Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 04 May 2017 11:13:56