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J Clin Microbiol. Aug 2010; 48(8): 2830–2835.
Published online Jun 16, 2010. doi:  10.1128/JCM.00185-10
PMCID: PMC2916577

Specific Detection of Unamplified Mycobacterial DNA by Use of Fluorescent Semiconductor Quantum Dots and Magnetic Beads[down-pointing small open triangle]

Abstract

Here we present the development of a specific DNA detection method using fluorescent semiconductor quantum dots (QDs) and magnetic beads (MBs) for fast detection of Mycobacterium spp., dispensing with the need for DNA amplification. Two biotinylated oligonucleotide probes were used to recognize and detect specific complementary mycobacterial target DNA through a sandwich hybridization reaction. Cadmium selenite QDs conjugated with streptavidin and species-specific probes were used to produce a fluorescent signal. MBs conjugated with streptavidin and a genus-specific probe were used to isolate and concentrate the DNA targets. The application of the proposed method to isolated bacteria produced the expected result in all cases. The minimum detection limit of the assay was defined as 12.5 ng of DNA diluted in a sample volume of 20 μl. In order to obtain an indication of the method's performance with clinical samples, we applied the optimized assay to the detection of Mycobacterium tuberculosis in DNA isolated from bronchoalveolar lavage specimens from patients with tuberculosis and Mycobacterium avium subsp. paratuberculosis in DNA isolated from feces and paraffin-embedded tissues in comparison with culture, Ziehl-Neelsen staining, and real-time PCR. The concordance of these methods compared to the proposed method with regard to positive and negative samples varied between 53.84% and 87.23% and between 84.61% and 100%, respectively. The overall accuracy of the QD assay compared to real-time PCR was 70 to 90% depending on the type of clinical material. The proposed diagnostic assay offers a simple, rapid, specific, and cost-effective method for direct detection and identification of mycobacterial DNA in clinical samples.

Mycobacterial infections have a high economic human and animal health impact. Diagnostic investigation of mycobacterial infections is hampered by the difficulty in detecting, in a specific manner, low populations of mycobacteria or the immunity markers associated with the infections they cause. The conventional methodology, which includes specimen treatment, microscopic examination for acid-fast bacilli, culture, and classification with biochemical tests, is laborious and time-consuming. Over the last few years, new molecular methods have been introduced, including PCR-restriction fragment length polymorphism (RFLP), real-time PCR, DNA sequencing, and DNA strip technology, leading to a considerable improvement in both the speed and accuracy of mycobacterial identification (10). All methods have advantages and limitations; in general, those with a high specificity and a low minimum detection limit are expensive and complex to perform (2, 11, 12, 14). The development of a sensitive and specific diagnostic assay that can be applied directly to clinical samples without the need for high-cost dedicated equipment will definitely improve diagnostic investigation of mycobacterial infections.

In recent years, many nanosystems have been integrated into pathogen detection. Nucleotide probes conjugated to gold nanoparticles (AuNP probes) have been used in research and routine applications (3, 7) for the detection of various bacteria, including Mycobacterium tuberculosis (1). Recently we developed a diagnostic assay that allows rapid and direct detection of members of the Mycobacterium genus (M. tuberculosis complex and M. avium complex) in clinical samples in a highly specific, easy-to-perform method that requires little infrastructure and expertise (9). One other, perhaps more promising approach to this involves the use of fluorescent semiconductor quantum dots (QDs), which have emerged as a more attractive class of fluorescent label probes due to their superior properties. The main advantages of QDs over conventional fluorescent dyes are their prolonged photostability and their broad excitation spectrum. QDs can be excited efficiently at any wavelength shorter than their emission peak, and yet they emit with the same characteristic, narrow, symmetric spectrum. Therefore, many sizes of QDs may be excited with a single wavelength of light, which makes it possible to detect many emission peaks simultaneously. Because of this unique property, the use of QDs as fluorescent probes has enabled multicolor imaging in demanding biological environments (13). QDs have found practical application in the detection of pathogens and their toxins and the definition of their characteristics, including virulence. Several different pathogens have been targeted so far: Cryptosporidium parvum, Giardia lamblia, Escherichia coli O157:H7, Salmonella enterica serovar Typhi, and Listeria monocytogenes (6).

In this article, we describe the development and evaluation of a DNA detection method using fluorescent semiconductor QDs and magnetic beads (MBs) with clinical samples for fast identification of two members of the Mycobacterium genus (M. tuberculosis and M. avium subsp. paratuberculosis), dispensing with the need for DNA amplification.

MATERIALS AND METHODS

Probe design.

Deposited sequences of some of the most common mycobacterial pathogens (GenBank accession numbers DQ445257, NC008769, NC008595, NC002944, and CP000325) were aligned using the ClustalW software program (EMBL-EBI). Five 30-bp-long genus-specific probes were designed for the detection of Mycobacterium based on the 23S rRNA gene, which is highly conserved among the mycobacterial species. For the detection of M. tuberculosis and M. avium subsp. paratuberculosis, 2 sets of five (n = 5) 30-bp-long probes were designed based on IS6110 and IS900, respectively. One probe was selected from each set based on specificity evaluation, which was performed through dot blot hybridization experiments (data not shown) using DNA isolated from several bacterial species (Table (Table1).1). Five adenine residues [(A)5] and a biotin group were added at the 5′ end of the probes in order to increase the distance between the QD and the capture sequence and thus facilitate hybridization. Probes were purchased from MWG, Germany, and were dissolved in distilled water (18 mΩ; Millipore) to a final concentration of 100 μΜ.

TABLE 1.
DNA probes used for the detection of mycobacteria

Functionalization of QDs and MBs with DNA probes.

Cadmium selenite (CdSe) quantum dots (15 to 20 nm in size) with a maximum emission wavelength of 655 nm, shelled with ZnS and a polymer coating presenting carboxylic groups, were purchased from Invitrogen (Q21321MP; Invitrogen, California). QDs were coated with streptavidin prior to use. Briefly, 50 μl of QDs were diluted in 400 μl of borate buffer (10 mM; pH 7.4), 96 μl of streptavidin solution (10 mg/ml) (Invitrogen, California), and 11.4 μl of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) (10 mg/ml) (Sigma-Aldrich, Missouri) and were incubated at room temperature (RT) for 90 min. Streptavidin-coated QDs were washed five times with 500 μl of borate buffer (50 mM; pH 8.3) on an Amicon Ultra-4 filter (Millipore, Massachusetts), dissolved in 50 mM borate buffer (pH 8.3) to a final volume of 50 μl, and stored at 4°C in the dark until use. Magnetic beads with a diameter of 2.8 μm were purchased with a monolayer coat of streptavidin (Dynabeads M-280 streptavidin 112-05D; Invitrogen, California).

For the functionalization of the oligonucleotides MTB1 and MAP3 with QDs, 50 μl of streptavidin-coated QDs were added to 200 μl of biotinylated oligonucleotide solution (100 pmol/μl), which was incubated for 15 min at RT and then washed three times with 50 Mm borate buffer (pH 8.3) and dissolved in 500 μl 50 mM borate buffer containing 0.1% bovine serum albumin (BSA). To confirm that the probes were immobilized on the surface of the QDs, 5 μl of the probe-conjugated QDs and an equal volume of nonconjugated QDs were loaded on a 1% agarose gel and electrophoresed for 90 min at 50 V.

For the functionalization of the probe MYCO4 on the surface of MBs, 50 μl of streptavidin-coated MBs (10 mg/ml) were washed twice with 100 μl of 2× binding and washing (B&W) buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 2.0 M NaCl) to remove any preservatives, and then they were resuspended in 100 μl of 2× B&W buffer. One hundred μl of the MYCO4 probe (100 μM) was added to the suspension of MBs, and they were incubated for 15 min at room temperature with gentle agitation. The probe-conjugated MBs were washed three times with 1× B&W buffer using a magnetic device (Dynal MPC-L; Invitrogen, Oslo, Norway). To confirm immobilization of the probe on the surface of MBs, the absorbance of conjugated and nonconjugated MBs was measured at 260 nm.

Hybridization and fluorescence detection.

The procedure followed for hybridization and fluorescence detection (European patent application no. 09008732.1-1223) is shown in Fig. Fig.1.1. Specificity was evaluated through hybridization experiments using DNA isolated from the bacterial species listed in Table Table2.2. DNA was denatured at 95°C for 5 min and added to 50 μl of MB probes. Hybridization was performed at 42°C for 30 min. MB probes conjugated to target DNA were separated and washed twice with 100 μl 2× SSC buffer (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) using a magnetic device and resuspended in 50 μl of hybridization solution (DIG Easy Hyb; Roche, Germany). The mixture was heated at 94°C for 2 min with 50 μl of QD probes. Hybridization was carried out at 42°C for 1 h. The resulting MB probe/target/QD probe conjugate was washed twice with 100 μl 2× SSC buffer using a magnetic device for its immobilization; it was resuspended in 150 μl of phosphate-buffered saline (PBS) and then transferred to a UV apparatus for visualization.

FIG. 1.
Schematic presentation of the fluorescent DNA detection method using DNA-functionalized QDs and MBs.
TABLE 2.
Bacteria used for specificity evaluation of QD probes

Minimum detection limit.

The minimum detection limit of the assay was assessed for both Mycobacterium tuberculosis and M. avium subsp. paratuberculosis as previously described (5, 9). Briefly, two series of 2-fold dilutions (1:2 to 1:512) were prepared with 180 ng/μl DNA of each pathogen in high-performance liquid chromatography (HPLC) water; 20 μl of each dilution (3,600 to 6.28 ng/reaction) was used for detection with the method described above. Blanks containing PBS instead of DNA were used as negative controls.

Application to clinical samples.

In order to assess the performance of the assay with clinical material, the optimized method was used for the detection of M. tuberculosis and M. avium subsp. paratuberculosis in DNA isolated from bronchoalveolar lavage (BAL) samples, formalin-fixed paraffin-embedded (FFPE) tissues, and feces. With regard to M. tuberculosis, the assessment relied on DNA isolated from BAL samples from 48 patients with clinical tuberculosis and 12 BAL samples from healthy individuals, both confirmed by culture and real-time PCR, with the exception of a BAL sample that reacted negatively by the latter method.

With regard to M. avium subsp. paratuberculosis, our assessment relied on DNA isolated from 14 positive and 6 negative caprine (Capra hircus) fecal samples confirmed by culture and real-time PCR. In addition to the above, we evaluated the performance of the proposed assay for the detection of M. avium subsp. paratuberculosis on DNA isolated from 26 Ziehl-Neelsen (ZN)-positive FFPE sections of lymph nodes and ileum collected from animals diagnosed with paratuberculosis and 7 negative controls, confirmed as such by ZN and real-time PCR.

For the isolation of DNA from BAL samples, 1 ml of sample was incubated with 1 ml of N-acetyl cysteine-NaOH (2%) for 25 min at RT with shaking. The sample volume was adjusted at 25 ml with sterile water; the sample was centrifuged at 4,000 × g for 30 min, and the supernatant was discarded. The pellet was resuspended in 500 μl of T1 buffer, and the sample was processed with the standard protocol for NucleoSpin tissue (Macherey-Nagel, Germany). Isolation of DNA from feces was performed as previously described (9).

DNA from FFPE was extracted using a commercial kit (Nucleospin tissue kit; Macherey-Nagel, Germany) according to the manufacturer's instructions, after a deparaffinization step using xylene followed by ethanol washes.

The real-time PCR assay for the detection and quantification of M. tuberculosis was performed using the TB real-time detection kit (Primer Design, Southampton, United Kingdom). The real-time PCR assay for the detection of M. avium subsp. paratuberculosis was previously described (8). Quantification of M. avium subsp. paratuberculosis was based on a standard curve constructed with serial dilutions of a known concentration of M. avium subsp. paratuberculosis DNA.

Accuracy and repeatability.

The method was assessed on the basis of binary classification (confusion matrixes). Accuracy was expressed as the ratio of the number of correct results divided by the total number of results, multiplied by 100 [(TP + TN)/n, where TP is true positives, TN is true negatives, and n is the total number of results] (15, 16). For the assessment of the method's repeatability, the proposed assay was applied on different days with new reagents five times on 10 randomly selected positive (n = 5) and negative (n = 5) control samples for each of the types of clinical material incorporated in this study (BAL samples, feces, FFPE tissue). The repeatability was expressed as the ratio of the number of identical results over the total number of samples tested (n = 150) multiplied by 100.

RESULTS

Functionalization of QDs and MBs.

Gel electrophoresis and absorbance measurements of probe-conjugated QDs and MBs clearly demonstrated the presence of oligonucleotides conjugated to QDs and MBs.

Binding of the QDs to the MBs.

As illustrated in Fig. Fig.2,2, in the presence of target DNA in the solution, the probes MB-MYCO4 and QD-TB1 or QD-MAP3 form complexes that fluoresce red when excited with UV radiation. No fluorescence is produced when the MB-MYCO4 probes are incubated with either of the QD conjugates (QD-TB1 and QD-MAP3) in the absence of target DNA. The results were confirmed using confocal microscopy, which indicated clearly that QD probes bind to MB probes only through the target DNA (Fig. (Fig.33).

FIG. 2.
Representative results recoded by the proposed assay: test tubes that contain MB-MYCO4 and only QD-TB1 or QD-MAP3 (blank), DNA isolated from cultivated Nocardia spp. (negative), and DNA isolated from cultivated (positive) M. tuberculosis (A) or M. avium ...
FIG. 3.
Representative confocal microscopy images: no fluorescence is detected in the absence of the target DNA (A); the MB probe incubated with target DNA and the QD probe fluoresced red (B).

Method's specificity and minimum detection limit.

The application of the proposed method to isolated bacterial DNA produced the expected result with all the positive- and negative-control samples (Table (Table2).2). Both QD probes (TB1 and MAP3) proved able to detect repeatedly as little as 12.5 ng of mycobacterial DNA per reaction.

Test performance on clinical samples.

The concordance of the positive results recorded by the proposed assay on BAL samples with those of culture and real-time PCR was, respectively, 85.41% (41 of 48) and 87.23% (41 of 47). The relevant percentage with regard to negative results was 100% in both cases. The method's accuracy compared to culture and real-time PCR was calculated as 88.33% (TP = 41 TN = 12 n = 60) and 90% (TP = 41; TN = 13; n = 60), respectively (Table (Table33).

TABLE 3.
Results recorded by culture, RT-PCR, and proposed method for detection of M. tuberculosis in clinical samplesa

With regard to FFPE tissues, the concordance of the results was again 100% for the samples that reacted negatively by Ziehl-Neelsen stain and real-time PCR. The relevant percentages with regard to the positive results were 53.84% for the ZN-positive samples (14 out of 26 ZN positive) and 84.61% for the real-time PCR-positive samples (22 out of 26 real-time PCR positive). The level of concordance for the samples that reacted positively both by the Ziehl-Neelsen stain and real-time PCR was 59.1% (13 of 22) (Table (Table4).4). The accuracy of the QD assay compared to the Ziehl-Neelsen stain and real-time PCR was 63.63% (TP = 14; TN = 7; n = 33) and 69.69% (TP = 13; TN = 10; n = 33), respectively.

TABLE 4.
Results recorded by ZN, culture, RT-PCR, and proposed method for detection of M. avium subsp. paratuberculosis in clinical samplesa

Finally, the relevant results recorded for the fecal samples cross-examined by real-time PCR were 78.57% (concordance of positive results for 11 of 14) and 100% (concordance of negative results for 6 of 6). The accuracy of the proposed method compared to real-time PCR applied on the specific type of clinical material was 85% (TP = 11; TN = 6; n = 20), (Table (Table44).

The concentration of M. tuberculosis DNA in the samples mentioned above ranged between 33 × 103 and 11 × 109 copies of target gene per reaction as calculated by absolute quantification (linear regression, 0.9865;, slope, −3.481; efficiency, 93.76%). The concentration of M. avium subsp. paratuberculosis DNA in the tested samples was 21.4 pg to 4.71 μg per reaction, as calculated again by absolute quantification (r2 = 0.9939; slope = −3.397; efficiency, 96.96%).

The repeatability of the method was defined as 100% since the results recorded for the samples included in this type of evaluation were identical for all assessments (n = 5).

DISCUSSION

In this article, we describe a newly developed technique for the detection of M. tuberculosis and M. avium subsp. paratuberculosis DNA which overcomes the need for DNA amplification. The methods incorporated in the cross-evaluation of our clinical material were selected on the basis of the routine diagnostic investigation of cases of tuberculosis and paratuberculosis. Therefore, certain types of clinical material, for which it was expected that their compatibility with our QD-based detection system would be low, were not excluded from this assessment since this would provide an indication of the applicability of the proposed methodology in practice. This was the case with the FFPE tissues, since the quality of the DNA isolated from the specific type of samples is often poor. However, the expectancy of a high level of discrepancy between the results recorded by the two methods (ZN staining and the QD assay) was not fully confirmed by our findings given that the concordance of the positive results exceeded 50%, whereas that of the negative results reached 100%. Notably, the relevant percentages for ZN and real-time PCR were defined as 84.61% and 100% with regard to positive and negative results, respectively. However, the fact that the relative concordance of the proposed assay with the reference methodologies that were incorporated in this study was the lowest with ZN has to be attributed to DNA fragmentation of the FFPE tissue samples. In more detail, given that the capture and detection probes of the QD assay are complementary to different genes of the mycobacterial genome, the chances of false-negative results due to DNA fragmentation are inevitably increased. This weakness of the proposed technique results from the approach that was followed for its construction and can be resolved by the use of a different set of DNA probes that anneal closer to each other. However, although the application of quality control on the isolation of DNA would increase the complexity of the procedure, it would be the obvious alternative solution to the problem mentioned above, especially since its significance is unquestioned for all diagnostic assays that target genetic material.

The accuracy of the QD detection system compared to culture and real-time PCR applied to fecal and BAL samples ranged between 85% and 90%. Admittedly, PCR is among the most reliable and useful techniques for the detection of nucleic acids. Many variants of the method, such as nested PCR, multiplex PCR, and real-time PCR, have been employed for the detection of mycobacterial DNA in various samples (2, 4, 11, 12). However, the application of PCR techniques requires trained personnel, dedicated space, and high-cost equipment. The proposed method can be used as an easily applicable, low-cost screening tool since it requires less than 2 h and is performed in a single tube, which reduces carryover contamination and facilitates simultaneous testing of many samples. Furthermore, the QD detection method avoids the drawback of PCR-based diagnostic assays that are prone to false-negative results generated by inhibitors commonly found in clinical samples such as feces. Indeed, this was the case with 2 ZN-positive samples that produced a positive real-time PCR result only after the DNA solutions they produced were diluted, which implies the presence of PCR inhibitors.

The fact that our QD detection system utilizes a combination of DNA probes allows an assessment that minimizes false-positive results associated with low specificity. This was demonstrated in our findings, since concordance of the negative results with cultivation and real-time PCR was 100%. Notably, the latter was confirmed even when the proposed assay was applied to DNA isolated from fecal samples that harbor mixed bacterial populations, the majority of which may exceed greatly that of the targeted bacteria.

The methodology described here provides a diagnostic alternative that can be used in a reliable manner even at the point of care. This is supported by the minimum detection limit of the QD detection system, the repeatability of the results it produces, and the fact that it does not require dedicated equipment. However, what may be more significant is that this QD detection system can be used with specifically constructed DNA-functionalized QDs and MBs for the fluorescence detection of various pathogens or genetic markers associated, for example, with drug resistance or infectivity. This implies the potential use of the proposed methodology as a diagnostic technology platform.

Acknowledgments

This work was performed within the context of the NANOMYC project, which is supported by the EU (LSHB-CT-2007-036812).

Footnotes

[down-pointing small open triangle]Published ahead of print on 16 June 2010.

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