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Applied and Environmental Microbiology, January 2008, p. 300-304, Vol. 74, No. 1
0099-2240/08/$08.00+0     doi:10.1128/AEM.01600-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Single-Nucleotide Primer Extension Assay for Detection and Sequence Typing of "Dehalococcoides" spp.{triangledown} ,{dagger}

Marcell Nikolausz,1* Antonis Chatzinotas,2 Márton Palatinszky,3 Gwenaël Imfeld,4 Paula Martinez,1 and Matthias Kästner1

Department of Bioremediation,1 Department of Environmental Microbiology,2 Department of Isotope Biogeochemistry, UFZ Helmholtz Centre for Environmental Research, Permoserstr. 15, D-04318 Leipzig, Germany,4 Department of Microbiology, Eötvös Loránd University of Science, Pázmány Péter sétány 1/c, 1117 Budapest, Hungary3

Received 13 July 2007/ Accepted 30 October 2007


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ABSTRACT
 
A single-nucleotide primer extension (SNuPE) assay in combination with taxon-specific 16S rRNA gene PCR analysis was developed for the detection and typing of populations of the genus "Dehalococcoides". The specificity of the assay was evaluated with 16S rRNA gene sequences obtained from an isolate and an environmental sample representing two Dehalococcoides subgroups, i.e., the Cornell and the Pinellas subgroups. Only one sequence type, belonging to the Pinellas subgroup, was detected in a Bitterfeld-Wolfen region aquifer containing chlorinated ethenes as the main contaminants. The three-primer hybridization assay thus provided a fast and easy-to-implement method for confirming the specificity of taxon-specific PCR and allowed rapid additional taxonomic classification into subgroups. This study demonstrates the great potential of SNuPE as a novel approach for rapid parallel detection of microorganisms and typing of different nucleic acid signature sequences from environmental samples.


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INTRODUCTION
 
Taxon-specific detection of distinct groups of microorganisms in environmental samples is crucial whenever a key process is linked to these taxa. However, such organisms are often overlooked by the so-called universal domain-specific diversity approach due to the limitations of the method used (10, 13, 16) and differences in genomic properties (2) or simply the low abundance of the target microorganism.

Examples of microorganisms presenting such a challenge are microbial communities involved in reductive dechlorination, in which the key player often belongs to the genus "Dehalococcoides" (proposed name) (3, 17), a bacterial taxon with members harboring only one copy of the 16S rRNA gene (15). Dehalococcoides spp. in many cases represent only a minor part of natural microbial communities and are therefore usually overlooked by PCR-based monitoring with universal primers targeting the evolutionarily highly conserved regions of the 16S rRNA gene (7, 12).

Recently, single-nucleotide primer extension (SNuPE) assays, which benefit from the fidelity of dideoxynucleoside triphosphate (ddNTP) incorporation catalyzed by a DNA polymerase, have been shown to allow highly specific detection and distinction of DNA sequence variants (18, 20). Primer extension methods are frequently used in forensic research (20) and routine medical diagnosis of genetic disorders (11, 19). In principle, primer extension can be used not only for the determination of a particular nucleotide position but also for the detection of a hybridization event via the labeling of the primers used. Therefore, we propose SNuPE as a promising tool for the detection of specific signature sequences of certain microbial taxa in PCR products obtained from environmental samples (see Fig. S1 in the supplemental material). Here, we report the development of a simple and fast primer extension assay for the detection of Dehalococcoides spp. in environmental samples and the typing of 16S rRNA gene sequences.

Phylogenetic analyses of 16S rRNA gene sequences of Dehalococcoides spp. have shown that these dehalorespiring microorganisms are separated into three subgroups, namely, the Cornell, Victoria, and Pinellas subbranches (3). The sequence from strain 195 of "Dehalococcoides ethenogenes", which is affiliated with the Cornell subgroup, and a 16S rRNA gene sequence (accession number AM399022), retrieved from an environmental sample, belonging to the Pinellas subgroup (9) served as positive controls for the development of an assay targeting the three subgroups. D. ethenogenes strain 195 was cultivated as described previously (8). Groundwater samples contaminated with chlorinated hydrocarbons were obtained from wells located in the Bitterfeld-Wolfen area, Germany. The sampling site and DNA isolation strategy were described in a previous study (4). DNA from the strain was isolated using a DNeasy tissue kit according to the instructions of the manufacturer (QIAGEN, Hilden, Germany) for gram-positive cells. DNA was eluted in 120 µl of RNase-free distilled water (the DNA concentration ranged between 0.2 and 0.6 ng µl–1). For Dehalococcoides-specific PCR analysis, the forward primer DHC1 and the reverse primer DHC 1377 were used in the first round and DHC1 coupled with reverse primer DHC 1212 (3) was used in the second round in a seminested PCR approach as described previously (9).

PCR products were purified using the QIAquick PCR purification kit (QIAGEN) and quantified using the Pico green double-stranded DNA quantification reagent (Molecular Probes, Eugene, OR). Unincorporated primers and deoxynucleoside triphosphates from a 42-µl sample of PCR products were removed by incubation with 12 U of shrimp alkaline phosphatase (SAP; Fermentas, Vilnius, Lithuania) and 6 U of exonuclease I (ExoI; Fermentas) in the SAP buffer provided by the manufacturer in a final volume of 60 µl. The 1-h incubation at 37°C was followed by enzyme inactivation at 75°C for 15 min. The estimation of gene copy numbers used in the SNuPE reactions was based on the length of the PCR amplicon (1,212 bp), the average weight of a base pair, and the measured concentrations of the products. Dehalococcoides-specific primers for primer extension assays were designed in this study based upon the sequence alignment of Hendrickson et al. (3). A triplex assay was developed to target three phylogenetic subgroups (Cornell, Victoria, and Pinellas) of the genus Dehalococcoides by priming three loci with primers Vic/Pin/Cor 962 (5' (T)3CGACCTGTTAAGTCAGGA), Vic/Pin 140 (5' (T)9TGTGGTGGRCCGACATA), and Vic/Pin 159 (5' (T)14GTTGGTTCACTAAAGCCG). The subscript numbers indicate the lengths of the poly-T mobility modifiers. Cyclic primer extension reactions were performed in a final volume of 10 µl containing 5 µl of SNaPshot multiplex kit reagent (Applied Biosystems), 4 µl of purified PCR products (the amount of DNA ranged between 9.9 and 20.8 ng per reaction), and 1 µl of primer solution or primer mixture (10 µM [each] primer). SNuPE reactions were carried out with 45 cycles of denaturation at 96°C for 10 s, annealing at 55°C for 5 s, and extension at 60°C for 30 s. In order to remove unincorporated ddNTPs, 1 U of SAP was added to each reaction mixture and the mixtures were incubated at 37°C for 1 h and then inactivated at 75°C for 15 min. SNuPE reactions were run in duplicate to ensure reproducibility, and a minimum of four replicate reactions were carried out in parallel when peak area ratios were calculated. One-half microliter of posttreated extension products was mixed with 9 µl of formamide and 0.5 µl of a 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 on an ABI PRISM 3100 genetic analyzer by using a 36-cm capillary filled with denaturing POP6 polymer with filter set E5 (Applied Biosystems). The following parameters were applied during the 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 area data were normalized according to an internal size standard (a peak corresponds to 35 nucleotides [nt]).

The signature base differences defining the three subgroups of the Dehalococcoides genus, namely, Cornell, Victoria, and Pinellas (Fig. 1), were used in this study for the primer design. The signatures allow differentiation among the subgroups by benefiting from the mismatch discrimination and signature nucleotide determination potential of SNuPE. The nucleotides expected to be incorporated, the assigned colors, and the patterns expected from the SNuPE assay for the different phylogenetic subgroups are shown in Table 1. One specific peak with the incorporation of the expected base (G [blue]) was obtained with the positive control used (D. ethenogenes strain 195, belonging to the Cornell group) (Fig. 2A). The PCR product of a 16S rRNA gene (accession number AM399022), obtained from an environmental sample, belonging to the Pinellas subgroup, obtained in a previous study (9), served as an additional positive control. The expected primer extension pattern (A, T, T [green, red, red]) was observed in accordance with the sequence data (Fig. 2B). The mixture of the DNA of the two positive controls also resulted in the expected pattern (Fig. 2C). The two Vic/Pin/Cor 962 primer extension products were always slightly displaced from one another due to the influence of the incorporation of different nucleotides and fluorescent dye on the migration. Such variation in the mobility behavior of differently labeled short extension products has also been described by others (6, 20).


Figure 1
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  		FIG. 1. Sequence alignment of the 16S rRNA gene variable regions 2 and 6 of Dehalococcoides strains representative of the three subgroups, Cornell, Victoria, and Pinellas. The alignment is based on data from Hendrickson et al. (3). The shaded letters indicate signature base differences defining Cornell, Victoria, and Pinellas phylogenetic branches. The primer design exploited the mismatch discrimination ability of SNuPE and the typing information from the incorporated labeled nucleotide (also see Table 1). Primer binding sites are underlined and aligned together with the primers designed in this study. During the enzymatic reaction, the primers hybridize to the opposite strands with the reverse complement sequences. seq., sequence. (Adapted from reference 3 with permission.)


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  		TABLE 1. Expected primer extension products and color codes for the three subgroups in Dehalococcoides-specific detection and typing


Figure 2
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  		FIG. 2. Results from primer extension assays for the detection and typing of Dehalococcoides 16S rRNA gene sequences. The 16S rRNA gene sequences from D. ethenogenes strain 195, belonging to the Cornell subgroup, and the environmental sequence dehaloc3072 (accession number AM399022), belonging to the Pinellas subgroup (9), were used as positive controls. (A) SNuPE pattern of the 16S rRNA gene PCR product (20 ng) of D. ethenogenes. (B) SNuPE pattern of the 16S rRNA gene PCR product (20 ng) of dehaloc3072. (C) SNuPE pattern of a mixture of both controls (10 ng of both PCR products). (D to G) Screening of groundwater samples taken from wells in Bitterfeld. The amount of PCR products used for SNuPE was between 9.9 and 20.8 ng per reaction. (H) Negative control containing sterile distilled water instead of a PCR product. The vertical axis represents fluorescence intensity (in relative light units); the horizontal axis represents the sizes of the extended products. Numbers in the top and bottom chromatograms indicate the lengths (in bases) of the internal standard (orange peaks).

The robustness of the SNuPE assay was evaluated by measuring the extents of the fluorescent signals generated with various initial amounts of Cornell and Pinellas type PCR products. The linear regression models significantly fit the data (P < 0.001), with coefficients of determination (R2) of 0.98 and 0.96 for the Cornell and Pinellas subgroups, respectively. The experimental range of linearity was 8.20 x 107 to 1.64 x 109 copies of PCR amplicons µl–1 for the Cornell group and 7.98 x 107 to 1.60 x 109 copies of PCR amplicons µl–1 for the Pinellas group (Fig. 3). The limits of detection were 3 x 106 and 8 x 106 copies of PCR amplicons µl–1 for the Cornell and Pinellas groups, respectively. When a mixture of amplicons corresponding to the two groups was analyzed, 4.1 x 107 copies of Cornell type PCR amplicons could be detected in the presence of 2.4 x 1010 copies of Pinellas type amplicons. This result corresponds to a 1:585 ratio, which substantiates a fair dynamic range of the method.


Figure 3
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  		FIG. 3. Fluorescent signals generated by labeled primer extension products (normalized peak areas) as a function of the amounts of the PCR product templates (copy numbers) obtained from Cornell (A) and Pinellas (B) type Dehalococcoides DNA. DHC1-DHC 1212 PCR products were serially diluted, and the peak areas were normalized according to the average signal strength of the applied internal standard (35 nt). Dashed lines represent upper and lower limits of 95% confidence intervals for the regression curves.

The screening of various wells in the Bitterfeld-Wolfen region, which comprises a contaminated area of about 25 km2 containing tetrachloroethene and trichloroethene as the main contaminants, revealed only one sequence type, belonging to the Pinellas subgroup (Fig. 2D to G). This result is in good agreement with the results of a previous study which detected only this sequence type from that region (9). Although other studies detected Dehalococcoides 16S rRNA gene sequences belonging to more than one sequence type at other sites (1, 3), our monitoring corroborated that the contaminated groundwater of the Bitterfeld-Wolfen region contains only one predominant subgroup of Dehalococcoides. However, it cannot be excluded that different populations, having the same or very similar 16S rRNA gene sequences but different physiological traits due to different inventories of dehalogenase genes (5, 17), coexist in the same contaminated aquifer.

Taxon-specific PCR detection is usually accomplished by verification via cloning and sequencing (3). Here, SNuPE was shown to provide a much faster and more cost-effective verification alternative. On the one hand, the multiplex hybridization and primer extension with three primers provide rapid and reliable proof of the presence of the target sequence, and on the other hand, the pattern code allows the taxonomic classification of the detected sequence. In this way, the multicolor detection system provides more flexibility than previous minisequencing-based detection methods using only one labeled ddNTP out of the four (14). Since the SNuPE reaction occurs in a solution, it can easily be optimized by altering the annealing temperature, cycle numbers, and template concentration by using a standard thermocycler. The separation and detection of the products using capillary electrophoresis and laser-induced fluorescence are very fast and can be highly automated, without the need for antigen-antibody reactions and lengthy posthybridization steps. Although there is a limitation on the number of primers (up to 10) used per reaction (20), the primers can be categorized according to their optimal annealing temperatures, and several SNuPEs can be run in parallel using a gradient thermocycler. The alteration of a SNuPE assay can be performed with minimal effort and cost. In conclusion, SNuPE has considerable potential in applied and environmental microbiology as a fast and easy-to-optimize tool to complement the broadly used molecular screening and detection methods.


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ACKNOWLEDGMENTS
 
This project was financially supported by the UFZ Helmholtz Centre for Environmental Research. Gwenaël Imfeld and Paula Martinez are supported by the Marie Curie Early Stage Training Project of the European Union (AXIOM; contract no. MEST-CT-2004-8332).

We thank Ivonne Nijenhuis for providing D. ethenogenes strain 195 and Ute Lohse for technical assistance with the capillary electrophoresis. We thank Anja Miltner and Hermann J. Heipieper for critical reading and suggestions.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Bioremediation, UFZ Helmholtz Centre for Environmental Research, Permoserstr. 15, 04318 Leipzig, Germany. Phone: 49 341 235 2053. Fax: 49 341 235 2492. E-mail: marcell.nikolausz{at}ufz.de Back

{triangledown} Published ahead of print on 9 November 2007. Back

{dagger} Supplemental material for this article may be found at http://aem.asm.org/. Back


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Applied and Environmental Microbiology, January 2008, p. 300-304, Vol. 74, No. 1
0099-2240/08/$08.00+0     doi:10.1128/AEM.01600-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Nikolausz, M., Chatzinotas, A., Tancsics, A., Imfeld, G., Kastner, M. (2009). Evaluation of Single-Nucleotide Primer Extension for Detection and Typing of Phylogenetic Markers Used for Investigation of Microbial Communities. Appl. Environ. Microbiol. 75: 2850-2860 [Abstract] [Full Text]  

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