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Journal of Clinical Microbiology, December 2005, p. 6189-6193, Vol. 43, No. 12
0095-1137/05/$08.00+0 doi:10.1128/JCM.43.12.6189-6193.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Linda Novarese,1,
Brunella Posteraro,1,
Stefania Ranno,1
Elena De Carolis,1
Giovanni Pecorini,1
Barbara Lucignano,2
Fausta Ardito,1 and
Giovanni Fadda1
Istituto di Microbiologia, Università Cattolica del Sacro Cuore,1 Laboratorio di Microbiologia, Ospedale Pediatrico "Bambino Gesù," Rome, Italy2
Received 30 May 2005/ Returned for modification 1 August 2005/ Accepted 6 September 2005
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In this study, we developed a new method, based on microelectronic chip array technology, to rapidly identify a variety of clinically relevant mycobacterial species by use of the Nanochip Molecular Biology Workstation (Nanogen, Inc., San Diego, CA). The instrument uses a proprietary semiconductor microchip, which incorporates a 10-by-10 array of microelectrodes coated with a permeation layer containing streptavidin, for the rapid transport and concentration of negatively charged nucleic acid molecules through the selective application of a positive electronic bias to selected test sites. The nucleic acid may then be immobilized by direct attachment to the permeation layer or by hybridization to a previously addressed nucleic acid. This technique has been used for single-nucleotide polymorphism assays using fluorescently labeled reporter probes and biotinylated amplified DNA samples (4).
A 205-bp highly conserved region of the Mycobacterium 16S rRNA gene was amplified from genomic DNA samples using the pMyc14 (5'-biotinylated) and pMyc7 primers and PCR conditions as previously described (7). A small amount of biotinylated PCR product was electronically deposited and anchored by streptavidin linkage on the microchip surface, denatured in situ, and hybridized with 13 dye-labeled oligonucleotide probes, seven of which were selective for M. tuberculosis complex, M. avium, M. xenopi, M. gordonae, M. terrae, M. triplex, and M. malmoense, respectively. The other six probes were each selective for two or multiple mycobacterial species, due to the high similarity between their PCR-amplified 16S rRNA gene fragments (Table 1).
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Amplicon addressing (the process by which each amplicon was electronically placed in the microchip) was performed following the Nanogen User's Guide instructions. Briefly, a 96-well microtiter plate was filled as follows: one well contained 60 µl of 0.3 M NaOH for sample passive (not electronic) pretreatment; another well contained 60 µl of 50 mM histidine (30 µl of 100 mM histidine plus 30 µl of high-performance liquid chromatography-grade water) as the background control; the other wells each contained one sample to be processed (15 µl of PCR product, 15 µl of high-performance liquid chromatography-grade water, and 30 µl of 100 mM histidine); the last well contained 60 µl of 0.1 M NaOH for sample passive denaturation. Then, the microtiter plate and Nanochip cartridge were put into the loader, and each purified amplicon was addressed to a specific test site on the cartridge and analyzed.
Prior to starting, the conductivity of the histidine buffer and each purified amplicon was checked; samples with a conductivity not exceeding 100 µS/cm were passed for loading. When addressing was completed, the cartridge was removed from the loader and processed for hybridization. Hybridization solutions were prepared by mixing in ice 2 µl each of Cy3- and Cy5-labeled probes (0.5 µM, final concentration) and 196 µl of high-salt buffer (50 mM sodium phosphate, pH 7.4, and 500 mM NaCl). Before hybridization, the Nanochip cartridge was washed four times with 200 µl of high-salt buffer.
The hybridization solution (200 µl) was accurately pipetted on the microchip surface. The cartridge was then put into the reader, heated at 56°C for 1 min, cooled until the discrimination temperature of each probe pair (Table 1), and left at this temperature for 3 min. Heating at 56°C was introduced to avoid the formation of secondary structures within the amplicons. After hybridization, three washing steps were performed with low-salt buffer (50 mM sodium phosphate, pH 7.0), and the cartridge was then cooled to 24°C and scanned using a two-laser system. Data were analyzed, and samples with a signal-to-noise ratio of >5 were registered as positive. To perform a new hybridization step with a different probe pair, bound probes were removed with NaOH (0.3 M). A single cartridge could be stripped and rehybridized at least 10 times.
To set up the assay, to evaluate probes efficacy and to discriminate hybridization thermal stringency, we performed preliminary experiments using mycobacterial reference strains M. tuberculosis ATCC 27294 and ATCC 35801; M. avium ATCC 25291; M. chelonae ATCC 35752; M. fortuitum ATCC 6841; M. intracellulare ATCC 13950; M. kansasii ATCC 12478; M. marinum ATCC 927; M. xenopi ATCC 19250; M. smegmatis ATCC 607; and M. terrae ATCC 15755 and clinical isolates of M. gordonae, M. triplex, and M. malmoense. Specific fluorescence signals were obtained for most of the probes tested in a series of independent experiments. Probes that failed to give reproducible hybridization results were redesigned by choosing another target sequence within the 16S rRNA gene region (Table 1).
We tested PCR products obtained from 270 primary liquid cultures of clinical specimens from patients with suspected tuberculosis or other mycobacterial infections that were detected as positive by the BACTEC Mycobacteria Growth Indicator Tube 960 system (Becton Dickinson, Sparks, MD). Briefly, a 1-ml aliquot from each Mycobacteria Growth Indicator Tube culture was incubated at 100°C to inactivate mycobacteria. Genomic DNA was then extracted as previously described (11). After amplification, the PCR products were purified and desalted by QIAquick PCR purification kit (QIAGEN, Hilden, Germany), according to the manufacturer's instructions with the following modifications: in step 5, two washes with buffer PE plus 1 wash with 0.75 ml of 70% ethanol were performed; in step 8, elution was performed using 30 µl of high-performance liquid chromatography-grade water instead of buffer EB.
Of the 270 mycobacterial PCR products tested, the Nanochip assay was able to identify 141 M. tuberculosis complex, 36 M. avium, 24 M. xenopi, 18 M. gordonae, 5 M. terrae, 1 M. triplex, and 1 M. malmoense. The results were compared to those obtained with the matched isolates by conventional biochemical tests (8) and molecular methods, such as PCR-restriction enzyme analysis and PCR-reverse hybridization (11, 16). A complete agreement was observed among these methods (data not shown). Among the remaining 44 PCR products, 12 hybridized with probe pInt2, 11 with probe pChel3, 9 with probe pFor3, 6 with probe pKan3, 5 with probe pSme2, and 1 with probe pMar2; correspondingly, the culture isolates were identified as M. intracellulare (12 isolates), M. chelonae (11 isolates), M. fortuitum (9 isolates), M. kansasii (6 isolates), M. smegmatis (5 isolates), and M. marinum (1 isolate), using biochemical and molecular tests (8, 16). Figure 1 shows the results of hybridization obtained with PCR products from 13 different mycobacterial species using three probe pairs. As expected, strong signals were observed only between the PCR products and their selective oligonucleotide probes.
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In this work, we applied the Nanogen Nanochip Molecular Biology Workstation to the field of bacterial diagnostics for the first time, focusing on the species identification of mycobacteria. We adapted this technology to our purpose by creating a hybridization assay in which multiple species-selective probes, matching a highly polymorphic region of the Mycobacterium 16S rRNA gene, were used in consecutive reactions on a single platform. This gene has been used in several molecular methods (11, 14), and, recently, in a microarray system developed by Troesch et al. (18), for the species identification of mycobacteria. Like the last method, the microelectronic chip has the potential answer to many diagnostic questions associated with the genus Mycobacterium, since it is possible to expand the spectrum of the mycobacterial species by testing additional probes on the same chip. In our assay, we included probes directed against either most clinically relevant mycobacterial species (i.e., M. tuberculosis complex and M. avium) or Mycobacterium species rarely causing human disease, such as M. triplex. The choice of these probes reflected the epidemiology of Mycobacterium infections in our hospital, but obviously, the panel of probes can be modified according to the patient population to which diagnostic investigation is referred.
In conclusion, the Nanochip system offers a great deal of promise for rapid identification, especially if used in combination with PCR detection methods. A similar approach has been ideated by Westin et al. (20), in which multiplex strand displacement amplification was combined with microelectronic chip array to detect the factor V Leiden mutation. Our results reconfirm the great flexibility of the Nanochip system and open new perspectives for its large-scale use in clinical diagnostics. In spite of the remarkable qualities of this system, such as accuracy, ability to select a minimal signal-to-noise ratio, need for only a small amount of DNA, reduced turnaround time, and easy performance, the Achilles' heel remains the attention in designing probes of interest to avoid additional synthesis costs, although this is of little importance compared to a 100-site array cost (approximately 500 Euros). As the system is capable of testing up to 100 samples simultaneously, we feel that the Nanochip system presented here is particularly suitable for routine use in mycobacteriology laboratories that process a wide number of clinical specimens.
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FOOTNOTES |
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These authors contributed equally to this work.
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