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Molecular and Cellular Probes 20 (2006) 60–63 www.elsevier.com/locate/ymcpr Optimized DNA microarray assay allows detection and genotyping of single PCR-amplifiable target copies Ralf Ehricht a, Peter Slickers a, Stefanie Goellner b, Helmut Hotzel b, Konrad Sachse b,* b a Clondiag Chip Technologies GmbH, Loebstedter Str. 105, 07743 Jena, Germany Institute of Bacterial Infections and Zoonoses at the Federal Research Institute for Animal Health (Friedrich-Loeffler-Institut), Naumburger Str. 96a, 07743 Jena, Germany Accepted for publication 20 September 2005 Available online 5 December 2005 Abstract This study was conducted to determine the detection limit of an optimized DNA microarray assay for detection and species identification of chlamydiae. Examination of dilution series of a plasmid standard carrying the target sequence from Chlamydia trachomatis and genomic DNA of this organism revealed that a single PCR-amplifiable target copy was sufficient to obtain a specific hybridization pattern. This performance renders the test suitable for routine testing of clinical samples. q 2005 Elsevier Ltd. All rights reserved. Keywords: DNA microarray; Sensitivity; Detection; Chlamydia trachomatis 1. Introduction While DNA microarray technology has been widely used in gene expression monitoring, genotyping has emerged as another area of application in the last few years. The highly parallel approach, i.e. the possibility to obtain precise sequence information on a variety of genomic loci renders DNA microarrays promising diagnostic tools. Given the complex nature of many bacterial virulence factors, the current PCRbased methods are not capable to fulfill the criteria required for highly informative diagnostic tests in the future. Multi-locus genotyping assays or even genomotyping [1] will supplant the ‘one-dimensional’ typing methods used nowadays. Recent applications of DNA microarrays in genotyping include detection of antibiotic resistance genes in grampositive bacteria [2,3], toxin typing of Clostridium perfringens [4], species differentiation among mixed bacterial communities [5], and identification of respiratory pathogens [6]. However, the suitability of DNA microarray assays for routine diagnosis has yet to be demonstrated as most studies have been dealing with bacterial cultures rather than direct examination of clinical specimens. To achieve this goal, any such test should * Corresponding author. Tel.: C49 3641 8040; fax: C49 3641 804228. E-mail address: [email protected] (K. Sachse). 0890-8508/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.mcp.2005.09.003 be easy-to-handle and cost efficient, as well as highly sensitive and specific. The recently developed ArrayTubee (AT) platform represents an interesting alternative to the widely used, but relatively expensive fluorescence-based glass slide microarray systems. It involves chips of 2.4!2.4 mm size placed on the bottom of 1.5-ml plastic micro-reaction tubes. Hybridization can be conducted on standard laboratory equipment without changing vessels. In a previous paper, we described the development of an AT microarray to differentiate among all nine species of Chlamydia (C.) and Chlamydophila [7]. In the present study, an optimized protocol of this assay was examined to determine detection limits and identify factors limiting sensitivity. 2. Materials and methods 2.1. DNA microarray The present version of the microarray includes 28 probes for species identification, three genus-specific probes, five probes for the closest relatives, i.e. Simkania negevensis and Waddlia chondrophila, as well as four positive controls (consensus probes), and one internal staining control (biotin marker). Each probe was spotted fivefold, yielding a total of 289 spots (Print pattern and probe identities, see Supplement 1; Barplot demonstrating specificity and discriminatory power, see Supplement 2). R. Ehricht et al. / Molecular and Cellular Probes 20 (2006) 60–63 2.2. DNA templates Recombinant plasmid pCR2.1-TOPOCDC38 (map see Supplement 3) served as a model target and was used from a stock solution containing 2.11!1010 copies per microlitre. It was prepared by cloning a 1086-bp insert comprising the 3 0 domain of the 16S rRNA gene, the intergenic spacer and domain I of the 23S rRNA gene of C. trachomatis, into vector pCR2.1-TOPO (invitrogen, Karlsruhe, Germany). The insert also contains the primer binding sites for biotinylation PCR and real-time PCR. Based on a DNA concentration of 0.111 mg/ml as measured in triplicate from UV absorption and a molecular mass of 3.11!106 g/mol, the present preparation was calculated to contain 2.11!1010 plasmid copies per microlitre. Chromosomal DNA of purified elementary bodies of C. trachomatis strain D was prepared from 100 ml of infected cell culture in BGM cells containing 6.38!109 inclusionforming units (ifu)/ml using standard methodology [8]. Based on a DNA concentration of 600 mg/ml as measured from UV absorption and a molecular mass of 6.42!108 g/mol for the C. trachomatis genome, the present preparation was calculated to contain 5.64!10 8 genome copies per microlitre. The proportion of residual mammalian DNA from host cells was lower than 0.5% as determined by ß-actin real-time PCR. 2.3. Amplification, labeling and quantitation of DNA templates used for the microarray test Target DNA was amplified and biotin labeled for the AT microarray assay in 40 cycles of 94 8C/30 s, 55 8C/30 s, and 61 72 8C/30 s, using primers U23F-19 (5 0 -ATTGAMAGGCGAWGAAGGA-3 0 ) and 23R-22 (5 0 -biotin-GCYTACTAAGATGTTTCAGTTC-3 0 ). Hybridization was conducted as described previously [7]. Hybridizing spots were visualized using 3,3 0 5,5 0 -tetramethyl benzidine (TMB) as substrate for streptavidine-conjugated horseradish peroxidase. Hybridization signals were processed using the Iconoclust version 2.3 software (Clondiag, Jena, Germany). Real-time PCR was conducted on a Mx 3000 (Stratagene, La Jolla, CA) using a modified version of the procedure of Everett et al. [9], which included primers Ch23S-F (5 0 CTGAAACCAGTAGCTTATAAGCGGT-3 0 ), Ch23S-R (5 0 ACCTCGCCGTTTAACTTAACTCC-3 0 ), and probe Ch23S-p (FAM-CTCATCATGCAAAAGGCACGCCG-TAMRA). Each dilution series was examined in triplicate by each test. 3. Results and discussion To evaluate the sensitivity of the microarray assay, we examined decimal dilution series of recombinant plasmid pCR2.1-TOPOCDC38. Fig. 1 illustrates that a single copy was sufficient to obtain a species-specific hybridization pattern on the microarray after PCR amplification. When chromosomal DNA of C. trachomatis was tested in an analogous trial, the detection limit was near 0.05 fg of DNA, which is equivalent to 56 genomic copies or 1.87 ifu (see Supplement 4). This prompted us to examine three different templates by quantitative real-time PCR (Fig. 2). The fact that chromosomal DNA was detected with lower sensitivity than Fig. 1. Examination of a dilution series of recombinant plasmid pCR2.1-TOPOCDC38 using the AT microarray assay. Upper line: images of microarray hybridization patterns obtained with plasmid copy numbers indicated on the abscissa. Diagram shows normalized signal intensities of C. trachomatis probes (pm perfect match; mm single mismatch) and its closest relatives, C. suis and C. muridarum, for comparison. Each array included an arbitrary biotinylated 26-mer oligonucleotide probe as internal staining control and four consensus probes representing genomic sequences conserved in all chlamydial species (hybridization controls). 62 R. Ehricht et al. / Molecular and Cellular Probes 20 (2006) 60–63 45 Ct value 40 35 30 25 chrom. DNA DNA digest Plasmid NTC 20 15 5,64E+04 5,64E+03 5,64E+02 5,64E+01 5,64E+00 5,64E-01 Number of template copies Fig. 2. Real-time PCR of serial dilutions of three different templates: (a) chromosomal DNA of C. trachomatis, (b) EcoRI-digested DNA of the same strain, and (c) plasmid pCR2.1-TOPOCDC38. The dotted line at the upper margin shows the average Ct value of the non-template control (NTC). Error bars represent standard deviations from nine individual measurements (except in the case of enzyme-digested DNA: 3 measurements). Increase in PCR product yield as a result of using the present primer pair, which reduced amplicon size from 1 kbp in the previous assay [7] to 176 bp without loss of discriminatory power (see Supplement 2), and (iii) Visualization of hybridization duplexes by enzyme-catalyzed TMB precipitation. It is known that precipitation methods surpass fluorescent reactions in terms of sensitivity by up to three orders of magnitude [10–12]. Detection limits of fluorescencebased microarray assays reported in the literature varied from 60 ng of bacterial DNA [13], 105 bacterial cells (50 ng genomic DNA)[14], to 102–103 cfu/ml from culture enrichment [15]. The present data demonstrate that the AT microarray assay for chlamydiae is sensitive enough to detect and genotype a single PCR-amplifiable copy of target DNA. As such a performance is required for examination of clinical samples the test can be considered for use in routine diagnosis. Acknowledgements We are grateful to Juergen Roedel, Jena, for providing the C. trachomatis culture. We also thank Simone Bettermann and Elke Mueller for excellent technical assistance. Table 1 Detection limits (in copy numbers) Detection method Chromosomal DNA C. trachomatis Plasmid DNA (pCR2.1TOPOCDC38) AT microarray assay Real-time PCR Conventional PCR (40 cycles) with agarose gel electrophoresis 56 56 1000 1 1 1000 plasmid DNA by both microarray and real-time PCR (Table 1) indicates that the actual number of target copies available for amplification was lower than the number of genome copies present in the sample. This may be a consequence of shear stress in the course of DNA extraction, which can lead to strand breaks and partial degradation, as well as the effect of steric hindrances during the enzymatic amplification reaction. In the case of plasmid template, these constraints would be far less relevant because of the high structural stability of circular plasmid DNA, which allows each copy to be PCR amplified and subsequently be involved in duplex formation on the microarray. Additionally, using EcoRI-digested chromosomal DNA as PCR template did not result in lower detection limits in the AT microarray assay (data not shown) nor in real-time PCR (Fig. 2). The data suggest that the inferior sensitivity of detection in the case of chromosomal DNA was not associated with the hybridization or visualization reactions on the AT microarray. From a general perspective, the findings of this study imply that other PCR detection tests would also require the presence of a sizeable number of genome copies in a sample for the theoretical detection limit of one copy to be realized. In the authors’ view, the high sensitivity of the AT microarray assay was attained because of: (i) Efficient probe design and further optimization by harmonizing probe melting temperatures through GCC values (Supplement 1), (ii) Appendix. Supplementary data Supplement 1Print pattern of the AT microarray for chlamydiae and probe identities. Supplement 2Barplot demonstrating specificity and discriminatory power of the microarray used in the present study. Supplement 3Map of recombinant plasmid pCR2.1-TOPOC DC38 and visualization by agarose gel electrophoresis of biotinylation PCR products of a dilution series of this plasmid. Supplement 4Examination of a dilution series of C. trachomatis chromosomal DNA using the AT microarray assay. Upper line: images of microarray hybridization patterns obtained with genome copy numbers indicated on the abscissa. Diagram: normalized signal intensities of C. trachomatis probes (pm, perfect match; mm, single mismatch) and its closest relatives, C. suis and C. muridarum, for comparison. Each array included an arbitrary biotinylated 26-mer oligonucleotide probe as internal staining control and four consensus probes representing genomic sequences conserved in all chlamydial species (hybridization controls). Supplementary data Supplementary data associated with this article can be found at doi:10.1016/j.mcp.2005.09.003 R. Ehricht et al. / Molecular and Cellular Probes 20 (2006) 60–63 References [1] Lucchini S, Thompson A, Hinton JC. Microarrays for microbiologists. Microbiology 2001;147:1403–14. [2] Perreten V, Vorlet-Fawer L, Slickers P, Ehricht R, Kuhnert P, Frey J. Microarray-based detection of 90 antibiotic resistance genes of grampositive bacteria. J Clin Microbiol 2005;43:2291–302. [3] Monecke S. Ehricht R. 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