Use of Microelectronic Array Technology for Rapid Identification of Clinically Relevant Mycobacteria

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0095-1137/05/$08.00⫹0 doi:10.1128/JCM.43.12.6189–6193.2005

Use of Microelectronic Array Technology for Rapid Identification

of Clinically Relevant Mycobacteria

Maurizio Sanguinetti,

1

*§ Linda Novarese,

1

§ Brunella Posteraro,

1

§ Stefania Ranno,

1

Elena De Carolis,

1

Giovanni Pecorini,

1

Barbara Lucignano,

2

Fausta Ardito,

1

and Giovanni Fadda

1

Istituto di Microbiologia, Universita` Cattolica del Sacro Cuore,1_{and Laboratorio di Microbiologia,} Ospedale Pediatrico “Bambino Gesu`,”2_{Rome, Italy}

Received 30 May 2005/Returned for modification 1 August 2005/Accepted 6 September 2005

We developed a new method based on the Nanochip microelectronic array technology for identification of various clinically relevant mycobacterial species. PCR-amplified rRNA genes obtained from 270 positive Mycobacteria Growth Indicator Tube cultures were successfully tested by hybridizing them with species-selective probes, and the results agreed with those of conventional identification methods. The system is rapid and accurate and opens new perspectives in clinical diagnostics.

Mycobacteria are slow-growing microorganisms causing a va-riety of infections that involve lung, skin, soft tissue, skeletal sys-tem, and lymph nodes in immunocompromised and immunocom-petent patients (5). Among them,Mycobacterium tuberculosisis the most common pathogen, causing 1.7 million deaths a year worldwide according to 2003 World Health Organization esti-mates (http://www.who.int/mediacenter/factsheets/fs104/en/print .html). Rapid and accurate identification of these microorganisms at the species level is important to establish a prompt and appro-priate therapy, considering that antibiotic treatment may vary according to the species encountered (2). Molecular assays that offer an alternative approach to time-consuming culture-based methods have recently been developed to improve identification of mycobacteria (14).

In this study, we developed a new method, based on micro-electronic chip array technology, to rapidly identify a variety of clinically relevant mycobacterial species by use of the Nano-chip Molecular Biology Workstation (Nanogen, Inc., San Di-ego, CA). The instrument uses a proprietary semiconductor microchip, which incorporates a 10-by-10 array of microelec-trodes 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 biotinyl-ated amplified DNA samples (4).

A 205-bp highly conserved region of theMycobacterium16S 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 oligonu-cleotide probes, seven of which were selective forM. tubercu-losiscomplex,M. avium,M. xenopi,M. gordonae,M. terrae,M. triplex, andM. 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).

Almost of these species are very closely related:M. intracel-lulareversusM. chimaera(17),M. chelonaeversusM. abscessus

andM. massiliense(1); andM. kansasiiversusM. gastri(8), or belong to the same taxonomic group:M. fortuitumversusM. porcinum,M. neworleansense, andM. boenickei(12) (Table 1). Probes were designed by the use of Vector NTI Advance 9.0 software (Invitrogen, Inc., Milan, Italy), taking into account their secondary structure, C⫹G content, length, and melting temperature. In addition, a general probe, specific for the genusMycobacterium, was included as a positive control. The specificity of these probes was assessed by hybridization with DNA from bacterial species belonging to the genera Coryne-bacterium, Nocardia, and Rhodococcus, which are closely re-lated to the genusMycobacterium. Probes were coupled on the basis of their melting temperature and synthesized so that the oligonucleotides of each pair contained either a 5⬘-Cy3 or 5⬘-Cy5 fluorophore (Table 1). The probe pairs were used in consecutive hybridization runs.

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␮l of 0.3 M NaOH for sample passive (not electronic) pre-treatment; another well contained 60␮l of 50 mM histidine (30␮l of 100 mM histidine plus 30␮l of high-performance liquid chro-matography-grade water) as the background control; the other wells each contained one sample to be processed (15␮l of PCR product, 15␮l of high-performance liquid chromatography-grade water, and 30␮l of 100 mM histidine); the last well contained 60

␮l of 0.1 M NaOH for sample passive denaturation. Then, the microtiter plate and Nanochip cartridge were put into the

* Corresponding author. Mailing address: Istituto di Microbiologia, Largo F. Vito, 1, 00168 Rome, Italy. Phone: 39 630154964. Fax: 39 63051152. E-mail: [email protected].

§ These authors contributed equally to this work.

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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␮S/cm were passed for load-ing. When addressing was completed, the cartridge was re-moved from the loader and processed for hybridization. Hybridization solutions were prepared by mixing in ice 2␮l each of Cy3- and Cy5-labeled probes (0.5␮M, final concen-tration) and 196␮l of high-salt buffer (50 mM sodium phos-phate, pH 7.4, and 500 mM NaCl). Before hybridization, the Nanochip cartridge was washed four times with 200 ␮l of high-salt buffer.

The hybridization solution (200␮l) was accurately pipet-ted 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 an-alyzed, 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 dis-criminate hybridization thermal stringency, we performed pre-liminary experiments using mycobacterial reference strainsM. tuberculosisATCC 27294 and ATCC 35801;M. aviumATCC 25291;M. chelonaeATCC 35752;M. fortuitumATCC 6841;M.

intracellulareATCC 13950;M. kansasiiATCC 12478;M. ma-rinum ATCC 927; M. xenopi ATCC 19250; M. smegmatis

ATCC 607; andM. terraeATCC 15755 and clinical isolates of

M. gordonae, M. triplex, andM. malmoense. Specific fluores-cence 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 de-tected as positive by the BACTEC Mycobacteria Growth In-dicator Tube 960 system (Becton Dickinson, Sparks, MD). Briefly, a 1-ml aliquot from each Mycobacteria Growth Indi-cator Tube culture was incubated at 100°C to inactivate myco-bacteria. Genomic DNA was then extracted as previously de-scribed (11). After amplification, the PCR products were purified and desalted by QIAquick PCR purification kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instruc-tions with the following modificainstruc-tions: 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␮l of high-performance liquid chromatography-grade water instead of buffer EB.

Of the 270 mycobacterial PCR products tested, the Nano-chip assay was able to identify 141M. tuberculosiscomplex, 36

[image:2.585.45.541.81.291.2]

M. avium, 24M. xenopi, 18M. gordonae, 5M. terrae, 1M. triplex, and 1 M. malmoense. The results were compared to those obtained with the matched isolates by conventional biochem-ical 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

TABLE 1. Oligonucleotides used in the study

Probe Target species Sequence (5⬘–3⬘) Probe pair

Tm(°C)

Probe pair designation

pGeneral3 Mycobacteriumspp. Cy5-CGGTATTAGACCC 38.5 Pair 1

pTub2 M. tuberculosiscomplex Cy3-AAGCGCTTTCC

pAvi1 M. avium Cy5-AGAAGACATGCG 37.4 Pair 2

pXen1 M. xenopi Cy3-CATGCGCAGAA

pInt2 M. intracellulare,M. chimaeraa _{Cy5-ACATGCGCCTA} _39.4 _{Pair 3}

pCheI3 M. chelonae,M. abscessus,M. massilienseb _{Cy3-ACCATGAAGTGTG}

pTripl2 M. triplex Cy5-GGACATGCGTC 37.7 Pair 4

pGord2 M. gordonae Cy3-CAGGACATGTGT

pMar2 M. marinum,M. ulcerans,M. liflandii,c_{M. pseudoshottsii}c _{Cy5-GAATCCCGTGG} _38.4 _{Pair 5}

pTer1 M. terrae Cy3-ACAGAACATGCAT

pKan3 M. kansasii,M. gastri Cy5-ATCCCGGTGTG 40.1 Pair 6

pFor3 M. fortuitum,M. fortuitumthird biovariant complexd _{Cy3-AGCGCGGTCCT}

pSme2 M. smegmatis,M. flavescens Cy5-TTCCCCTACCAG 37.5 Pair 7

pMal2 M. malmoense Cy3-GGGGTCCTATTC

a_{Included in the}_{M. avium}_complex,_{M. chimaera}_{has recently been proposed as a new species (17).}

b_{Having identical 16S rRNA gene hypervariable region sequences, like}_{M. abscessus}_,_{M. massiliense}_{can be discriminated from}_{M. chelonae}_{by biochemical tests}

(1, 8) or by PCR restriction analysis of thehsp65Telenti fragment (16).

c_{These species are pathogenic for animals (9, 10).}

d_{Among the species included in this group, only 16S rRNA gene hypervariable region sequences of}_{M. porcinum}_,_{M. neworleansense}_{, and}_{M. boenickei}_{matched probe}

pFor3 (12).

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[image:3.585.52.533.53.698.2]

FIG. 1. Examples of Nanochip hybridization results for three sequence-selective fluorescently labeled probe pairs against the 13 mycobacterial species tested. Graphs show fluorescence signal values obtained in each of the three experiments. Histidine was used as a negative control.

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not shown). Among the remaining 44 PCR products, 12 hy-bridized 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 iden-tified as M. intracellulare (12 isolates), M. chelonae (11 iso-lates), M. fortuitum (9 isolates), M. kansasii (6 isolates), M. smegmatis(5 isolates), andM. marinum(1 isolate), using bio-chemical 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.

Microelectronic chips or arrays are innovative methods for DNA hybridization analysis of point mutations, single-nucle-otide polymorphism, short tandem repeats, and gene expres-sion. Electronic molecule addressing and hybridization are car-ried out on these devices, where the electric field and the use of low-conductance buffers (histidine, etc.) greatly accelerate the hybridization reactions on the selected test sites (4, 15). The feasibility of these devices, initially developed for geno-typing purposes (6, 13, 20), makes them adaptable for other clinically important applications (3, 19).

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 hybrid-ization assay in which multiple species-selective probes, matching a highly polymorphic region of the Mycobacterium16S 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 Mycobacte-rium, since it is possible to expand the spectrum of the mycobac-terial 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. tuberculosiscomplex and

M. avium) or Mycobacteriumspecies rarely causing human dis-ease, such asM. 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 combina-tion with PCR deteccombina-tion methods. A similar approach has been ideated by Westin et al. (20), in which multiplex strand dis-placement 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 suit-able for routine use in mycobacteriology laboratories that pro-cess a wide number of clinical specimens.

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