Download Optimized DNA microarray assay allows detection and genotyping

Molecular and Cellular Probes 20 (2006) 60–63
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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
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