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J Mol Diagn. Jan 2009; 11(1): 42–48.
PMCID: PMC2607564

Multi-Probe Real-Time PCR Identification of Common Mycobacterium Species in Blood Culture Broth

Abstract

Mycobacterium tuberculosis complex, M. avium, and M. intracellulare are the most common causes of systemic bacterial infection in AIDS patients. To identify these mycobacterial isolates in primary blood culture broths, we developed a multiple hybridization probe-based real-time PCR assay using the LightCycler system. The primers were designed to amplify a 320-bp fragment of Mycobacterium 16S rRNA genes. Reaction specificity was evaluated using PCR amplification curves along with specific melting temperatures of probes on DNA extracted from 13 Mycobacterium species. In this study, results showed 100% accuracy for the selected bacterial panel. Detection limits were 350, 600, and 650 colony-forming unit (CFU)/ml blood culture broths for M. tuberculosis complex, M. avium, and M. intracellulare, respectively (1 to 2 CFU/reaction). To evaluate clinical applicability, 341 acid-fast bacilli in blood culture broths were analyzed. In total, 327 (96%) were positively identified: 54.5% M. tuberculosis complex, 37.5% M. avium, and 3.8% M. intracellulare. Results can be available within 3 hours of receiving a broth sample, which makes this rapid and simple assay an attractive diagnostic tool for clinical use.

Mycobacterium tuberculosis and M. avium-intracellulare complex are now the most common causes of systemic bacterial infection in AIDS patients. Automated broth culture rapidly recovers mycobacteria from infected blood. Molecular methods offer advantages over conventional methods such as improved accuracy in identification of Mycobacterium species and shorter turnaround time. Currently, molecular methods are widely used in many microbiological laboratories for pathogenic identification. There are commercial kits and in-house assays based on DNA hybridization probes for detecting mycobaterial DNA or RNA with or without nucleic acid amplification. Although these assays have been useful for identifying mycobacteria, most were applicable only for detecting one species per reaction and not recommended for use on blood-contaminated samples.1,2,3,4,5,6

The 16S rRNA gene is highly conserved and contains genus- and species-specific sequence variations useful for identifying different mycobacterial species. Among mycobacterial isolates from the blood of 385 AIDS patients at Siriraj Hospital and Bamrasnaradura Institute in 2001, 148 (38%) were M. tuberculosis and the rest were non-tuberculosis mycobacteria (NTM).7 Additionally, we sequenced the 16S rRNA gene from NTM isolates, which revealed 180 (46%) M. avium, 39 (10%) M. intracellulare, 13 (3.3%) M. simiae, 11 (2.8%) M. sherrisii, 3 (0.8%) M. kansasii, and 1 (0.3%) M. chelonae/abscessus cases. Analysis of 39 M. intracellulare strains sequenced using hypervariable region A yielded 3 sequivars (distinct nucleic acid sequences), of which 18 (50%), 15 (38.5%), and 6 (15.5%) were identical to strain sequence database accession numbers AJ536036, AY652954, and AY652960, respectively. Both M. tuberculosis and M. avium did not contain sequivars in this region.

We designed a multihybridization probe real-time PCR assay targeting the 16S rRNA gene to facilitate identification of M. tuberculosis complex, M. avium and M. intracellulare in primary blood culture broth using the LightCycler PCR system (Roche Applied Science, Mannheim, Germany). This system acquired amplification fluorescence along with melting temperature (Tm) curves for the probe-PCR product complex. Tm analysis not only validates the fluorescence data but strengthens the assay especially in the case of M. intracellulare sequivars. Additionally, the assays have been validated on routine clinical blood culture broths.

Materials and Methods

Mycobacterial Strains and DNA Extraction

Mycobacterial strains included in this studies were either ATCC strains or clinical isolates (S) identified by 16S rRNA sequencing. These included M. tuberculosis (ATCC 27294, H37Ra, H37Rv), M. bovis BCG Tokyo, M. bovis (ATCC 19210), M. avium (ATCC 700898), M. intracellulare (ATCC 13950 and 8 clinical isolates; S6, S9, S33, S34, S58, S77, S90, and S105), M. kansasii (ATCC 12478), M. scrofulaceum (ATCC 19981), M. terrae (ATCC 15750), M. simiae (ATCC 25275), M. szulgai (ATCC 35799), M. xenopi (ATCC19250), M. sherrisii (5 clinical isolates; S24, S53, S75, S81, and S155), M. chelonae (clinical isolate S12), and M. abscessus (clinical isolate S16). They were cultured on Löwenstein- Jensen (L-J) medium. Cell suspensions were prepared in PBS. DNA was extracted using High Pure PCR Template Preparation Kit (Roche Molecular Biochemicals, Mannheim, Germany) according to manufacturer's instructions. Extracted DNA was stored at −20°C.

Blood Culture Broth Samples and DNA Extraction

A total of 341 individual routine acid-fast bacilli (AFB)-positive blood culture broths, which were either in 129 BACTEC Myco/F-lytic broths (Becton Dickinson, Sparks, MD) or 212 BacT/ALERT MB broths (bioMérieux, Durham, NC) were obtained from the Mycobacteria Laboratory, Department of Microbiology, Faculty of Medicine Siriraj Hospital and the Microbiology Laboratory, Bamrasnaradura Institute, Department of Disease Control, Ministry of Public Health, Thailand. Once automated machines confirmed growth, broth aliquots were used to inoculate a L-J solid medium and to prepare a smear for Ziehl-Neelsen staining. The remaining portion was used for DNA extraction. L-J mediums were incubated at 37°C and inspected weekly for growth. Conventional biochemical tests identified the acid-fast bacilli from L-J mediums for M. tuberculosis. PCR–restriction fragment-length polymorphism of the hsp65 gene detected individual NTM species. Lastly, unidentifiable bacilli were sequenced.8,9

DNA was extracted from 200 μl of a blood culture broth sample using the High Pure PCR Template Preparation Kit according to the manufacturer's instructions with modifications. Briefly, the sample was centrifuged and the pellet was suspended in 200 μl of PBS. Subsequently, lysis buffer with proteinase K was added. The sample underwent five freeze thaw cycles (boiled for 5 minutes, vortexed, and incubated for 2 minutes in a −70°C ethanol bath) followed with a final 5 minutes boiling step. DNA was eluted with 100 μl pre-warmed elution buffer. The rest of the steps were according to manufacturer's instructions. The elution was stored at −20°C.

Primers and Probes

The primer and hybridization probe designs were based on alignments of the 16S rRNA genes of Mycobacterium spp. as shown in Figure 1, which included sequences from various mycobacterial species from the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov) and the clinical isolates: M. tuberculosis (BX842576), M. avium (FE059905), M. intracellulare 1 (AJ536036), M. intracellulare 2 (AY652954), M. intracellulare 3 (AY652960), M. simiae (X52931), M. scrofulaceum (AF480604), M. sherisii (AY353699), M. kansasii (X15916), M. gastri (AF080602), M. genavens (X60070), M. hemophilum (U06638), M. marinum (AY513243), M. ulcerans (AF547979), M. microti (AF480584), M. szulgai (X52926), M. triplex (U57632), M. fortuitum (AY457068), M. chelonae (AF547909), M. abscessus (DQ866776), M. godonae (EF428556), and M. xenopi (AF547982). The designations in parentheses are GenBank accession numbers.

Figure 1
Alignment of 16S rRNA gene fragments of various mycobacterial species from the NCBI database and the clinical isolates. Nucleotides corresponding to the primers are in bolds. Sequences for the upstream and downstream hybridization probes are in shadows. ...

The LightCycler Probe Design software 2.0 from Roche Applied Science was used to design optimal primers and probes for the system. The PCR assay included multiple species-specific probes to facilitate species identification by means of amplification curve detection and specific melting peaks. The sequences of the corresponding species and the sequences of the primers and probes are shown in Figure 1 and Table 1. A BLAST search (http://www.ncbi.nlm.nih.gov/BLAST/) was performed to check the specificity of primers and probes to the target DNA. The sequences of the forward and reverse primers recognized a ~320-bp region of the 16S rDNA. Sequence specific hybridization probes were designed Tb-fl/705-T and M-fl/640-A for M. tuberculosis complex and M. avium, respectively. Probe designated for M. intracellulare, 610-I, had A/G wobble at position 15 to perfectly match sequivars 1 and 3 and only one mismatch with sequivar 2 as shown in Figure 1 and Table 1. The upstream oligonucleotide probes (Tb-fl and M-fl) were labeled with fluorescein at the 3′ terminus. The downstream oligonucleotide probes (705-T, 640-A, and 610-I) were labeled with different shades of LightCycler Red at the 5′ terminus and phosphorylated at the 3′ terminus (TIBMOLBIOL, Berlin, Germany).

Table 1
The Primers and Hybridization Probes Sequences for the Real-Time PCR Assays

Real-Time PCR Assay

The real-time PCR mixture contained 4 μl of commercial ready-to-use reaction mix for PCR (LightCycler FastStart DNA MasterPLUS Hybridization Probes kit; Roche Molecular Biochemicals, Mannheim, Germany), 0.5 μmol/L of each primer, 0.1 μmol/L of each probe and 2 μl of DNA extract with nuclease-free water added to a final volume of 20 μl. Each set of experiments included negative and positive controls. Nuclease-free water replaced DNA template for negative controls. Positive controls were prepared by adding 2 μl of template DNA from a M. tuberculosis H37Rv and M. avium ATCC 700898 mixture or M. intracellulare ATCC 13950. Reactions were performed on the LightCycler Real-Time PCR system, version 2.0. The cycling conditions included initial FastStart TaqDNA polymerase activation phase at 95°C for 10 minutes followed by a 40-step amplification procedure delineated into 10 seconds at 95°C, 20 seconds at 50°C, and 13 seconds at 72°C. Subsequently, melting analysis of the probe-PCR product duplex consisted of 95°C for 30 seconds followed by cooling to 40°C for 30 seconds before the temperature was raised to 75°C at a rate of 0.1°C/s with continuous fluorescence acquisition. The final cooling step was 40°C for 10 seconds. Downstream oligonucleotide probes were labeled with varying LightCycler Red dye and for that reason, amplified products were measured through different channel modes of emitted energy such as 640 nm, 705 nm, and 610 nm. Tm was recorded and the presence of ~320-bp amplification products was verified by 2% agarose gel electrophoresis.

Determination of Sensitivity

To determine detection limits of our assay, 2 ml blood culture negative broth or EDTA-blood of health volunteers were spiked with M. tuberculosis H37Rv, M. avium ATCC 700898, or M. intracellulare ATCC 13950 to a final concentration of 106 CFU/ml. These samples were serially diluted tenfold down to 102 CFU/ml and performed in duplicates. DNA was extracted using 200 μl of each sample.

Direct Identification of Mycobacteria in Positive Blood Culture Broth

Initially, the positive blood culture broth was screened for the presence of M. tuberculosis or M. avium. M. tuberculosis complex probes (Tb-fl and 705-Tb) and M. avium probes (M-fl and 640-A) were added in one reaction mixture. Fluorescence signals from specific probes for M. tuberculosis complex and M. avium were detected in channels 705 and 640, respectively. Amplification curves in the 705 channel indicated positive for M. tuberculosis complex while amplification in the 640 channel denoted positive for the M. avium species. The Tm curves were simultaneously analyzed to confirm findings. In cases that were negative for these species, the samples were tested for presence of M. intracellulare using a second reaction tube containing M-fl and 610-I probes. Both amplification in channel 610 and Tm peaks within the range 57°C to 67°C must be observed for positive identification of M. intracellulare.

Results

Specificity of Probes and Melting Temperature Values

The BLAST searches revealed the M. tuberculosis probes, TB-fl and 705-T, were 100% sequence specific to all M. tuberculosis complex sequences in GenBank database, which includes 11 M. tuberculosis, 7 M. bovis, 3 M. africanum, 2 M. microti, 1 M. canettii, and 1 M. pinnipedii. The M. avium probes, M-fl and 640-T, were 100% specific to all 39 M. avium strains in the database. The blast search with the M. intracellulare probes, M-fl and 610-I, revealed 100% specificity to M. intracellulare as well as M. chimaera. This is a novel species in the Mycobacterium avium complex with 16S rRNA sequence similar to M. intracellulare 3 or ATCC 13950 as shown in Figure 1.10

The specificity of all primers and probes was tested on DNA from 12 mycobacterial species shown in Table 2. We confirmed ~320 bp PCR products on gel electrophoresis (data not shown). Fluorescence signals were detected in respective channel, which depended on the shade of LightCycler Red dye on the probe. Tm measurements were collected ranging from 40°C to 75°C. At temperatures lower than expected probe-PCR product complex Tm, melting peaks of non-specific products were observed in some cases. In positive samples, distinct secondary peaks occurred, which were more pronounced in samples with high concentrations of specific PCR products (Table 2 and Figure 2).

Figure 2
Real-time PCR amplification curves and the melting curves at channel 705 (A), 640 (B), and 610 (C) detections: 1. M. tuberculosis ATCC 27294; 2. M. avium ATCC 700898; 3. M. intracellulare ATCC 13950; 4. M. kansasii ATCC 12478; 5. M. scrofulaceum ATCC19981; ...
Table 2
Mycobacterium Species Tested by the Real-Time PCR Procedure

For assays using probes specific for the M. tuberculosis complex or M. avium, only the amplification products of M. tuberculosis complex or M. avium were detected in 705 or 640 channels, respectively. The target-specific Tms were 57.4°C/65.2°C (corresponds to primary/secondary peaks) for M. tuberculosis complex and 56°C/58.5°C for M. avium shown on Table 2. No cross-reactivity was observed with other mycobacterial species. In some cases where non-specific melting peaks were observed, the Tms were at least 8°C to 10°C lower as illustrated in Figure 2, A and B.

M-fl and 610-I probes detected all 3 M. intracellulare sequivars: 1) M. intracellulare 1 (S6, S33, S90) 2) M. intracellulare 2 (S9, S58, S77) 3) M. intracellulare 3 (ATCC 13950, S34, S105). The corresponding Tm values were 57°C/65.5°C, 57.5°C, and 60.7°C/67.3°C, respectively (Table 2). These probes showed cross-reactivity with M. avium at Tm value of 57.9°C. Positive signals were present in M. simiae, M. kansasii, M. scrofulaceum, M. szulgi and M. xenopi with non-specific melting peaks between 51°C to 54°C (Figure 2C).

Sensitivity

Dilutions of mycobacterial culture in broth samples were prepared to determine the lowest mycobacterial load for a positive signal. Detection limits for M. tuberculosis, M. avium, and M. intracellulare were 350, 600 and 650 CFU/ml (0.7, 1.2 and 1.3 CFU per reaction), respectively. The assay sensitivity in mycobactria spiked blood samples for M. tuberculosis and M. avium was 800 and 2000 CFU/ml (1.6 and 4 CFU per reaction), respectively. Analytical sensitivity was not compromised when the M. tuberculosis and M. avium probes were combined in one reaction mixture.

Clinical Application in Positive Blood Culture Broth

Multiprobe real-time PCR assay was used to identify 341 positive clinical specimens from the automated blood broth system. Results from biochemical and PCR assays (amplification and Tm values) are presented in Table 3 and Figure 3. Initial reaction correctly identified M. tuberculosis complex (n = 186, 54.5%) and M. avium (n = 128, 37.5%) positive samples, which matched corresponding results from conventional identification assays. The negative samples were further investigated for M. intracellulare in a second reaction. Thirteen samples were positive for M. intracellulare where 8 samples with corresponding Tm values of M. intracellare sequivar 1, 3 of M. intracellare sequivar 2, and 2 of M. intracellare sequivar 3 were observed. One sample showed weak positive for M. intracellulare with Tm peak at 57.7°C but it's Tms with M. tuberculosis complex and M. avium probes were not in ranges of M. intracellulare. This sample was identified by sequencing as M. hemophilum. NTM isolates that were unidentifiable demonstrated lower Tms than expected Tms. These samples were identified by sequencing as M. kansasii (n = 5, 1.5%), M. sherrisii (n = 4, 1.2%), M. simiae (n = 3, 0.9%), and M. triplex (n = 1, 0.5%).

Figure 3
Real-time PCR amplification curves and melting curves channel 705-M. tuberculosis (A) and channel 640- M. avium (B) in the same reaction tube. 1. M. tuberculosis positive control; 2. M. avium positive control; 3. through 13., samples; and 14. negative ...
Table 3
Mycobacterial Samples in the Primary Blood Culture Broth Identified by Real-Time PCR Compared with the Results of Culture Analysis

However, the 610-I probe designed for M. intracellulare sequivar 3 or ATCC 13950 had cross similarity with the recently published species, M. chimaera.9 We performed hypervariable 16S rRNA gene sequencing on clinical isolates S34 and S105, which were positive for M. intracellare 3 to establish definitive identifications. Sequences were found to be identical to M. intracellulare (GenBank accession number AJ356036), which consisted of C at position 459 in hypervariable B region and not M. chimaera (T at position 459).

Discussion

Rapid identification of disseminated mycobacterial infections in AIDS patients is important for early detection and treatment to improve patient outcome. A development of a high-throughput, sensitive diagnostic test performed on primary blood broth is crucial. Therefore, we sought to develop rapid molecular diagnostic assay, which is simple and specific for the detection of common pathogenic mycobacteria in primary blood culture broth.

Traditional identification methods are time-consuming from slow growing subculture colonies on solid media to using biochemical tests. Recently, molecular techniques such as nucleic acid probes, species-specific PCR, reverse hybridization, and 16S rRNA sequencing have been introduced that offer rapid and highly sensitive assays.1,2,5,6,11,12,13 Many of these assays require up-front sample processing and are not tested directly on blood or blood culture broths. Commercial kits are also available for species identification but some kits are expensive and presence of blood in samples may interfere or compromise efficacy.14,15,16 One such kit is the commercial acridinium-ester-labeled probe assay, which is easy to use and highly specific for the M. tuberculosis complex, but the probe has a high detection limit (approximately 106 CFU/ml) and requires a separate reaction tube for M. avium-intracellulare complex identification.17 Although multispecies identification is possible with the multiprobe reverse hybridization commercial assay, the procedure is laborious with high risk for PCR amplicon contamination, which is not ideal for a clinical laboratory.

The LightCycler system has been developed for rapid identification of M. tuberculosis complex and M. avium but not for M. intracellulare.1 However, the assay used one reaction per species and did not apply to use on blood containing samples. This system has also been developed to differentiate M. tuberculosis and NTM by using probe-PCR product Tm ranges in a single probe set.2 Nevertheless, the assay could not identify the species of NTM because of the wide variety of NTM with Tm overlapping. The system has also been developed for rapid detection of rifampin and isoniazid resistance in M. tuberculosis.3

We designed the PCR assay on a LightCycler for several reasons. It is one of the fastest real-time PCR systems. Additionally, the ability to collect data through multiple channels allows for discrimination of various probe-amplicon complexes and reports their Tms. Two sets of hybridization probes were included in a single reaction tube to provide an efficient, simultaneous differentiation of M. tuberculosis complex and M. avium in one LightCycler run. When the samples were negative for M. tuberculosis complex and M. avium, they were subsequently tested for M. intracellulare. Tm analysis was essential in identifying not only the mycobacterial species but to differentiate the three sequivars of M. intracellulare.

Modifications were made to the DNA extraction protocol with the High Pure PCR Template Preparation Kit. We determined that following this modified process is imperative in successfully detecting mycobacterial DNA from samples with blood. The most significant change was the use of mechanical force to lyse the cells via the freeze-thaw cycles. In this study, detection limits were 1 to 2 CFU per test, and 2 to 4 CFU per test, for blood culture broth and whole blood, respectively. This highly sensitive assay is a direct result of the high quality DNA template. Bacterial concentration in the blood circulation is inconsistent, thus the exact blood volume necessary for direct testing on blood is difficult to ascertain.

Our multiprobe real-time PCR assay was successful in identifying samples from primary blood broths without reduction in sensitivity. The probes specific for M. tuberculosis complex and M. avium showed 100% accuracy in the first reaction. Although M. intracellulare probe in the second reaction could not differentiate between M. intracellulare 2 sequence and M. avium (Tms in 610 was ~57°C), since M. avium is confirmed in the first run positive samples were not included in the second reaction pool. Even if Tm of M. hemophilum was in the same range of M. intracellulare, we could discriminate by its Tms in 705 and 640 that were higher than M. intracellulare. In total, 96% of the samples were correctly identified making this simple PCR assay a robust, highly specific diagnostic tool in differentiating commonly isolated mycobacteria from blood cultures of AIDS patients.

In addition, our method provides a cost-effective and efficient alternative to conventional techniques and commercial kits currently in use. The ability to work on primary blood culture broth and blood decreases the up-front sample preparation time. On receiving a patient sample, it takes approximately 3 hours (DNA extraction 1 hour, reaction preparation 20 minutes, and LightCycler 50 minutes) to identify these three common species of mycobacteria by this semiautomated system. The ability to modify this assay such as designing new detection probes for other species is paramount to expand its use to account for endemic diseases and new outbreaks in various clinical laboratories.

This assay will be evaluated to detect mycobacterial pathogen directly on patient specimens such as blood, sputum, cerebrospinal fluid and stool. In order to identify active disease, targeting RNA that degrades rapidly compared to DNA will help to evaluate current disease activity in a patient even after drug therapy.

Within the limitations of this study, it may be concluded that this multiprobe real-time PCR assay is an accurate, rapid and simple method for identifying the most common mycobacteria species directly in positive blood culture broths. This assay is useful for the diagnosis of M. tuberculosis and M. avium-intracellulare complex from AIDS disseminated mycobacteriosis but alternate molecular techniques such as DNA sequencing should be used to identify other infrequent species.

Acknowledgements

We thank Dr. Mami Taniuchi, Professor Dr. John J Boitano and Associate Professor Dr. Eric Houpt for their advice and English editing.

Footnotes

Supported by Siiraj Grant Research and Development and Medical Education, Faculty of Medicine Siriraj Hospital, Mahidol University.

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