Abstract
A PCR-microarray assay was developed in which PCR primers and hybridization probes were designed for the 16S rRNA genes of 16 clinically relevant mycobacteria and for IS6110 of Mycobacterium tuberculosis. The assay, based on a multiplex PCR followed by hybridization with oligonucleotide probes, was tested against 16 Mycobacterium species and 70 clinical samples.
TEXT
Tuberculosis, which is caused by members of the Mycobacterium tuberculosis complex, is one of the most common human infectious diseases (13, 18). Infections caused by nontuberculous mycobacteria (NTM) are of growing clinical interest, especially in individuals with reduced or compromised immune function. NTM infect many sites within the body but primarily cause pulmonary diseases, cervical lymphadenopathies, and localized skin and soft tissue lesions (4, 7, 17). Prompt and efficient identification of mycobacteria at the species level is extremely important not only for correct management of the diseases caused by NTM but also because differentiation of M. tuberculosis from NTM is important for infection control and choice of antitubercular therapy (12). Here we describe novel sets of PCR primers and hybridization probes to be used for development of PCR-microarray assays for detection and identification of clinically relevant mycobacteria.
PCR primers and hybridization probes.
Primers and probes were designed for IS6110 of M. tuberculosis and the 16S rRNA genes of 11 mycobacterial species (see Table S1 in the supplemental material), which were aligned using ClustalW (16). PCR primers MYC1p and MYC2p were designed for conserved regions of the 16S rRNA gene to amplify a 235-bp fragment containing hypervariable region A. PCR primers MYC13p and MYC14p were designed for orfB of insertion sequence IS6110 (GenBank accession number X17348), yielding an amplicon 122 bp in size. Species-specific probes were designed on the segment of hypervariable region A of the 16S rRNA genes spanning nucleotides (nt) 140 to 178, whereas genus-specific probe MYC10a matched the adjacent segment spanning nt 100 to 137. Probes MYC15a and MYC16a, specific for the M. tuberculosis complex, were also designed for the segment of orfB of insertion sequence IS6110 spanning nt 795 to 869. For the prediction of thermodynamic stability of all probes, the nearest-neighbor method (15) was used. Probes were checked using BLAST (1) to avoid unwanted cross-homology to nontargeted sequences.
Mycobacterial DNA microarray.
Epoxy-modified glass slides (Genetix) and a manual microarray system (V&P Scientific) were used for printing. Oligonucleotide probes with a 5′ C6 amino linker (MWG Biotech) were spotted in three replicates. Procedures for slide printing, labeling of PCR products with Cy5, hybridization, and data analysis were essentially as described previously (3). Slides were read using an Agilent GMS 428 scanner (Genetics MicroSystems), evaluating the absolute signal from each spot. Probes were between 19 and 27 nucleotides, with a melting temperature (Tm) of 56°C to 65°C. The array contained 3 probes specific for the M. tuberculosis complex, MYC15a and MYC16a designed on IS6110, and MYC3a designed on 16S rRNA genes. Four different species-specific probes were designed on 16S rRNA genes to identify M. avium (MYC4a), M. intracellulare (MYC5a), M. xenopi (MYC17a), and M. haemophilum (MYC19a), whereas probe MYC6a was used for identification of M. kansasii, as well as M. simiae and M. scrofulaceum, since in these 3 species the hypervariable region A of the 16S rRNA genes is identical. Also, the rapidly growing M. abscessus and M. chelonae species have an identical hypervariable region A and could be identified by a single probe (MYC8a). Probe MYC31a was specific for M. fortuitum, by far the most clinically relevant species of the M. fortuitum group (2). Multiple attempts to design probes for M. malmoense/szulgai, M. marinum/genavense, and Mycobacterium ulcerans were unsuccessful due to generation of unspecific positive signals (data not shown). For this reason, detection of these mycobacterial species relied on the genus-specific MYC10a probe, followed by identification by DNA sequencing.
Specificity of the multiplex PCR.
In silico analysis showed that the MYC1p/MYC2p primer pair had the potential to amplify (i) all the 16S rRNA genes of the genus Mycobacterium present in GenBank and (ii) the 16S rRNA genes of some species belonging to phylogenetically related genera, such as Brevibacterium, Corynebacterium, and Nocardia, whereas MYC13p/MYC14p were specific for the M. tuberculosis complex. Primer pairs MYC1p/MYC2p and MYC13p/MYC14p combined in a multiplex PCR were tested for their capacity to amplify the genomic DNA from the 21 strains described in Table 1. Multiplex PCR results (data not shown) confirmed the in silico predictions.
Table 1.
Complex and/or straina | Sourceb |
---|---|
Mycobacterium tuberculosis complex | |
Mycobacterium tuberculosis H37 Rv | ISS (Rome) |
Mycobacterium bovis BCG | Institut Pasteur (Paris) |
Mycobacterium africanum Myc83 | Clinical isolate (Florence) |
Mycobacterium avium complex | |
Mycobacterium intracellulare Myc48 | Clinical isolate (Florence) |
Mycobacterium intracellulare 551 | Clinical isolate (Naples) |
Mycobacterium avium 15445 | Clinical isolate (Siena) |
Mycobacterium kansasii 97758 | Clinical isolate (Siena) |
Mycobacterium xenopi 44382 | Clinical isolate (Siena) |
Mycobacterium chelonae A2 | Clinical isolate (Naples) |
Mycobacterium fortuitum Myc56 | Clinical isolate (Florence) |
Mycobacterium haemophilum 44634 | DSMZ |
Mycobacterium fortuitum/peregrinum complex C6 | Clinical isolate (Naples) |
Mycobacterium marinum Myc42 | Clinical isolate (Naples) |
Mycobacterium malmoense Myc39 | Clinical isolate (Florence) |
Mycobacterium ulcerans 19423 | ATCC |
Mycobacterium gordonae 14832 | Clinical isolate (Siena) |
Mycobacterium szulgai A4 | Clinical isolate (Naples) |
Corynebacterium glutamicum 44475 | DSMZ |
Nocardia asteroides 43255 | DSMZ |
Brevibacterium ravenspurgense 21258 | DSMZ |
Escherichia coli 25922 | ATCC |
Identifications of all strains used in this study were confirmed by sequencing of the 16S rRNA gene.
Clinical strains were kindly provided by Lanfranco Fattorini (ISS, Rome, Italy), Brigitte Gicquel (Institut Pasteur, Paris, France), Giulia Santoro (Ospedale Monaldi, Naples, Italy), and Enrico Tortoli (Laboratorio di Microbiologia e Virologia, AOU Careggi, Florence, Italy). Abbreviations: ATCC, American Type Culture Collection (Manassas, VA); DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany).
Validation of the primer-probe sets.
Primer-probe combinations were tested using genomic DNA of control strains (Table 2). All mycobacterial species were detected by probe MYC10a. As predicted by in silico analysis, the DNA of B. ravenspurgense, C. glutamicum, and N. asteroides also reacted, albeit weakly, with MYC10a. Each of the 3 probes specific for the M. tuberculosis complex reacted with M. tuberculosis, M. bovis, and M. africanum, whereas each of the 7 species-specific probes reacted only with one Mycobacterium species. Primer-probe combinations were also tested on 70 nonconsecutive clinical samples from various body sites, including 41 from the respiratory tract (Table 3). Clinical samples were processed according to standard procedures (8, 9, 11) at the Bacteriology Unit of the Siena University Hospital (UOC Batteriologia, AOUS). Each sample was analyzed for the presence of mycobacteria by (i) smear microscopy with the Ziehl-Neelsen method, (ii) culture in liquid medium (Bactec MIGIT 960), and (iii) real-time PCR (home-brew assay). Identification of all mycobacterial isolates was confirmed by sequencing of the 16S rRNA genes. Results of the PCR-microarray (i) were in good agreement with results obtained by standard laboratory diagnosis, (ii) were more reliable than microscopy alone, and (iii) confirmed the presence of M. tuberculosis, M. avium, M. intracellulare, M. xenopi, and M. fortuitum in positive-control samples.
Table 2.
Strain | MYC10a | MYC15ab | MYC16ab | MYC3a | MYC4a | MYC5a | MYC6a | MYC8a | MYC17a | MYC19a | MYC31a |
---|---|---|---|---|---|---|---|---|---|---|---|
Mycobacterium tuberculosis H37 Rv | +++ | +++ | +++ | +++ | |||||||
Mycobacterium africanum Myc83 | +++ | +++ | +++ | +++ | |||||||
Mycobacterium bovis BCG | +++ | +++ | +++ | +++ | |||||||
Mycobacterium avium 15445 | +++ | + | |||||||||
Mycobacterium intracellulare 551 | +++ | +++ | |||||||||
Mycobacterium kansasii 97758 | +++ | +++ | |||||||||
Mycobacterium xenopi 44382 | +++ | +++ | |||||||||
Mycobacterium haemophilum 44634 | +++ | +++ | |||||||||
Mycobacterium fortuitum/peregrinum complex C6 | +++ | +++ | |||||||||
Mycobacterium marinum Myc42 | +++ | ||||||||||
Mycobacterium malmoense Myc39 | +++ | ||||||||||
Mycobacterium ulcerans 19423 | +++ | ||||||||||
Mycobacterium gordonae 14832 | +++ | ||||||||||
Mycobacterium szulgai A4 | +++ | ||||||||||
Mycobacterium chelonae A2 | +++ | +++ | |||||||||
Mycobacterium fortuitum Myc56 | +++ | +++ | |||||||||
Escherichia coli 25922 | |||||||||||
Brevibacterium ravenspurgense 21258 | + | ||||||||||
Nocardia asteroides 43255 | + | ||||||||||
Corynebacterium glutamicum 44475 | + |
All strains were tested with all probes. For clarity, only positive results are reported here, whereas negative strain-probe combination results are not shown. +++, very strong signal; ++, strong signal; +, positive signal.
Probes MYC15a and MYC16a target IS6110.
Table 3.
Clinical sample source | Positive samples |
No. of negative samples | Total no. of samples | ||
---|---|---|---|---|---|
n | Species detected | No. of smear-positive samples | |||
Sputum | 6 | M. tuberculosis complex | 3 | 15 | 25 |
2 | M. intracellulare | 1 | |||
1 | M. avium | 0 | |||
1 | M. xenopi | 1 | |||
Bronchoalveolar lavage | 1 | M. tuberculosis complex | 1 | 8 | 9 |
Bronchial aspirate | 2 | M. tuberculosis complex | 2 | 5 | 7 |
Bone biopsy | 1 | M. tuberculosis complex | 1 | 0 | 1 |
M. avium | 0 | ||||
Fistulas | 1 | M. tuberculosis complex | 1 | 6 | 8 |
1 | M. fortuitum | 0 | |||
Urine | 11 | 11 | |||
Blood | 2 | 2 | |||
Total | 18 | 52 | 70 |
Many different assays have been developed for the molecular diagnosis of mycobacterial infections (5, 6, 10, 14). This work provides proof of concept that oligonucleotide probes on a microarray can discriminate amplicons of the 16S rRNA genes of different mycobacterial species, and it validates IS6110 and mycobacterial 16S rRNA genes as targets for DNA-based detection and identification of M. tuberculosis complex and NTM by the PCR-microarray approach. The primer-probe combinations produced in this study could be the basis for development of new automated PCR-microarray assays for laboratory diagnosis of mycobacterial infections.
Supplementary Material
ACKNOWLEDGMENT
This study was supported by FP7 EU grant TM-REST (HEALTH-F3-2008-202145).
Footnotes
Published ahead of print 12 September 2012
Supplemental material for this article may be found at http://jcm.asm.org/.
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