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Molecular Microbiology: Diagnostic Principles and Practice, 2nd Ed. Edited by David H. Persing et al. 䉷2011 ASM Press, Washington, DC Maiwald M. Broad-range PCR for detection and identification of bacteria. pp. 491-505. In: Persing DH, Tenover FC, Tang YW, Nolte FS, Hayden RT, van Belkum A (eds.). Molecular microbiology: diagnostic principles and practice. 2nd edition. American Society for Microbiology, Washington DC, 2011. ISBN 978-1-55581-497-7. 31 Broad-Range PCR for Detection and Identification of Bacteria MATTHIAS MAIWALD BACKGROUND ecules, though, provide suitable priming sites as well as phylogenetically informative sequences (see chapter 9). For example, sequences of genes coding for conserved proteins (e.g., heat shock proteins and RNA polymerases) can be very useful for identification within bacterial families (99) but generally do not provide sufficiently conserved sites for primers across the domain Bacteria. rRNAs, on the other hand, fulfill many of the necessary requirements (104). rRNA molecules have been described as the ultimate molecular chronometers (163); they reflect evolutionary changes, while functional constraints in protein translation prevent these changes from being too extensive. In the 1980s, oligonucleotides from conserved regions were first used for reverse transcriptase sequencing of bacterial 16S rRNA (77). Subsequently, such conserved oligonucleotides were utilized as primers in PCR (23, 38). The two main molecules that are suitable for bacterial broad-range PCR are the 16S rRNA gene, consisting of approximately 1,540 bp (in Escherichia coli, 1,542 bp), and the 23S rRNA gene, consisting of approximately 2,900 bp (in E. coli, 2,904 bp). The 5S rRNA gene, the third structural rRNA gene with about 120 bp, is too small for this purpose. While the 23S rRNA gene possesses greater information content and more potential priming sites due to its larger size, the 16S rRNA gene was adopted earlier, has become a quasistandard, and has many more reference sequences available for comparison. For example, the SILVA online resource (111) contained 324,342 different smallsubunit (16S rRNA-like) sequences of 1,200 bp or greater (900 bp or greater for Archaea) and 12,506 different largesubunit (23S rRNA-like) sequences of 1,900 bp or greater in its release of 14 October 2008 (http: / / www.arb-silva.de). The numbers of small partial 16S and 23S rRNA sequences of ⬎300 bp are much greater. Broad-range PCR is based on the recognition that there are a number of broadly conserved molecules across a range of many different organisms. rRNA genes, for example, are present in all cellular forms of life, namely the domains Bacteria, Archaea, and Eukarya (163). rRNA genes possess highly conserved regions that are suitable as sites for PCR primers that recognize large, diverse groups of organisms (e.g., all members of the Bacteria) and possess variable regions that provide distinct signatures for identification at phylogenetic levels below the level initially targeted by the primers. Commonly, broad-range PCR are aimed at members of the domain Bacteria, although broad-range PCRs have also been developed for other large relevant groups of organisms or combinations of such groups. For example, broad-range PCRs may target Eukarya (91, 119), Archaea (1, 4, 143), Eukarya and Archaea (10), fungi (70, 130, 159), or fungi and protists (44). Fungal broad-range PCRs are often termed panfungal PCRs in the medical literature. Below the level of broad groups of organisms, PCRs may be constructed to target organisms at various other phylogenetic levels; for example, within the Bacteria it is possible to design phylum-, family-, or genus-specific PCRs, depending on the availability of phylogenetically informative signature sequences. The strength of broad-range PCR for diagnostic microbiology lies in the relative absence of selectivity, assuming little or no prior knowledge of an infecting organism, so that—in principle—any bacterium (in the case of bacterial assays) can be detected and identified. This is an area of analogy to the detection by culture and is in contrast to typical organism-specific PCR assays. The aim of most broad-range PCRs is to amplify as broadly as possible within the domain Bacteria and, at the same time, to obtain sufficient portions of variable sequence for identification. Not all broadly conserved mol- PATHOGEN DISCOVERY Broad-range ribosomal PCR and sequence analysis have played key roles in the characterization of novel bacterial pathogens, particularly in settings where detection by culture has failed. The first agent discovered by this technique Matthias Maiwald, Department of Pathology and Laboratory Medicine, KK Women’s and Children’s Hospital, Singapore 229899, Singapore. 491 492 ■ MAIWALD was the agent of bacillary angiomatosis in AIDS patients (118), which was microscopically visible in clinical samples but had not been cultivated. The 16S rRNA sequence revealed a relationship to Rochalimaea (subsequently named Bartonella) quintana, the agent of trench fever. The novel bacterium was subsequently cultivated, named Bartonella henselae, and found also to be an agent of cat scratch disease (13, 17, 157). Other examples of pathogen discoveries by broad-range PCR and sequencing are Ehrlichia chaffeensis, an agent of human tick-borne monocytic ehrlichiosis (5), Tropheryma whipplei, the agent of Whipple’s disease (120, 160), and Mycobacterium genavense, a cause of disseminated infections in AIDS patients (15). Thus, broad-range PCR joined several other molecular methods that were capable of gathering insight into distinguishing features of pathogens by obtaining molecular signatures before other characteristic traits were known (46). It was also recognized that the mere identification of a microbial molecular signature in a person with a disease does not establish a causal relationship between microbe and disease, and a set of nucleic acid-based criteria for causality was proposed, by analogy with the historic Koch’s postulates (46). Broad-range PCR and sequencing is not only suitable for the characterization of uncultivated bacteria; 16S rRNA sequence deposition has become a mainstay of established procedures whenever a new bacterial species is described. One review article (165) counted 215 newly described bacterial species (obtained mostly by culture) from human specimens between the years 2000 and 2007, of which 29 were assigned to novel genera and 100 were found in four or more patients. PRIMER SYSTEMS FOR BROAD-RANGE PCR rRNA molecules consist of conserved as well as variable regions; a depiction of these regions and the corresponding priming sites in the bacterial 16S rRNA gene is given in Fig. 1A. A selection of broad-range primers for the 16S and 23S rRNA genes is provided in Table 1. Further primer and probe sequences for 16S and 23S rRNA gene can be found in other articles (1, 4, 6, 9, 61, 63, 76, 88, 123, 133, 150, 155), as well as in the European rRNA database (http: / / bioinformatics.psb.ugent.be / webtools / rRNA) (166) and in the ProbeBase database (http: / / www.microbial-ecology. net / probebase) (84). When looking at rRNA secondary structure, conserved regions tend to lie in loops, while variable regions tend to lie in stems, where nucleotides are paired with those of the opposite strand (76). Primers for diagnostic broad-range PCR need to amplify sufficiently long stretches of variable sequence to be able to identify bacteria at or close to species level; generally, targets of ⬃300 to 900 bp are used in most diagnostic broad-range PCRs (e.g., primers 533 forward [f] plus 806 reverse [r], 27f plus 556r, 27f plus 806r, or 533f plus 1371r). Targets towards the shorter end of this range may be preferable when the extracted DNA is likely to be damaged (e.g., formalinfixed, paraffin-embedded tissue); longer targets (e.g., primers 27f / 1492r) may be used when bacteria in samples are abundant. Generally, longer targets provide more variable sequence for more accurate identification. Primers that anneal to eukaryotic nuclear or mitochondrial DNA (‘‘universal’’ primers; e.g., 533f plus 1492r) should not be used as pairs in the same assay when working with human samples (117). The distinction between conserved and variable regions in rRNA molecules is not as sharp as the depiction in Fig. 1A suggests and is instead better reflected in Fig. 1B. With an increasing number of rRNA sequences in databases, it has been recognized that some nucleotide positions that are less conserved are interspersed in highly conserved sequence stretches, and some taxa that are part of an intended target group of organisms have mismatches to commonly used broad-range primers (9, 42). This is in part accounted for by the ambiguous nucleotides in the primers FIGURE 1 (A) Schematic drawing of the bacterial 16S rRNA gene, with conserved (open bars) and variable (shaded bars) regions. The location of broad-range primers (Table 1) is shown. Modified from reference 117, with permission from the American Society for Microbiology. (B) Conservation profile of 16S rRNA across the domain Bacteria, constructed with the software package ARB (85) and the SILVA (111) data set. The graph shows the relative frequency (y axis) of the most common nucleotide for each position (x axis) in 16S rRNA. Courtesy of Frank Oliver Glöckner and Elmar Pruesse (Max Planck Institute for Marine Microbiology, Bremen, Germany). 31. Broad-Range PCR for Bacterial Identification ■ 493 TABLE 1 Commonly used primers and probes for broad-range PCR with bacterial 16S and 23S rRNA genes Name a Sequence b Location c Specificity d Reference(s) b 16S rRNA 27f 355f 338r 533f 515r 556r 806f 787r 926f 907r 930f 911r 1194f 1175r 1371r 1391r 1492r 1525r 5⬘-AGAGTTTGATYMTGGCTCAG 5⬘-ACTCCTACGGGAGGCAGC 5⬘-GCTGCCTCCCGTAGGAGT 5⬘-GTGCCAGCMGCCGCGGTAA 5⬘-TTACCGCGGCKGCTGGCAC 5⬘-CTTTACGCCCARTRAWTCCG 5⬘-ATTAGATACCCTGGTAGTCC 5⬘-GGACTACCAGGGTATCTAAT 5⬘-AAACTYAAAKGAATTGACGG 5⬘-CCGTCAATTCMTTTRAGTTT 5⬘-TCAAAKGAATTGACGGGGGC 5⬘-CCGTCAATTCMTTTRAGTTT 5⬘-GAGGAAGGTGGGGATGACGT 5⬘-ACGTCATCCCCACCTTCCTC 5⬘-AGGCCCGGGAACGTATTCAC 5⬘-TGACGGGCGGTGWGTRCA 5⬘-GGTTACCTTGTTACGACTT 5⬘-AAGGAGGTGWTCCARCC 8–27 338–355 33–355 51–533 515–533 556–575 787–806 787–806 907–926 907–926 911–930 911–930 1175–1194 1175–1194 1390–1371 1391–1408 1510–1492 1525–1541 Most Bacteria Most Bacteria Most Bacteria Universal Universal Most Bacteria Most Bacteria Most Bacteria Most Bacteria Most Bacteria Most Bacteria Most Bacteria Most Bacteria Most Bacteria Most Bacteria Universal Universal Most Bacteria and Archaea 38 3 3 77, 120 77, 120 13 162 162 77 77 120 120 23 23 23 77 37, 156 38 23S rRNA 1077f 1950r 5⬘-AGGATGTTGGCTTAGAAGCAGCCA 5⬘-CCCGACAAGGAATTTCGCTACCTTA 1054–1077 1950–1926 Most Bacteria Most Bacteria 73 73 a Names of 16S rRNA primers are based on the 3⬘ nucleotide position and the orientation (f, forward; r, reverse), according to the numbering system by Lane (76). b Sequences may have been modified from the indicated references by subsequent authors. References are given for the original publications of the priming sites. Location in E. coli 16S rRNA or 23 S rRNA. d Universal includes Bacteria, Archaea, and Eukarya. c in Table 1, but in some instances it may necessitate the redesign of commonly used broad-range primers or hybridization probes to achieve more complete coverage of bacterial phyla (30, 42). While the impact of this appears to be somewhat smaller for diagnostic broad-range PCR in cases of suspected monomicrobial infections, it can be dramatic for polymicrobial infections, microbial flora studies, or environmental studies, where certain bacterial taxa may become significantly over- or underrepresented after PCR amplification (11, 39, 59, 63, 137, 142, 155). In a general sense, the performance of PCR assays may be affected by (i) length of the PCR product, (ii) annealing temperature, (iii) number of cycles, (iv) template concentration, (v) internal composition (e.g., G⫹C richness) of the target, and (vi) primer base composition (G⫹C richness and internal base pairing). Longer distances between primer pairs generally result in less analytical sensitivity, but this may be partially offset by the finding that DNA contaminations in PCR reagents (see below) appear to be fragmented into smaller sizes (132), so that the ratio between target and contaminations becomes more favorable. A large study of broad-range PCR has in fact shown quite robust amplification of an ⬃850-bp target from the 23S rRNA gene with diverse clinical samples (115). In addition to the possible mismatches of rRNA gene primers (see above), broad-range PCR has also been found to be affected by rRNA gene copy numbers in different bacterial genomes and even by interference from bacterial DNA outside the PCR target region (53, 154). In some instances, different primer sets show different amplification efficiency despite perfect primer-to-target matches (39). As a consequence of performance and coverage differences between broad-range PCR primers, they may have to be redesigned and reevaluated, depending on the needs of particular diagnostic or scientific applications. There are a number of tools to establish and check the matches of primers and probes against large 16S rRNA and 23S rRNA data sets. The Ribosomal Database Project (RDP) website (http: / / rdp.cme.msu.edu) (27) has a probe match tool for that purpose. A computer program called PRIMROSE can also be used to construct primers and probes in conjunction with the RDP data set (8). Other web interface tools are the probeBase (http: / / www.microbial-ecology. net / probebase) (84) and probeCheck (http: / / www. microbial-ecology.net / probecheck) (83) resources. In addition, the ARB phylogeny program package (http: / / www.arb-home.de) (85) has probe design and probe match functions that can be used, for example, in conjunction with the SILVA 16S and 23S rRNA data sets (111). CONTAMINATION ISSUES OF BROAD-RANGE PCR Broad-range PCR is subject to much of the same contamination problems and requires much of the same prevention measures as specific PCRs (see chapter 8). In addition, the inherent nonselective nature of broad-range PCR makes it 494 ■ MAIWALD susceptible to minute amounts of any bacterial DNA that might be encountered along the various steps of testing. While specific PCR does not amplify DNA from nontarget organisms, broad-range PCR, by its design, can amplify DNA from almost any bacterium. Soon after broad-range PCR was originally implemented, it was found that negative controls often yielded amplified DNA of the appropriate size. Such false-positive results would not be avoided by common anticontamination measures. Several types of reagents have been implicated as being sources of DNA contamination: Taq polymerase was commonly implicated (14, 113, 132), but also other reagents, such as water and oligonucleotide solutions (49) and buffers or enzymes for sample preparation (144). The application of restriction analysis (113) suggested that Thermus aquaticus and E. coli (the host species for native and recombinant Taq polymerase) are unlikely sources of contamination in Taq polymerase. By contrast, the columns and buffers used for the industrial purification of Taq polymerase were thought to be likely sources. Sequencing methods identified contaminating DNA as originating from within the pseudomonad group of organisms (86, 118), which are known waterborne contaminants. The amount of contaminating DNA was estimated to be in the range of 100 copies of 16S rRNA gene per Taq portion needed for a single reaction (113). Taq polymerases from different manufacturers were found to be affected (14, 62, 132), and there appears to be manufacturer-to-manufacturer and even batch-to-batch variation in the amount of contaminating DNA (113, 128, 132). As a consequence, the detectable limit (i.e., analytical sensitivity) of PCR assays using such Taq preparations lies well above 100 copies per PCR, if one wants to distinguish ‘‘real’’ from contaminating DNA. Shorter PCR targets tend to be more susceptible to the presence of background DNA, since this DNA appears to be fragmented into small sizes (132). There also seems to be a physiochemical affinity between Taq polymerase and contaminating DNA, since they appear to be strongly associated with each other (113). Several protocols have been developed to avoid or reduce contaminating DNA in PCR reagents. There are a few commercial Taq polymerase preparations, such as AmpliTaq LD or AmpliTaq Gold LD (Applied Biosystems), where LD stands for low DNA, and these enzymes have been purified to contain 10 or fewer bacterial rRNA gene copies in a standard 2.5-U aliquot. Published protocols also include the treatment of PCR mixes, including Taq polymerase, with DNase I (56, 124, 136, 139, 147, 148), restriction enzymes (20), or UV light (47, 105). DNase I and restriction enzymes need to be heat inactivated before the addition of samples and commencement of cycling (20, 124). Some authors pointed out that UV treatment should take place prior to adding deoxynucleoside triphosphates (dNTPs), because dNTPs absorb UV light and prevent exposure of other PCR mix contents (47). Several others (62, 92, 128) described a combined protocol of 8methoxypsoralen and UV treatment of PCR mixes. 8Methoxypsoralen acts as UV-induced cross-linker of DNA in contaminated reagents. However, it was pointed out that the 8-methoxypsoralen concentration and time of UV exposure need to be carefully adjusted, because intense exposure damages Taq polymerase and leads to reduced PCR sensitivity. Other published approaches include the ultrafiltration of PCR reagents (136) and treatment with ethidium monoazide or propidium monoazide (55, 129). Ultrafiltration with a 100-kDa exclusion size (e.g., Microcon YM-100; Millipore) lets Taq polymerase (94 kDa) pass through the filter, while typical contaminating DNA is retained; however, some Taq polymerase loss may be observed. Ethidium monoazide and propidium monoazide are photoreactive DNA-intercalating substances that are activated by visible light. Some protocols involve splitting PCR mix components for different treatments. For example, Steinman et al. (139) subjected the PCR mix without primers to DNase I treatment and treated the primers separately with UV light before combining the mix. Table 2 contains an overview of the different anticontamination methods for broad-range PCR. Several authors have examined different anticontamination procedures in parallel (28, 71, 136). It became clear that most anticontamination measures provide a trade-off between reducing contaminating DNA and retaining PCR TABLE 2 Methods to reduce bacterial background DNA in PCR reagents Measure(s) Comment(s) Low-DNA Taq polymerase (e.g., AmpliTaq LD, AmpliTaq Gold LD) DNase I treatment of PCR mix Commercially available, contains 10 or fewer bacterial rRNA gene copies per 2.5 U Effective in DNA elimination; inactivation critical; minor loss of PCR sensitivity; some authors treat PCR mix without primers Mixed results concerning DNA elimination and retained sensitivity; some authors treat PCR mix without dNTPs and primers Taq polymerase may be sensitive to UV action; dNTPs can impede UV action; probable loss of sensitivity 8-Methoxypsoralen concn and UV exposure have to be fine-tuned; longer exposure reduces PCR sensitivity Concn of ethidium monoazide in PCR mix is critical; good elimination of contaminating DNA and retained sensitivity Type of filter and centrifugation time and speed are critical; good elimination of contaminating DNA and retained sensitivity Restriction enzyme treatment of PCR mix UV irradiation of PCR mix 8-Methoxypsoralen and UV light treatment of PCR mix Treatment of PCR mix with ethidium monoazide or propidium monoazide and light exposure Ultrafiltration of PCR mix with exclusion size of 100 kDa Reference(s) 147 28, 56, 57, 71, 124, 136, 139, 147, 148 20, 28, 71 28, 47, 71, 105 28, 62, 71, 92, 128 55, 129 136 31. Broad-Range PCR for Bacterial Identification ■ sensitivity. Corless et al. (28) examined UV irradiation, 8methoxypsoralen plus UV, DNase I digestion, restriction enzyme digestion, and DNase I plus restriction enzyme treatment, using broad-range PCR in a quantitative realtime format; this format facilitates the monitoring of DNA contamination (see below). A significant reduction in analytical sensitivity accompanied the reduction of contaminating DNA in most of the treatment protocols, except DNase I treatment, for which the reduction in sensitivity was relatively small, with only a 1- to 2-log reduction. The combination of DNase I and restriction enzyme digestion suffered from problems with test-to-test reproducibility. The authors concluded that DNA contamination in broadrange PCR assays will continue to be a problem. Klaschik et al. (71) compared DNase I treatment, restriction enzyme digestion, UV irradiation, and 8-methoxypsoralen plus UV and found that all methods inhibited PCR. Only DNase I was able to eliminate contaminating DNA while retaining a useful distinction between negative controls and low levels of template DNA. Silkie et al. (136) compared DNase I digestion and ultrafiltration with Microcon YM-100 (Millipore) filters. It was found that both methods eliminated contaminating DNA and retained sensitivity, but for DNase I digestion, the concentration of DNase and the presence of Ca2⫹ ions in the digestion mix was critical, as was thorough inactivation of DNase in the presence of dithiothreitol. Complementing the specific protocols to avoid or eliminate DNA in PCR reagents, Millar et al. (95) proposed a set of broad, general guidelines for laboratory practice in order to avoid contamination in the setting of broad-range PCR assays. These included guidelines for the sampling of clinical specimens, separation of rooms for pre- and postPCR work, and the use of high-quality reagents. SAMPLE PREPARATION FOR BROAD-RANGE PCR In contrast to microbial culture, where a sample in a volume of up to about 10 ml can be collected and used for inoculation (e.g., into a blood culture bottle) and one single viable bacterium may lead to a positive result, the typical sample volume added to a PCR tube is only about 5 l. Sample preparation for PCR is therefore extremely important; its aims are to crack microbial wall structures, remove PCR inhibitors, and purify and concentrate the template DNA, but also to avoid DNA overload (see chapter 7). There are two important issues concerning broad-range PCR: (i) sample preparation should be kept simple in order to reduce the risk of introducing external contamination, and (ii) some reagents used for sample preparation may contain intrinsic DNA. Proteinase K is one example of a potentially contaminated reagent; it is commonly used in sample preparation and requires purification during manufacturing, in analogy to Taq polymerase. There are a number of in-house and commercial sample preparation products and protocols; these include, among others, classical proteinase K digestion and phenolchloroform extraction, crude lysis with proteinase K and boiling with Chelex resin, simple alkaline lysis of blood samples, DNA spin column kits (e.g., Qiagen kits), and fully automated sample preparation workstations, such as the MagNA Pure LC Instrument and Kit (Roche) (35, 93, 94, 114, 135, 140). There are relatively few published sideby-side evaluations of different techniques; however, they may differ considerably in DNA recovery and the resultant 495 analytical sensitivity (114). It is also clear that any of these protocols can introduce DNA contamination. For example, some lots of Qiagen kits have been found to contain Legionella DNA (151). Reagents for the MagNA Pure LC workstation have also been found to contain bacterial DNA (98, 109), and subsequently, the company (Roche) released ‘‘M-grade’’ MagNA Pure reagents that were free of detectable bacterial DNA. Interesting observations were published by Tanner et al. (144); the authors conducted broad-range PCRs exposed to common sample preparation reagents and prepared a PCR product clone library from amplified DNA, presumed to be from reagents for sample preparation or PCR. They found that the obtained sequences matched published sequences from a range of ‘‘uncultured environmental bacteria’’ well. These sequences had been amplified from very diverse environmental samples, all with low total biomass in common, and had been deposited by various authors in databases. This suggests that some published sequences from ‘‘environmental bacteria’’ were in fact due to PCR contamination. There are also unusual examples of contamination involving sample acquisition and preparation, affecting not just bacterial broad-range PCR. Zymolyase, an enzyme digesting fungal cell walls and commonly used for panfungal PCRs, was found to contain fungal DNA; it is manufactured by a brewery company that also handles yeast (122). Another group found an unusually high number of positive Bordetella pertussis PCRs from one particular pediatric examination room; this room had previously been used for administration of cellular pertussis vaccine, and environmental swabs from the room also yielded positive PCRs (146). Another study aimed at broad-range PCR diagnosis of bacterial endophthalmitis from vitreous fluid samples; all 14 tested samples revealed Pseudomonas spp. sequences. The povidone-iodine antiseptic solution used for preoperative eye antisepsis was subsequently found to be contaminated (149). DETECTION OF PCR PRODUCTS AND SEQUENCE ANALYSIS The classical way to detect and identify bacteria by broadrange PCR is via visualization of PCR products by standard gel electrophoresis, followed by sequencing, preferably for both DNA strands of the products (26). This is the most accurate method. Direct sequencing of PCR products is feasible when it can be assumed that a sample contains a single species of microorganism. However, when direct sequencing reveals ambiguous reads or when multiple organisms are present, it is generally necessary to clone PCR products (e.g., by using commercial plasmid vectors for PCR product cloning) and to sequence an adequate number of clones (45, 116). The latter technique can also be applied to the construction of 16S rRNA gene clone libraries, in which a single PCR product from a polymicrobial sample may result in a few hundred plasmid clones with different sequences, representing different organisms (74, 107, 108). There are a number of tools for analysis of 16S or 23S rRNA gene sequences from broad-range PCR. The simplest method is to perform BLAST similarity searches in public sequence databases, such as in GenBank at the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov). These searches provide a percentage of similarity to the closest sequences in the database. 496 ■ MAIWALD However, this may not suffice for accurate species identification; searches may reveal no identical sequences, or they may reveal identical sequences from different species, and there is no defined similarity cutoff above which a new sequence can be assigned to the same genus or species (26, 66, 165). In addition, public databases contain many sequence errors, incorrect species and strain assignments, and rRNA gene sequence chimeras. A chimera is an artificial sequence that may be generated from a mixed microbial sample when PCR, after partial amplification of a DNA strand of one species, continues to amplify the homologous strand of another species, so that a phylogenetically heterogenous sequence is generated (154). Thus, it is advisable to check sequences obtained with broad-range PCR from polymicrobial samples; available tools are the programs Bellerophon (60) or Pintail (7). More accurate rRNA gene sequence assessment is possible with curated rRNA databases, web-based or commercial identification tools, and phylogenetic analysis. BIBI (http://pbil.univ-lyon1.fr / bibi) is a web-based identification tool aimed at clinically relevant bacteria (33). MicroSeq (Applied Biosystems) is a commercial kit, with PCR reagents included, that aims at 16S rRNA-based identification of cultivated bacterial isolates in clinical microbiology. It comes with its proprietary database of quality-checked sequences from reference strains and is available in two versions, covering either about 500 bp or almost the full length of the 16S rRNA gene (164). The Ribosomal Database Project website (http: / / rdp.cme.msu.edu) contains a curated 16S rRNA database with over 715,000 partial and full-length sequences in its 2008 release (27). It can be searched via a web interface that generates provisional taxonomic assignments, and a set of aligned sequences can be downloaded and imported into a phylogeny program. Greengenes (http: / / greengenes.lbl.gov) is a taxonomically and chimera-checked 16S rRNA database with over 290,000 sequences of ⬎1,250 bp in length (in its 2008 release) that can be interrogated via a web interface, and it has a fully aligned data set for download (31). SILVA (http: / / www.arb-silva.de) is a large database of qualitychecked, aligned 16S and 23S rRNA sequences with ‘‘Ref’’ and ‘‘Parc’’ data sets for each gene, where Ref stands for near-full-length sequences and Parc for all sequences over 300 bp (111). While the majority of entries in these data sets are comprised of environmental or microbial flora sequences from uncultivated organisms, SILVA contains nearly 20,000 16S rRNA and nearly 2,000 23S rRNA sequences from cultivated isolates (2008 release). Both the data sets from Greengenes and SILVA can be downloaded and imported into ARB (http: / / www.arb-home.de), which is a large phylogeny program package with a graphical user interface (85). Quantitative real-time PCR provides an alternative detection method that is rapidly replacing gel-based detection of broad-range PCR amplicons (28, 35, 36, 68, 100, 102, 127, 168). It offers several advantages. In gel-based assays, cycle numbers and other conditions need to be carefully adjusted, so that negative controls show no bands while sufficiently small amounts of positive control DNA become visible. In real-time PCR, the product is detected during cycling, so that negative controls (e.g., water controls, sample preparation controls, and known negative samples), positive controls with known copy numbers of 16S rRNA gene, and actual samples can be continuously monitored, and the quantification cycle (Cq [see chapter 4]) can be used as a criterion to compare the amount of DNA in controls to that in samples. As a consequence, real-time PCR offers much improved distinction between ‘‘true’’ and background DNA. Detection of amplicons can be achieved with fluorescent broad-range probes that simultaneously hybridize to the target sequence during each cycle (102), or with nonspecific nucleic acid dyes, such as SYBR Green (168). Products from real-time broad-range PCR can be sequenced and analyzed using standard procedures. Realtime PCR products can be subjected to melting (i.e., thermal DNA dissociation) analysis, and a variant technique, high-resolution melting analysis, may be used for a provisional species assignment among a limited range of medically relevant bacteria (24). As with gel-based assays, background DNA in PCR mixes or sample preparation reagents causes most negative controls to reach the threshold of amplified DNA at high numbers of cycles (Fig. 2). Pyrosequencing is an emerging alternative to standard Sanger type sequencing. It is much more rapid and cheaper per base than standard sequencing, detects the incorporation of nucleotides by light emission, and can be highly automated. Earlier generations of pyrosequencing instruments were limited to short sequence reads (e.g., 30 bp) and could be used in conjunction with broad-range PCR to interrogate short, informative sequence stretches that allowed preliminary identification of bacteria, such as gram negative or gram positive (72), Streptococcus sp., Staphylococcus sp., enteric gram-negative rod, etc. (67). More recent instruments allow for massively parallel pyrosequencing with increased read lengths (up to 400 bp) and can be used for accurate characterization of broad-range PCR products from highly complex polymicrobial communities (64, 82). Broad-range PCR can also be combined with amplicon detection by DNA microarrays. Several formats have been developed, ranging from low-density arrays that contain a set of probes for a limited number of medically relevant pathogens (96) to high-density arrays that cover large numbers of diverse taxa across a wide phylogenetic range (18). Such high-density arrays are commonly termed ‘‘phylochips’’ or ‘‘phyloarrays.’’ For example, a high-density microarray has been developed on the Affymetrix platform that contains 500,000 oligonucleotide probes covering about 9,000 distinct prokaryotic taxa (18). This array has been used, in a research setting, to characterize bacterial populations in urban atmospheric aerosols (19) and in uranium-contaminated soils (18). Another type of highdensity array has been used to characterize the development and diversification of intestinal flora of infants during the first year of life (107). High-density microarrays after broad-range PCR can be used for the quantitative analysis of PCR products across a wide range of taxa. CLINICAL APPLICATIONS Clinical microbiology has a constant need for alternative diagnostic methods that complement or expand on the existing ones. Most diagnostic methods have limitations: microscopy requires a significant number of organisms present in samples to become positive, and identification based on morphology is often not possible, serologic testing often only provides retrospective information, and culture may fail when fastidious or ‘‘culture-resistant’’ microorganisms cause infections. For many common pathogens, the rate of isolation is significantly reduced after the initiation of antibiotic therapy. Some pathogens may not be difficult to cultivate but may require special media, growth conditions, 31. Broad-Range PCR for Bacterial Identification ■ 497 FIGURE 2 Logarithmic amplification plot of a broad-range bacterial 16S rRNA gene PCR in real-time format. This amplification pattern has arisen in a study in which blood from healthy subjects was compared with negative controls (102). Samples were analyzed in triplicate and for 40 cycles. The Cq value is derived from the number of cycles needed for the amplified DNA to reach the quantification cycle (threshold). ⌬Rn, relative fluorescence. Reprinted from reference 102, with permission from the American Society for Microbiology. or lengthy incubation times; thus, they normally need to be considered in order to become detected (45). Broadrange PCR offers two potential benefits: it lacks selectivity for particular groups of bacteria, and it can detect as well as identify culture-resistant, fastidious, damaged, and slowgrowing microorganisms. Since the inception of broad-range PCR in the late 1980s, many clinical microbiology laboratories have implemented this technique for research and clinical applications. In diagnostic settings, broad-range testing usually takes place in selected cases, with selected specimen types, on specific request by physicians, and by using in-house assays (115). When implemented, broad-range PCR testing is usually done with samples from primary ‘‘sterile’’ sites, such as blood, cerebrospinal fluid, synovial fluid, and tissue. Clinical applications of broad-range PCR have been published in multiple case reports, small case series, and a number of large prospective and retrospective evaluation studies. Published case reports include instances where the diagnosis was missed because all standard tests using conventional diagnostic methods (culture, serology, and microscopy) were unrevealing. Examples are the detection of B. quintana in culture-negative endocarditis (65), T. whipplei in spondylodiscitis (2) and endocarditis (51), Mycoplasma pneumoniae in osteomyelitis (78), and Granulicatella elegans in endocarditis (22). A number of clinical scenarios and specimen types have attracted particular interest and more thorough evaluation studies for broad-range PCR. These include excised heart valves in suspected bacterial endocarditis (16, 50, 89, 153), bloodstream infections in neonates or hematologyoncology patients (69, 80, 103, 110, 138), cerebrospinal fluid in suspected meningitis or shunt infections (32, 73, 134, 158, 167), bone samples or synovial fluid samples in osteomyelitis or septic arthritis (40, 41, 127), vitreous or aqueous fluid in intraocular infections (152), tissue or pus from brain or spinal abscesses (75, 115), amniotic fluid in chorioamnionitis or preterm labor (34, 58), and various other scenarios. In one of the earlier studies, Goldenberger et al. (50) applied broad-range PCR to resected heart valves in a series of 18 endocarditis cases. Cultures of the valves were positive in only 4 cases, blood cultures were positive in 15 cases, and PCR was positive in 15 cases. Tentative bacterial identification based on 16S rRNA sequences was possible in 13 cases. Two PCR-positive cases were missed by cultures: DNA sequences of a Streptococcus sp. and of T. whipplei were detected in these valves. Similar findings were made in other studies of heart valve PCR: at the time of operation, most patients were treated with antibiotics and valve cultures were often negative, and among the cases detected by PCR but not by culture were pretreated cases and infections by fastidious bacteria (16, 89, 153). Ley et al. (80) published findings of broad-range PCR directly from EDTA-anticoagulated blood samples from 111 episodes of fever and neutropenia in cancer patients and compared the results with those of conventional blood cultures. Broad-range PCR was positive in 9 of 11 culturepositive episodes but also in 20 culture-negative episodes, 18 of which occurred under antibiotic treatment. It was concluded that broad-range PCR can augment culture in patients under antibiotic treatment. Another study (69) examined 1,233 blood samples from neonates being evaluated for early-onset sepsis. Whole blood samples taken for cell count analysis were preincubated for 5 h in broth and examined by PCR, and the results were compared to conventional blood cultures. Seven samples were concordantly positive. PCR failed to detect 10 of 17 culture-positive samples, some of which yielded skin organisms, and was positive in 30 culture-negative cases. The authors pointed out that due to ethical reasons for study design, samples for 498 ■ MAIWALD PCR were often taken by heel pricks, which may have introduced skin organisms that were then multiplied by preincubation. Bloodstream infections have also been addressed by the design of a commercial kit, the SeptiFast test (Roche). This kit is based on real-time PCR with different sets of primers and probes for a limited range of 25 gram-positive, gram-negative, and fungal organisms commonly involved in bloodstream infections and requires about 6 h to obtain results (21). Kotilainen et al. (73) examined 56 cerebrospinal fluid samples from patients with suspected meningitis and found that PCR detected Neisseria meningitidis sequences in 5 samples, 4 of which were also culture positive. One PCRpositive sample from a patient under antibiotic treatment was negative by microscopy and culture, but autopsy findings were consistent with meningococcal disease. One additional case of culture-positive Listeria monocytogenes meningitis was missed by broad-range PCR. Similar findings were made in larger studies evaluating broad-range PCR for the diagnosis of meningitis: PCR failed in some cases that were positive by culture but was often successful when patients were pretreated with antibiotics (134, 158). A large study of 525 bone and joint samples, comprising tissues and fluid aspirates, was published by Fenollar et al. (41). Concordantly negative results between culture and PCR were obtained in 386 cases, concordantly positive results in 89 cases, and discrepant results in 50 cases. Culture and PCR each had similar rates of false-positive and falsenegative results. Among 21 culture-negative, PCR-positive cases were 7 cases with antibiotic treatment and 2 cases with fastidious organisms. Seven cases revealed mixed sequences by PCR; some of the sequences in these cases belonged to established bone and joint pathogens, others to environmental organisms rarely involved in bone and joint infections. A large study from Finland (115) applied broad-range PCR to 536 diverse clinical samples from 459 hospitalized patients and compared PCR with culture. Samples consisted of body fluids and tissues and were collected over a 4-year period in a routine diagnostic setting. Results of culture and PCR were concordant in 447 (83%) specimens. PCR-positive but culture-negative results occurred in 19 samples, which included specimens taken during antibiotic treatment or infections caused by bacteria with unusual growth requirements. For 11 patients in the study, broadrange PCR was the only method that provided evidence of the etiologic agent of the infection, and for 10 of these patients, the agent identified by sequencing was considered significant in the clinical context. The tendency of broadrange PCR to be positive after the initiation of antibiotic therapy, as opposed to culture, was statistically significant. A large, population-based study of unexplained deaths and critical illnesses due to possible infectious causes was initiated by the Centers for Disease Control and Prevention (CDC) and conducted in four states of the United States (52, 101). Included were cases in which all conventional diagnostic methods had yielded negative results, although the patients demonstrated clinical signs and symptoms suggestive of infection. A total of 137 cases were identified, and broad-range PCR was performed in 46 cases. The PCR results were positive for eight cases, and bacterial sequences (e.g., N. meningitidis and Streptococcus pneumoniae) recovered from six patients were consistent with the clinical symptoms. Seven of the eight patients had been pretreated with antibiotics. Bacterial broad-range PCR has also attracted interest in transfusion medicine, in order to assess blood products for bacterial contamination (35, 36, 97, 126). Platelet concentrates are of particular concern, because they require storage and agitation at room temperature, thus making them prone to bacterial growth from very small inocula. Culture in blood culture bottles requires several days, and slowgrowing bacteria such as Propionibacterium spp. may require 4 to 5 days, so that cultures may become positive at a time when platelets have been transfused. Several broad-range real-time PCR assays have been designed and evaluated for that purpose (35, 36, 97, 126). One study evaluated 2,146 platelet concentrate samples in parallel with culture and PCR (97). The test had a turnaround time of 4 h and showed concordant results between the two methods, thus showing the feasibility of PCR testing for platelet concentrates. ANALYSIS OF POLYMICROBIAL INFECTIONS AND MICROBIAL COMMUNITIES Broad-range PCR can also be applied to mixed infections or to determine the composition of complex polymicrobial communities, such as in samples from the environment or samples containing microbial flora of humans and animals. This type of analysis has originally been established in environmental microbiology (48). Such studies as well as in situ hybridization experiments (4) have provided estimates that ⬎99% of microorganisms in environmental communities are not detectable by standard culture techniques. PCR-based community studies usually involve the construction and phylogenetic analysis of 16S rRNA gene clone libraries and commonly reveal many sequences corresponding to new species (‘‘phylotypes’’ or ‘‘operational taxonomic units’’) that are not detected by culture. Analysis of human microbial flora has been performed on specimens such as feces (141, 161) and subgingival plaque material (74, 108). These studies revealed a considerable fraction (40 to 75%) of sequences that did not belong to known bacterial species. The diversity of sequences was greater for PCR-amplified clone libraries than for bacterial colonies from cultures that were set up in parallel (74, 161), but larger numbers of cycles (35 cycles versus 9) in the initial PCRs reduced diversity, probably due to preferential amplification (161). Based on clone library data, it was estimated that the number of different bacterial species in subgingival plaque communities exceeds 400 (108). Another study, combining broad-range PCR with both clone libraries and microarray analysis, examined the stool flora development of 14 infants during the first year of life (107). Each of the babies had and retained a distinct community profile, and by the end of the first year, the profiles had diversified into patterns that were similar to those of the adult gastrointestinal tract. Other studies have focused on the association between human microbial flora disturbances and conditions of impaired health. One study examined samples from women with bacterial vaginosis and women with normal vaginal flora by broad-range PCR followed by clone libraries, as well as by in situ hybridization (43). Bacterial vaginosis samples contained much greater microbial diversity, including 16 presumptive new species, among which three species were very strongly associated with bacterial vaginosis and were only distantly related to any other known bacteria. Another set of interesting findings was reported by Ley et al. (81). The authors examined the stool flora of 31. Broad-Range PCR for Bacterial Identification ■ obese and lean people, as well as obese people during the course of diet. For the two dominant phyla in stool, the Firmicutes and the Bacteroidetes, they found that the number of Bacteroidetes was smaller in obese people than in lean people and that obese people under diet underwent a shift towards fewer Firmicutes and more Bacteroidetes, approaching the ratios of lean people. It was pointed out that these changes constitute a major shift of bacterial phyla that is associated with health disturbance. Broad-range PCR has also been applied to polymicrobial infections, such as destructive periodontitis (25), prostatitis (145), dental root canal infections (125), and destructive keratitis (131). These studies also revealed the unexpected diversity of bacteria associated with polymicrobial infections, as well as sequences corresponding to unknown species. 499 enormous implications on the analysis of samples from supposedly sterile sites by broad-range PCR. Among the clinical evaluation studies of broad-range PCR, there were some that reported largely consistent results between PCR and culture, with moderate numbers of samples positive by PCR but negative by culture, and vice versa (50, 73, 89, 115, 134). Others reported a greater proportion of samples that were positive by PCR and negative by culture (54, 80, 138, 167). As pointed out by Peters et al. (110), the interpretation of such studies is difficult, with culture being the reference. A positive PCR may be the result of greater sensitivity of PCR, or it may be a falsepositive result. It may be the result of true pathogen DNA or of presumed normal background DNA in human blood or tissue, or it may be due to bacterial DNA in reagents. Interpretation in the context of clinical findings and other laboratory results is necessary. This is feasible in thorough scientific evaluation studies, but it may not be feasible in a routine diagnostic setting. UNANSWERED QUESTIONS Interesting questions were raised in a study by Nikkari et al. (102). By use of a real-time broad-range PCR assay, blood samples from healthy volunteers were compared with negative controls (i.e., water treated as for sample preparation), human tissue culture cells, and known amounts of extracted bacterial DNA (Fig. 2). Negative controls consistently gave rise to amplification products, reaching the quantification cycle around cycle 38. The quantification cycles of healthy blood samples were four to six cycles earlier, indicating a higher load of bacterial DNA. Clone libraries were prepared from both a negative control and a blood sample and revealed sequences that belonged to phylogenetic groups similar to those of sequences previously reported from negative controls (144), but blood samples yielded additional sequences from phylogenetic groups that were not represented in negative controls. The origin and significance of the blood-derived sequences remained unclear, but the possibility was raised that supposedly sterile body compartments may harbor a ‘‘normal’’ burden of bacterial DNA. This appears consistent with previous findings of culture-positive bacteremia after toothbrushing (12) and with pneumococcal DNA found in the blood of healthy children (29). Apparently similar findings were made by another group after the incidental observation of pleomorphic bacteria by dark-field microscopy in the blood of one healthy subject (90). The findings were confirmed by in situ hybridization and broad-range PCR in several more healthy subjects; 16S rRNA gene and gyrB gene sequences were similar but not identical to those of Stenotrophomonas maltophilia, but cultures on standard media remained negative. The significance of these findings remained unclear. Interesting observations were also made in two other studies, examining coronary arteries after death from various causes (79) and atherosclerotic lesions from abdominal aortas (121) by broad-range PCR. In the second study (121), the authors took particular care to avoid background DNA from reagents by cloning and subtracting sequences obtained after extensive cycling of negative controls, but despite this, sequences from typical oral commensals and low-level pathogens were detected in both studies. The significance remained unclear, but the authors speculated that commensals may become embedded in vascular tissues during transient bacteremias. Again, these studies point towards the possibility that human blood and tissue may have a normal background of bacterial DNA, and this may have CONCLUSIONS Conducting broad-range PCR analysis at a level of high analytical and clinical sensitivity is a complex task and remains one of the most difficult and challenging PCR applications. Implementation of broad-range PCR in a diagnostic setting requires appropriate laboratory facilities and staff, familiarity with the theoretical underpinnings and practical aspects, extensive precautions to avoid contamination, and thorough in-house evaluations of analytical as well as clinical sensitivity and specificity. The presence of ubiquitous bacterial background DNA and the problems associated with its elimination suggest that dealing with a certain, low level of interfering DNA will be unavoidable, at least in the foreseeable future. Some of the decontamination methods in Table 2 describe the reduction of contaminating DNA to levels of about 10 copies of rRNA gene per PCR test, which means that the amount of real target DNA that can be distinguished will have to be significantly above that level, and broad-range PCRs will not achieve the same level of sensitivity as organism-specific PCRs. Careful implementation of positive and negative controls is necessary. The presence of bacterial DNA in PCR mixes, in sample preparation reagents, and presumably in some normal human tissues mandates that negative controls should include DNA-free controls in the PCR mix, sample preparation controls, and control samples from patients without the disease in question, in order to detect and be aware of the levels of background DNA in all of these settings. Positive controls must contain serial dilutions of known copy numbers of bacterial DNA or bacterial cells, both in the PCR mix and spiked into negative samples, to determine the threshold of detectable bacterial DNA that is clearly above the background level. Sequence-based identification of positive diagnostic broad-range PCR products is generally advisable, even when real-time technology is used. Some authors have proposed using a second, independent, broad-range primer system in order to confirm results (73, 112, 115). Additional controls may include PCRs using a human gene (e.g., the beta-globin gene) in order to assess the presence of amplifiable DNA in a sample or inhibition of PCR. Some PCR tests use internal, coamplified DNA controls (126). In addition to the specific measures to eliminate background DNA (Table 2), a wide range of general anticontamination precautions is necessary, including separate 500 ■ MAIWALD rooms for pre- and post-PCR work, UV decontamination of surface areas, use of high-quality reagents, and adequate sampling techniques and vials for clinical specimens (95). It is important to understand that the traditional concepts of sterile laboratory techniques from culture-based microbiology do not apply in the same way in the setting of molecular tests, as shown by experiments in which extensive autoclaving failed to eliminate amplifiable DNA (87). Clinical studies have shown a number of situations in which bacterial broad-range PCR can provide considerable benefits. Clear benefits exist when bacteria are seen by microscopy but fail to be recovered in culture, when infections may be caused by fastidious or slow-growing organisms, and when patients have been pretreated with antibiotics. The examination of resected heart valves exemplifies these benefits very well: endocarditis can be caused by various organisms, including fastidious ones, valve resection is a highly invasive sampling procedure that warrants considerable diagnostic efforts, the examination by blood cultures may fail to detect the pathogen, and patients have often been pretreated with antibiotics, so that heart valves at the time of operation are often culturenegative (50). However, diagnostic broad-range PCR, with full amplicon sequencing, is expensive and time-consuming and requires appropriate laboratory facilities (115). The primary diagnostic utility of broad-range PCR lies in the testing of specimens from primary sterile sites, especially if they are difficult to obtain, and in clinical situations in which other methods are known to pose difficulties. The analysis of polymicrobial infections and complex communities becomes feasible with the aid of clone libraries and the emerging techniques of pyrosequencing and high-density microarrays, but it still requires considerably more effort and resources and is currently done almost exclusively in research laboratory settings. Other emerging technologies may take the current place of broad-range PCR as a nonprescient tool for the examination of unknown infections. This has been exemplified by the discovery of a novel transplant-associated arenavirus that caused fatal infection in three organ recipients (106). The virus was discovered by random amplification of RNA transcripts from infected organs, followed by unbiased highthroughput pyrosequencing. 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