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Applied and Environmental Microbiology, May 2009, p. 2850-2860, Vol. 75, No. 9
0099-2240/09/$08.00+0     doi:10.1128/AEM.01910-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Evaluation of Single-Nucleotide Primer Extension for Detection and Typing of Phylogenetic Markers Used for Investigation of Microbial Communities{triangledown}

Marcell Nikolausz,1* Antonis Chatzinotas,2 András Táncsics,3 Gwenaël Imfeld,4 and Matthias Kästner1

UFZ, Helmholtz Centre for Environmental Research, Department of Environmental Biotechnology, Permoserstr. 15, D-04318 Leipzig, Germany,1 UFZ, Helmholtz Centre for Environmental Research, Department of Environmental Microbiology, Permoserstr. 15, D-04318 Leipzig, Germany,2 Eötvös Loránd University of Science, Department of Microbiology, Pázmány Péter Sétány 1/c, 1117 Budapest, Hungary,3 UFZ, Helmholtz Centre for Environmental Research, Department of Isotope Biogeochemistry, Permoserstr. 15, D-04318 Leipzig, Germany4

Received 18 August 2008/ Accepted 20 February 2009


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ABSTRACT
 
Single-nucleotide primer extension (SNuPE) is an emerging tool for parallel detection of DNA sequences of different target microorganisms. The specificity and sensitivity of the SNuPE method were assessed by performing single and multiplex reactions using defined template mixtures of 16S rRNA gene PCR products obtained from pure bacterial cultures. The mismatch discrimination potential of primer extension was investigated by introducing different single and multiple primer-target mismatches. The type and position of the mismatch had significant effects on the specificity of the assay. While a 3'-terminal mismatch has a considerable effect on the fidelity of the extension reaction, the internal mismatches influenced hybridization mostly by destabilizing the hybrid duplex. Thus, carefully choosing primer-mismatch positions should result in a high signal-to-noise ratio and prevent any nonspecific extension. Cyclic fluorescent labeling of the hybridized primers via extension also resulted in a significant increase in the detection sensitivity of the PCR. In multiplex reactions, the signal ratios detected after specific primer extension correlated with the original template ratios. In addition, reverse-transcribed 16S rRNA was successfully used as a nonamplified template to prove the applicability of SNuPE in a PCR-independent manner. In conclusion, this study demonstrates the great potential of SNuPE for simultaneous detection and typing of various nucleic acid sequences from both environmental and engineered samples.


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INTRODUCTION
 
Fast detection, differentiation, and identification of bacteria are crucial tasks in clinical, food, and environmental microbiology. Cultivation-independent tools not only save time compared to cultivation-based techniques but also allow access to the difficult-to-cultivate part of a microbial community. Molecular detection methods are usually based on hybridization of oligonucleotide probes to signature sequences (phylogenetically informative regions) in the nucleic acids (RNA or DNA) of the target microorganisms. Verification of the hybridization event can be accomplished by detection of hybridized labeled probes in situ (e.g., fluorescence in situ hybridization [FISH]) or ex situ (dot blot hybridization). Combining two specific oligonucleotides in a PCR increases the sensitivity of specific detection, while real-time monitoring of the amplification product formed allows quantification of the original template (for a review, see reference 17). Multiple detection can be achieved by using more than one primer pair targeting several loci in multiplex PCR assays (for a review, see reference 32). However, the main disadvantages of FISH are its restricted capability for parallel analysis of several target groups in the same sample and limitations in probe design due to differences in accessibility of the probes to their target sites (3, 7). Moreover, detection of slowly growing bacteria with low ribosome contents requires labor-intensive techniques (30, 36). Multiplex PCR also has limitations for multiplexing and challenges for primer design (32).

Recently, single-nucleotide primer extension (SNuPE) was proposed as a fast, semiquantitative multiplex detection tool for analyzing sequence variants. This method is frequently used for determination of single-nucleotide polymorphisms and benefits from the fidelity of dideoxynucleoside triphosphate (ddNTP) incorporation catalyzed by a DNA polymerase. When primer extension takes place on a solid support, the method is called minisequencing (35, 37), while a reaction in solution is referred to as SNuPE (34) or single-base extension (15). These methods were originally developed for routine medical diagnosis of genetic disorders (23, 35) or for use in forensic research (38). Different versions of the primer extension technique have also been used recently for fast identification and genotyping of microbial strains (9, 31). Recent studies showed that detection of a hybridization event via labeling of the hybridized primer in the extension reaction is possible. However, the use of this method as a detection tool in applied and environmental microbiology has not been fully exploited so far. Rudi and coworkers were the first workers who used a minisequencing approach with PCR products from environmental DNA to detect toxic cyanobacteria by labeling only one of the four ddNTPs used in the reaction (27). Multiplexing was accomplished by hybridizing the labeled products to complementary oligonucleotides in an array format. In combination with antibody-based chromogenic visualization, genetic profiles of cyanobacterial diversity (28), microbial communities in vegetable salads (25), and Listeria strains (26) were obtained. However, this approach is labor-intensive and time-consuming and requires specific equipment; furthermore, the primer is restricted to certain positions since only one terminator nucleotide is labeled.

An alternative strategy for multiplexing in solution benefits from incorporation of four differently labeled ddNTPs and attachment of mobility modifiers to the different primers. Subsequent separation using capillary electrophoresis and laser-induced fluorescence detection results in a very fast assay that is easy to interpret. Determination of the incorporated nucleotide provides additional proof of the assay specificity or may even provide extra phylogenetic information. The first application of primer extension with four differently labeled ddNTPs in environmental microbiology was the use of this method by Wu and Liu (41) for multiplex detection of different Bacteroides spp. This study also addressed different methodological issues and aspects, such as the effects of noncomplementary tail length, annealing temperature, cycle number, and primer-to-template ratio on extension efficiency. In a previous study, Nikolausz et al. (19) reported development and application of a multiplex SNuPE assay for detection and typing of "Dehalococcoides" sp. sequences obtained from chloroethene-contaminated groundwater samples. However, there still has not been a systematic evaluation of factors that affect primer design and the discriminatory power of primer extension. Moreover, quantitative aspects of the method have not been thoroughly addressed so far.

The present study focused on these crucial issues by investigating the effects of the type, number, and position of primer mismatches on the extension efficiency and hence the specificity. Furthermore, quantitative aspects of SNuPE were investigated in a model community experiment by using defined template mixtures of 16S rRNA gene PCR products or reverse-transcribed RNA.


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MATERIALS AND METHODS
 
Bacterial strains and cultivation.
The following strains were used in this study: Pseudomonas putida mt-2, Pseudomonas stutzeri DSM50238, Thauera aromatica K172, and Bacillus subtilis DSM402. Strains were cultivated on nutrient agar (DSMZ medium 1).

DNA and RNA isolation, reverse transcription-PCR, PCR, and nucleic acid quantification.
DNA was isolated from strains using a DNAeasy tissue kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions for gram-positive cells. DNA was eluted in 120 µl of RNase-free distilled water. RNA was isolated from strains using an RNeasy mini kit (Qiagen) as described previously (20) and was quantified by using the Ribo Green RNA quantification reagent (Molecular Probes, Eugene, OR) along with the rRNA standard supplied with the kit. Fluorescence was determined using a fluorimeter (Viktor 2 1420 multilabel counter; Wallac, Finland). Each PCR was performed using a 50-µl (final volume) mixture with HotStar Taq polymerase with the supplied buffer (Qiagen) on a Mastercycler gradient (Eppendorf, Hamburg, Germany) and primers 27F (13) and 1378R (8). The conditions used for PCR amplification of the 16S rRNA genes were as follows: initial denaturation at 95°C for 15 min, followed by 30 cycles of denaturation for 30 s at 95°C, primer annealing at 52°C for 30 s, and chain extension at 72°C for 70 s and then a final extension at 72°C for 30 min.

PCR products were purified (QIAquick PCR purification kit; Qiagen) and quantified by using the Pico Green double-stranded DNA quantification reagent (Molecular Probes) and a lambda phage DNA standard supplied with the kit. Unincorporated primers and deoxynucleoside triphosphates from 42 µl of purified PCR products were treated with 12 U shrimp alkaline phosphatase (SAP) (Fermentas, Vilnius, Lithuania) and 6 U exonuclease I (Fermentas) in the SAP buffer provided in a 60-µl (final volume) mixture. Reaction mixtures were mixed and incubated at 37°C for 1 h, which was followed by enzyme inactivation at 75°C for 15 min.

RNA isolated from different strains were mixed at defined ratios and subjected to reverse transcription. The reaction was carried out using an Omniscript reverse transcription kit (Qiagen) with primer 1378R in a 20-µl (final volume) mixture according to the manufacturer's instructions, including a denaturation step at 65°C for 5 min. The amount of RNA used per reaction ranged from 60 ng to 180 ng. Reverse transcription was carried out at 42°C for 2 h. Treatment of the cDNA was performed by using only SAP as described above.

Primer design.
Oligonucleotides that were specific for the genera of the reference strains and targeted 16S rRNA were selected using the search engine and database of probeBase (http://www.microbial-ecology.net/probebase/) (16). The specificities of the primers were further evaluated by using the Probe Match function of Ribosomal Database Project II (5). The reverse complements of the original probes were hybridized to reverse-transcribed RNA. The target positions and sequences of the primers used in the model community experiments are shown in Fig. 1 and 4 and Table 1.


Figure 1
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  		FIG. 1. Detection of target sequences obtained from strains of T. aromatica (Ta), P. putida (Pp), and B. subtilis (Bs) via hybridization with specific primers and labeling by SNuPE. The AmpliTaq FS polymerase incorporates a labeled dideoxynucleotide, which terminates the reaction and results in a fluorescently labeled product. Multiplex reactions are carried out with different target-specific primers that also differ in length. The primers and products are subsequently separated by capillary electrophoresis, and the extended products are detected with laser-induced fluorescence.


Figure 4
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  		FIG. 4. Primer design strategy for demonstration of the one-mismatch discrimination potential of SNuPE. Differences between the 16S rRNA genes of two closely related Pseudomonas species (P. putida and P stutzeri) are highlighted with colors in the sequence alignment of the target region of the gene. The colors are the colors assigned for the nucleotide analogues used in the SNuPE assays. The primer design benefited from the fidelity of nucleotide incorporation of the Taq polymerase (primer Pseud_SNP) or from the discrimination potential due to the detrimental effect of 3' end mismatch on extension efficiency (primers Pp_SNP and Ps_SNP). The expected products for both microorganisms are also shown. The colors of the incorporated ddNTPs are the colors of the expected peaks.


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  		TABLE 1. Primers used to assess the effects of mismatch type, position, and number on the primer extension efficiency

SNuPE.
Cyclic primer extension reactions were performed in 10-µl (final volume) mixtures containing 5 µl of SNaPshot multiplex kit reagent (Applied Biosystems), 4 µl of purified PCR products or cDNA, and 1 µl of primer solution or primer mixture (10 µM of each primer). The SNuPE reactions were carried out using 35 cycles of denaturation at 96°C for 10 s, annealing at 55°C for 5 s, and extension at 60°C for 30 s. The volume of PCR products added and the annealing temperature were different in different experiments. In order to remove unincorporated ddNTPs, 1 U of SAP was added to each reaction mixture and incubated at 37°C for 1 h, which was followed by inactivation at 75°C for 15 min. The copy number of the PCR product for each SNuPE reaction was calculated based on the measured concentration and average length of the amplicons. SNuPE reactions were performed in duplicate to ensure reproducibility, while at least triplicate reactions were carried out when peak area ratios were calculated; 100 relative fluorescence units was used as the detection limit.

Capillary electrophoresis and analysis.
A 0.5-µl portion of posttreated extension products was mixed with 9 µl of formamide and 0.5 µl of the GeneScan-120 LIZ internal size standard (Applied Biosystems). The mixture was denatured at 95°C for 5 min and quickly cooled on ice. DNA fragment separation was performed with an ABI PRISM 3100 genetic analyzer using a 36-cm capillary filled with denaturing POP6 polymer with an E5 filter set (Applied Biosystems). The following parameters were used for electrophoresis: injection time, 10 s; electrophoresis voltage, 15 kV; run temperature, 60°C; and run time, 24 min. Data analysis was performed using the GeneMapper software (version 3.5; Applied Biosystems). Peak areas were normalized based on the average signal strength of the applied internal standard (35 nucleotides), and the area data were used for comparison of different samples.

Statistical analysis of single and multiplex SNuPE reaction data.
The robustness of the single SNuPE assay was assessed by measuring the extents of the fluorescent signals generated (peak areas) with dilution series of the PCR products from the different strains (T. aromatica, P. putida, and B. subtilis). Ninety-five percent confidence intervals were determined for the slopes of the corresponding linear regressions. The linear models were considered to significantly fit the data at a P value of <0.001 using F statistics (analysis of variance). All data were found to significantly fit the linear models. The linear models were further used as calibration curves to evaluate defined template mixtures of PCR amplicons of the three strains at various ratios in multiplex SNuPE assays. The calculated frequency distributions of various ratios of the PCR products were compared to the distribution of the retrieved ratios of the mixed target templates in the multiplex SNuPE assays using a one-dimensional {chi}2 "goodness-of-fit" test at a significance threshold ({alpha}) of 0.5.


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RESULTS AND DISCUSSION
 
Specificity of the SNuPE assay and effect of primer mismatch.
One of the most important requirements for an efficient and reliable molecular detection technique is the ability to specifically detect one type of sequence in the presence of closely related types of sequences. Our previous study showed that discrimination between closely related sequences could be achieved with two mismatches in the SNuPE primer annealing region of the nontarget sequence (19). In order to further investigate the effect of the nature and position of mismatches on the efficiency, specificity, and the discrimination potential of SNuPE, 16S rRNA gene PCR amplicons of P. putida were used as templates in single reactions. Instead of alteration of the template used for the SNuPE reaction, the Pseudomonas-specific primer (PAE997rc) was redesigned to obtain 14 further primer variants (Table 1). Single and multiple base alterations were introduced at different positions in the primer sequence. First, variants with primer-template mismatches at the 3' end of the primer sequence were synthesized, since primer extension is expected to be most sensitive to a terminal mismatch. In addition, internal single and multiple mismatches were introduced close to the 3' end, as well as in the middle of the primer, to investigate to what extent internal mismatches potentially affect the specificity of SNuPE.

In the first experiment, primer extension with single primer-template mismatches at the 3' end was investigated at two different annealing temperatures (Fig. 2) since early studies of the fidelity of DNA polymerases indicated the importance of mismatched primer termini (22, 40). SNuPE with the specific primer resulted in a strong specific peak, whereas only very low signals were obtained with the mismatch primers. A higher annealing temperature (55°C) further decreased the very low nonspecific signals, as expected. Various non-Watson-Crick base pairings destabilize duplex formation at different ratios. Previous studies showed that G-T and G-A mismatches are relatively stable (11, 14), which is in good agreement with our results. When the 3' end of the primer was changed to G, resulting in a terminal G-T mismatch, a product peak area that was 2.3% of the specific product peak area was obtained at an annealing temperature of 55°C. On the other hand, a terminal T-T mismatch (very weak base pairs [11]) resulted in signals under the detection limit, similar to the results obtained with the primer with four internal mismatches. This is in accordance with several previous studies that showed the importance of a 3' end mismatch for specific discrimination in primer extension during PCR amplification (2). However, there is little agreement on which 3' terminal primer-template mismatches are more readily extended by Taq DNA polymerase. Kwok et al. (12) observed a minimal effect of T-G or T-T terminal mismatches on the PCR product yield, while Okayama and coworkers have not obtained any PCR products with T-G and T-T terminal mismatches (21). The major difference between PCR and SNuPE is that SNuPE mismatch products are not effective templates for the next cycles of enzymatic reactions, while the mismatch products introduced in the early PCR cycles are readily amplified in the later cycles.


Figure 2
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  		FIG. 2. Effects of terminal and internal primer mismatches at annealing temperatures of 50°C (A) and 55°C (B) on the specificity of the primer extension assays. The P. putida PCR product was targeted with perfectly matching primer PAE997rc (panels a and f). Mismatch discrimination was achieved by replacement of the perfectly matching terminal base adenine by cytosine (panels b and g), guanine (panels c and h), or thymine (panels d and i). Primer PAE997_BTEX designed to detect Pseudomonas veronii had four internal mismatches compared with the target DNA (panels e and j). The vertical axis indicates fluorescence intensity (in relative fluorescence units); the horizontal axis indicates the sizes of the extended products. The numbers indicate the lengths (in bases) of the internal standards (black peaks).

In addition, little is known about the effect of internal mismatches on primer extension. The effect of nonterminal and multiple mismatches on the stability of primer annealing was investigated in detail by introducing strong and weak mismatches at different positions and in different numbers (Table 1). In this second experiment, the cycle number was increased from 35 to 55 in order to increase the probability of nonspecific extension events (2) and to facilitate further evaluation. The peak areas of the mismatched extension products were relatively small compared to the peak area of the perfectly matching product (Fig. 3). Three independent SNuPE reactions were performed in all cases, and the average values are shown in Fig. 3. The 3'-terminal position again had a pronounced detrimental effect on the extension efficiency, and there was a considerably larger signal in the case of the more strongly binding G-T mismatch (Fig. 3A) than in the case of the T-T mismatch (Fig. 3B). The primer extension efficiency increased when the mismatch was moved toward the 5' end of the primer sequence. However, the opposite trend was observed with primers having a mismatch at position –9 with respect to the 3'-terminal position (i.e., the middle of the primer sequence). A similar result was obtained by Bru and coworkers (4) when they investigated the effect of mismatch positions on real-time PCR efficiency. The mismatches closer to the 5' end did not significantly affect the PCR efficiency, while the mismatches closer to the 3' end decreased the efficiency by 1 to 3 orders of magnitude. Mismatch positions in the middle part of the primer had a detrimental effect on PCR efficiency greater than the general trend. This was hypothesized to occur as a result of changes in the secondary structure of the primer. On the other hand, when the target DNA was altered instead of the primer, some base conversions in the middle of the primer binding site of the template had a greater effect than expected from the trend, which cannot be explained by alteration of the secondary structure of the primer.


Figure 3
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  		FIG. 3. Effects of the type and position of (A) single strongly binding (G-T), (B) single weak (T-T), and (C) double mismatches on the primer extension efficiency. Primer sequences are shown in Table 1. For easier measurement of nonspecific extension products, 55 cycles were used. The results are expressed as averages for triplicate experiments and are based on comparisons with the results for perfectly matching primer extension. The error bars indicate standard deviations for average values. U.D., under the detection limit (100 relative fluorescence units).

The specificity of the primer extension in both SNuPE and PCR depends on the primer annealing and the fidelity of the DNA polymerase. While the formation and the thermal stability of a primer-target duplex are influenced mainly by mismatches in the middle part, strand extension and hence PCR amplification efficiency are influenced more by mismatches at the 3' end of the primer. Our results demonstrate that both factors are likely to be responsible for the discriminating power of SNuPE, as shown in Fig. 3C. With two mismatches, one close to the 3' end and one close to the middle part of the primer, no detectable signal was observed, not even in the case of strongly binding G-T mismatches. Thus, two well-chosen primer mismatch positions result in a high signal-to-noise ratio and prevent any nonspecific extension regardless of the mismatch type even under nonstringent conditions.

To demonstrate the efficient mismatch discrimination potential of the SNuPE method, new assays were designed (Fig. 4) to discriminate two closely related strains, a P. putida strain and a P. stutzeri strain. Primer Pseudo_SNP was designed to hybridize to both sequences in the same target region, which, however, could be extended with different signature nucleotides specific for each target strain. Primers Pp_SNP and Ps_SNP are specific for P. putida and P. stutzeri, respectively. Only one nucleotide difference was targeted in this second assay, while the nucleotides that were potentially incorporated were also different in the two target sequences to allow unambiguous evaluation of the extended products. The single-nucleotide incorporation proved to be very specific, since a nonspecific product could not be detected when primer Pseudo_SNP was used alone (Fig. 5A). When two specific primers (Pp_SNP and Ps_SNP) and only one of the target DNA templates were used in the extension reaction (Fig. 5B), only specific signals were detected. The lack of even traces of a nonspecific signal indicates that in precise primer design absolute one-mismatch discrimination can be achieved. In this case this meant that there was a perfectly matching 3'-terminal position with strong base pairing (C-G) compared with a weakly binding mismatch (C-A) or a moderate mismatch (G-T).


Figure 5
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  		FIG. 5. Primer extension assays for discrimination of P. putida and P. stutzeri 16S rRNA gene sequences using either primer Pseud_SNP (A) or a mixture of primers Pp_SNP and Ps_SNP (B). (Panels a to c) SNuPE patterns obtained with 65 ng of P. stutzeri PCR product (a), 65 ng of P. putida PCR product (b), or mixed PCR products from P. stutzeri and P. putida (16 and 48 ng per reaction, respectively) (c), using primer Pseud_SNP. (Panels e to g) SNuPE patterns obtained with 65 ng of P. stutzeri PCR product (e), 65 ng of P. putida PCR product (f), or mixed PCR products from P. stutzeri and P. putida (48 and 16 ng per reaction, respectively) (g), using a mixture of primers Pp_SNP and Ps_SNP. Negative controls (panels d and h) contained sterile distilled water instead of a PCR product. The vertical axis indicates fluorescence intensity (in relative fluorescence units); the horizontal axis indicates the sizes of the extended products. The numbers indicate the lengths (in bases) of the internal standards (orange peaks).

Assessment of sensitivity.
Besides the specificity, the sensitivity of a detection method is a critical issue. The overall sensitivity of SNuPE strongly depends on the preceding PCR amplification. The sensitivity of the primer extension assay was evaluated with a dilution series containing known quantities of PCR products as templates and a mixture of three primers (Fig. 1). As expected, only the specific signal was observed (data not shown). Moreover, the signal strength decreased proportionately with decreasing template concentration. The detection limit was estimated to be between 2 x 106 and 2 x 107 copies of PCR amplicons µl–1 for the P. putida target sequence. The limits of detection were slightly higher for T. aromatica and B. subtilis and were estimated to be around 1.4 x 108 and 6.2 x 107 copies of PCR amplicons µl–1, respectively. However, the overall sensitivity of the approach is much higher, since the method includes preceding PCR amplification of the target sequence. Compared to ethidium bromide-stained agarose gels (separating approximately 1.5-kbp PCR amplicons), where bands containing as little as approximately 10 ng of DNA can be detected (29), a detection sensitivity that is 2 to 3 orders of magnitude higher can be expected. The increased sensitivity of SNuPE compared to gel-based detection is even more pronounced when a shorter target sequence is amplified, since primer extension depends only on the copy number of the target, whereas nucleic acid staining also depends on the length of the sequence. This finding implies that lower cycle numbers can be used for preamplification for SNuPE in order to avoid biases associated with high cycle numbers (24). With more than 40 cycles of SNuPE, formation of side products and nonspecific extension products was observed in some cases (data not shown); therefore, the use of higher cycle numbers is not recommended. The sensitivity of SNuPE is expected to increase with the development of more sensitive detection sensors, as well as with the improvement of fluorophores and discovery of new fluorophores. In addition, primer extension was demonstrated to detect less than 0.1% of the target template in the presence of nontarget PCR amplicons (41) and was able to detect a minor target population in the presence of dominant population at a ratio of 1:585 (19), which corresponds to good sensitivity and a good dynamic range.

Single and multiplex SNuPE reactions with mixed target templates.
Experiments were designed to evaluate SNuPE for multiplex detection of members of an artificial community established by mixing PCR products of 16S rRNA genes derived from target strains. Figure 1 shows the target sequences of the microorganisms (T. aromatica, P. putida, and B. subtilis) used in this study aligned with the specific primers designed for detection. In the presence of four differently labeled ddNTPs, the type of the labeling provides information about the base located downstream of the hybridization site (Fig. 1.). This may provide extra information for additional phylogenetic typing of the detected sequence (19). In the first step, single reactions using only one type of purified PCR product of a target microorganism were performed. No apparent cross-reactivity was observed for the target-specific primers, and primer extension events were confirmed by detection of incorporated fluorescent dye (data not shown). In each case, the expected nucleotide was incorporated, which indicates that there was a high level of specificity for each target sequence tested. The three different taxon-specific primers also varied in length due to attachment of a noncomplementary tail (polythymidine) to the 5' end of the primers. Polythymidine tails were used as non-acid-labile mobility modifiers, since a previous study showed that these tails were more stable than, e.g., polyadenine tails (37). The extended products were subsequently separated by capillary electrophoresis, and the incorporated label was detected by laser-induced fluorescence. Including internal standards labeled with a fifth color allowed further verification of the specificity by very accurate determination of the sizes of the extended products.

Furthermore, the quantitative power of the SNuPE assay was investigated by mixing template PCR products at defined ratios. Because the single reactions had revealed differences in the extension efficiencies of the different template-primer pairs, calibration of the SNuPE was performed. Dilution series of the PCR products from the different strains (T. aromatica, P. putida, and B. subtilis) were used to obtain calibration curves for the extension efficiencies (Fig. 6). Subsequently, defined template mixtures containing PCR amplicons of the three strains at various ratios were used for multiplex SNuPE. The results of a quantitative analysis of the resulting peak areas were compared to the relative abundance of the target templates in the mixture by taking into account the different calibration curves (Table 2). The {chi}2 test revealed no statistically significant differences between the frequency distribution of ratios corresponding to the added PCR products and the distribution of ratios retrieved for the mixed target templates in the corresponding multiplex SNuPE assays (P > 0.95). This emphasizes the similarity between the observed and expected ratio patterns at a high level of confidence. To investigate in detail the relationships between the theoretical and retrieved ratios, seven different template mixtures of T. aromatica and B. subtilis PCR products (ratios of T. aromatica products to B. subtilis products of 10, 5, 3, 1, 0.3, 0.2, and 0.1) were used separately in duplicate in multiplex assays (Fig. 7). No statistically significant differences were found between the distribution of theoretical ratios and the distribution of retrieved ratios for each of the two replicate experiments (P > 0.7). Furthermore, the fit of the linear regression model with the data was highly significant (P < 0.001), with a coefficient of determination (R2) of 0.99, whereas the slope of the regression line was around 0.7. Overall, this indicates that there was high goodness of the fit between the theoretical ratio values and the expected ratio values retrieved for the multiplex assay reactions up to a T. aromatica/B. subtilis threshold ratio of 5. For higher T. aromatica/B. subtilis ratios the experimental ratio values tend to underestimate the expected ratios. Rehybridization of the products (single-nucleotide extended primers) to the target may interfere with the primer binding and extension. This competition may particularly affect the predominant target sequence in the later cycles, as the detection of the less abundant template is not significantly influenced. This phenomenon might be similar to the "C0t effect" described for PCR, resulting in overrepresentation of the rare template types in the final PCR products (18).


Figure 6
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  		FIG. 6. Calibration of the extension efficiency for different primer-template pairs. The fluorescent signal generated by primer extension (normalized peak area) is expressed as a function of the amount of template PCR products (copy number) obtained from (A) B. subtilis DNA, (B) T. aromatica DNA, and (C) P. putida DNA. The dashed lines indicate the ower 95% confidence intervals of the regression curves.


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  		TABLE 2. Quantitative analysis of multiplex SNuPE reactions for simple model communities consisting of mixtures of PCR amplicons from strains of P. putida, T. aromatica, and B. subtilis


Figure 7
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  		FIG. 7. Experimental template mixture ratios (observed) expressed as a function of the defined template mixtures ratios (theoretical) of T. aromatica (Ta) and B. subtilis (Bs) PCR products (at defined ratios of T. aromatica to B. subtilis of 0.1, 0.2, 0.3, 1, 3, and 5). For information concerning the experimental setup see Table 2. The dashed lines indicate the 95% confidence intervals of the regression curves.

The results presented here suggest that SNuPE can be used as a tool for multiplex quantification of different PCR amplicons in a certain range. However, due to the well-known bias inherent in PCR (33), current SNuPE protocols cannot be used for absolute quantification of microorganisms. Nevertheless, our results suggest that SNuPE can be used as a semiquantitative tool for following spatial and temporal changes in the relative abundance of target microorganisms in a given set of samples. In a previous study, Wu and Liu used a different strategy for normalization of primer extension reactions (41). Calibration factors for individual primers were obtained by comparison of the extension efficiency to the results obtained with a primer at a higher hierarchical level (e.g., domain specific). This primer was included in all reaction mixtures and was used as an internal standard for comparison of Bacteroides populations in a wastewater treatment plant, in stools (10), and in wastewater samples (41).

Use of reverse-transcribed 16S rRNA as a template in a PCR-independent manner.
Until now, SNuPE had to be coupled with PCR preamplification of the target gene for screening environmental samples due to limitations in sensitivity. In general, quantitative biases are associated with the PCR approach due to preferential amplification (33, 39) and differences in the genomic properties, such as genome size, the copy number of 16S rRNA genes, and the G+C content of the target microorganisms (6). As an alternative approach, rRNA was considered as a potential naturally amplified (i.e., abundant) target. Bacterial cells harbor around 103 to 105 ribosomes per cell (1), which may suggest that the predominant members of a microbial community can be targeted by SNuPE in a PCR-independent manner. Since rRNA is not a template for the enzymatic system used for SNuPE reactions, RNA was first reverse transcribed, and the cDNA was used for further reactions. Different ratios of total RNA were used to obtain mixtures of P. putida and B. subtilis RNA, as shown in Fig. 8. A decrease in the P. putida-specific signal corresponded proportionally to a decrease in the P. putida RNA concentration in preparations with constant amounts of B. subtilis RNA. However, the P. putida-specific peak area was approximately twice the B. subtilis-specific product area when the RNA templates were used at a P. putida/B. subtilis ratio of 1:5.


Figure 8
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  		FIG. 8. Use of reverse-transcribed rRNA as a template for SNuPE. The RNA used for the reverse transcription reactions were as follows: (A) total RNA from B. subtilis (60 ng), (B) total RNA from P. putida (60 ng), (C) a 2:1 mixture of total RNA from P. putida and B. subtilis (120 ng and 60 ng, respectively), (D) a 1:1 mixture of total RNA from P. putida and B. subtilis (60 ng and 60 ng, respectively), and (E) a 1:5 mixture of total RNA from P. putida and B. subtilis (12 ng and 60 ng, respectively). (F) Negative control containing sterile distilled water instead of cDNA.

Although this result demonstrates the potential of SNuPE to target reverse-transcribed rRNA, it also illustrates that the PCR-independent procedure does not necessarily result in a solution without bias. The Pseudomonas rRNA was preferentially detected, and use of this rRNA resulted in signal strength that was significantly higher than the signal strengths obtained with the other templates (Fig. 7). When P. putida RNA was mixed with T. aromatica RNA, even more pronounced preferential detection of the P. putida target was observed (data not shown). The secondary structure of the cDNA or primer annealing of the reverse transcript may significantly influence the results of PCR-independent RNA SNuPE, which should be investigated further.

In conclusion, SNuPE has excellent mismatch discrimination potential, and the linear signal amplification contributes to the increased sensitivity of detection. Since SNuPE reactions take place in solution, they are easy to optimize by altering the annealing temperature, cycle number, and template concentration using widely used thermocyclers. Not only PCR products but also reverse-transcribed RNA are suitable templates for primer extension, as long as a sufficient number of variable sites are present in the phylogenetic or functional marker gene analyzed. The separation and detection of the products using capillary electrophoresis and laser-induced fluorescence are very fast and can be highly automated without a need for antigen-antibody reactions and lengthy posthybridization steps. Although there is a limit to the number of primers used per reaction (38), the primers can be grouped according to the optimal annealing temperature, and several SNuPE reactions can be run in parallel using a gradient thermocycler. Therefore, SNuPE has considerable potential in applied and environmental microbiology for filling the gap between the performance of FISH and the performance of oligonucleotide microarrays.


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ACKNOWLEDGMENTS
 
This project was financially supported by the Helmholtz Centre for Environmental Research—UFZ. G.I. was supported by the Marie Curie Early Stage Training Project of the EU (AXIOM, contract MEST-CT-2004-8332).

We thank Ute Lohse for technical assistance with the capillary electrophoresis.


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FOOTNOTES
 
* Corresponding author. Mailing address: UFZ Helmholtz Centre for Environmental Research, Department of Environmental Biotechnology, Permoserstr. 15, 04318 Leipzig, Germany. Phone: 49 341 235 1763. Fax: 49 341 235 2492. E-mail: marcell.nikolausz{at}ufz.de Back

{triangledown} Published ahead of print on 27 February 2009. Back


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Applied and Environmental Microbiology, May 2009, p. 2850-2860, Vol. 75, No. 9
0099-2240/09/$08.00+0     doi:10.1128/AEM.01910-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.




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