| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Abstracts of Oak Ridge Posters |
1
BioStar, Inc., 6655 Lookout Rd., Boulder, CO 80301
a author for correspondence: fax 303-581-6405, e-mail rmo{at}csn.net
The reported incidence of methicillin-resistant Staphylococcus aureus (MRSA) isolates in hospitals has increased from 2% in 1974 to 50% in 1997 (1). In addition, MRSA is emerging as a community-acquired pathogen. Resistance is most often mediated by the mecA gene, which encodes an altered penicillin-binding protein (PBP-2A) with low affinity for ß-lactam antibiotics. Rapid identification of the mecA gene is important for implementation of appropriate antibiotic therapy.
MRSA is identified by either culture or molecular methods. Routine culture methods require two sequential steps, one to isolate S. aureus and the second to determine antibiotic susceptibility (2). Recently, molecular methods, including PCR, branched DNA, and cycling probe assays, have been described that identify mecA sequences from individual S. aureus colonies (3)(4)(5)(6)(7). These methods are sensitive and specific, but they all require instrumentation to interpret the results.
In this report, we describe a thin film biosensor for qualitative
visual detection of mecA either directly from a single
S. aureus colony or from a PCR amplification reaction. The
technology allows direct visual detection of the interaction of target
DNA sequences with complementary oligonucleotide probes immobilized to
an optically coated silicon chip (8). The key assay steps
for thin film formation are: (a) simultaneous hybridization
of the target sequence to covalently attached surface capture probe and
solution-phase biotinylated detector probes; (b) binding of
anti-biotin antibody enzyme conjugate to the annealed detector probes;
and (c) mass deposition through enzyme-catalyzed
precipitation (Fig. 1A
). The thin film alters the interference pattern of light on
the surface, producing a perceived color change. Composition of the
optical layers is designed such that small increases in thickness
produce a color change from gold to purple, producing contrast in the
range where the human eye is most sensitive. Thickness changes as small
as 10Å can be detected visually.
|
The thin film biosensor may be interpreted visually for a qualitative result. Alternatively, quantification may be obtained by either of two instrumented methods, charge-coupled device (CCD) imaging or ellipsometry (9)(10). CCD imaging software measures color intensity, and ellipsometry measures thin film thickness. Each method provides a measurement that is proportional to the amount of target bound to the biosensor surface.
The nucleic acid detection surface described in these studies was a multilayer optical surface composed of a silicon wafer layered with 515Å of silicon nitride applied by plasma vapor deposition and 130Å of a hydrophobic polymer (polydimethylsiloxane; United Chemical Technologies) applied by spin coating. A final layer of polyphenylalanine-polylysine (Sigma) was applied by passive adsorption to provide functional amines. Covalent attachment of the mecA capture probe (5'-GTCATTTCTACTTCACCATTACCAAC-3') was accomplished by reacting the homobifunctional cross-linker disuccidimidyl suberate (Pierce) with the 3' amine of the capture probe and amines on the biosensor surface, producing a stable amide linkage. Hybridization of the target sequence to the biosensor surface and to biotinylated detector probes is followed by reaction with an anti-biotin antibody conjugated to horseradish peroxidase (HRP) and precipitating substrate. This precipitation event deposits additional mass onto the surface, triggering a color change from gold to purple. This thin film deposition effectively transduces the hybridization reaction into a visible result.
The mecA biosensor determined the correct genotype of
methicillin-sensitive S. aureus (MSSA) and MRSA strains from
a single colony in a simple 3-h procedure (Fig. 1B
). MRSA (ATCC strain
33592) and MSSA (ATCC strain 11632) were incubated at 37 °C for
24 h on a trypticase soy-blood agar plate (Remel). A
single colony was suspended in lysis buffer (10 mg/L
lysostaphin (Sigma), 75 mmol/L NaCl, 25 mmol/L EDTA, 20 mmol/L Tris, pH
7.5) and incubated at room temperature for 20 min. Samples were then
denatured at 95 °C for 10 min in the presence of three biotinylated
detector probes (1.33 µmol/L each):
The sample was diluted with an equal volume of hybridization
buffer [10x standard saline citrate (SSC), 2 g/L sodium
dodecyl sulfate, 10 g/L
BlockAidTM (BioStar reagent)], incubated for 10
min at 95 °C, and then immediately added to the biosensor surface.
Simultaneous hybridization of the S. aureus genomic DNA to
the biosensor surface and the biotinylated detector probes was carried
out for 2 h at 53 °C, followed by 10 min at 23 °C. The
surfaces were washed with 0.1x SSC containing 1 g/L sodium dodecyl
sulfate, followed by 0.1x SSC. Anti-biotin antibody conjugated to HRP
(1 mg/L) was incubated on the surface for 10 min at room temperature;
the surface was washed, and a precipitating
3,3',5,5'-tetramethylbenzidine (TMB) substrate (BioFX) was added
for 15 min. The mecA gene was specifically detected from the
MRSA strain, illustrating the utility of the mecA biosensor
for direct detection of mecA sequences from a single
S. aureus colony without purification or amplification of
the genomic DNA (Fig. 1B
).
A second rapid biosensor format was developed to analyze the products of mecA PCR amplification. In this 10-min assay, the mecA hybridization target was a 617-bp PCR amplicon rather than total genomic DNA. Because the PCR amplicon target was less complex and more abundant than chromosomal DNA, hybridization times were shortened from 2 h to 3 min. In addition, the increased copy number of the specific targeted sequences allowed for reduction in timing of all assay steps.
PCR amplification was performed with purified MRSA genomic DNA, (forward primer, 5'-TAATAGTTGTAGTTGTCGGGTTTG-3'; reverse primer, 5'-GGTTTTAAAGTGGAACGAAGGTAT-3'). PCR conditions were as follows: 95 °C for 4 min, followed by 25 cycles of 95 °C for 45 s, 53 °C for 45 s, 72 °C for 60 s, which amplified a 617-bp fragment from the mecA gene. The PCR reaction mixture was diluted in hybridization buffer, mixed with 1 µmol/L each of the three biotinylated detector probes, and denatured at 95 °C for 3 min. The sample was incubated on the biosensor surface at 50 °C for 3 min, washed, and reacted with the anti-biotin antibody HRP conjugate for 2 min at room temperature. The precipitating TMB substrate was then added for 2 min at room temperature. The assay was complete in 10 min.
We compared the relative sensitivity of the rapid mecA
biosensor PCR amplicon detection protocol with traditional agarose gel
electrophoresis and ethidium bromide staining (Fig. 1C
). The
mecA biosensor was 10-fold more sensitive than the
electrophoretic method with results available in 10 min. The lower
limit of detection for the mecA biosensor corresponded to
30–50 fmol of amplified target. Because a positive result with the
biosensor requires specific nucleic acid hybridization, amplification
of the correct sequence was confirmed.
The thin film biosensor provides rapid, noninstrumented detection of nucleic acid target sequences. Detection of mecA from a single S. aureus colony eliminates the need for confirmatory culture methods and reduces the time for MRSA determination from 24–48 h to <3 h. Sequence-specific detection of mecA PCR amplicons was complete in 10 min with sensitivity exceeding electrophoretic analysis. The thin film biosensor is a rapid, sensitive alternative to traditional methods for PCR amplicon detection such as gel electrophoresis or microwell hybridization assays. Because the biosensor sensitivity provides for detection of relatively few surface capture hybridization reactions, targets as complex as bacterial chromosomal DNA can be analyzed without purification. The assays may be configured for multitarget detection simply by attaching multiple capture probes to the silicon surface in discrete locations. The thin film biosensor can be formatted for single-gene or multigene detection to provide information for multiple antibiotic resistance mutations with one biosensor surface.
Acknowledgments
We thank Chris High for preparation of the figures.
References
The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  R. Ramakrishnan, W. Buckingham, M. Domanus, L. Gieser, K. Klein, G. Kunkel, A. Prokhorova, and P. V. Riccelli Sensitive Assay for Identification of Methicillin-Resistant Staphylococcus aureus, Based on Direct Detection of Genomic DNA by Use of Gold Nanoparticle Probes Clin. Chem., October 1, 2004; 50(10): 1949 - 1952. [Full Text] [PDF]
|
|
|
|
|
|
C. M. Strom, D. D. Clark, F. M. Hantash, L. Rea, B. Anderson, D. Maul, D. Huang, D. Traul, C. Chen Tubman, R. Garcia, et al. Direct Visualization of Cystic Fibrosis Transmembrane Regulator Mutations in the Clinical Laboratory Setting Clin. Chem., May 1, 2004; 50(5): 836 - 845. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Jenison, H. La, A. Haeberli, R. Ostroff, and B. Polisky Silicon-based Biosensors for Rapid Detection of Protein or Nucleic Acid Targets Clin. Chem., October 1, 2001; 47(10): 1894 - 1900. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
