Detection of Pathogenic Mycobacteria Based on Functionalized Quantum Dots Coupled with Immunomagnetic Separation

Mycobacteria have always proven difficult to identify due to their low growth rate and fastidious nature. Therefore molecular biology and more recently nanotechnology, have been exploited from early on for the detection of these pathogens. Here we present the first stage of development of an assay incorporating cadmium selenide quantum dots (QDs) for the detection of mycobacterial surface antigens. The principle of the assay is the separation of bacterial cells using magnetic beads coupled with genus-specific polyclonal antibodies and monoclonal antibodies for heparin-binding hemagglutinin. These complexes are then tagged with anti-mouse biotinylated antibody and finally streptavidin-conjugated QDs which leads to the detection of a fluorescent signal. For the evaluation of performance, the method under study was applied on Mycobacterium bovis BCG and Mycobacterium tuberculosis (positive controls), as well as E. coli and Salmonella spp. that constituted the negative controls. The direct observation of the latter category of samples did not reveal fluorescence as opposed to the mycobacteria mentioned above. The minimum detection limit of the assay was defined to 104 bacteria/ml, which could be further decreased by a 1 log when fluorescence was measured with a spectrofluorometer. The method described here can be easily adjusted for any other protein target of either the pathogen or the host, and once fully developed it will be directly applicable on clinical samples

Validation of a Real-Time PCR for the Detection of Mycobacterium tuberculosis Complex Members in Bovine Tissue Samples

Although the post-mortem diagnosis of bovine tuberculosis is mainly achieved through microbiological culture, the development of other techniques to detect Mycobacterium tuberculosis complex (MTBC) members directly from tissue samples has been pursued. The present study describes the development, optimization and validation of a Real-Time PCR based on the mpb70 gene to detect MTBC members in clinical tissue samples from cattle. Specific primers and a hybridization probe were used to amplify MTBC-specific sequences in order to avoid cross-reaction with non-MTBC species. An Internal Amplification Control (IAC) was included in order to assess the presence of PCR inhibitors in the samples. The PCR was optimized to achieve maximum efficiency, and the limit of detection, limit of quantification and dynamic range of the reaction were determined. The specificity of the reaction was tested against 34 mycobacterial and non-mycobacterial species. The diagnostic sensitivity, specificity and positive and negative predictive values (PPV and NPV) of the method were assessed on 200 bovine tissue samples in relation to bacteriological culture. The dynamic range of the reaction spanned from 5 ng/reaction (106 genome equivalents) to 50 fg/reaction (10 genome equivalents). The efficiency of the reaction was 102.6% and the achieved R2 was 0.999. The limit of detection with 95% confidence was 10 genome equivalents/reaction. No cross-reactions with non-MTBC species were observed. The diagnostic sensitivity and specificity values of the mpb70 specific Real-Time PCR respect to culture were 94.59% (95% CI: 86.73–98.51%) and 96.03% (95% CI: 90.98–98.70%), respectively, with a PPV of 93.33% (95% CI: 85.55–97.07%) and a NPV of 96.80% (95% CI: 92.10–98.74%). The concordance of the Real-Time PCR based on mpb70 is comparable to that of culture (K = 0.904) showing a great potential for the detection of members of the MTBC in animal tissues

Epigenetic changes of hepatic glucocorticoid receptor in sheep male offspring undernourished in utero

The aim of this study was to characterise the effects of maternal undernutrition during gestation on hepatic gluconeogenic enzyme gene expression and to determine whether such effects are mediated through epigenetic changes in the glucocorticoid receptor (GR). Pregnant ewes were fed a 50% nutrient-restricted diet from Day 0 to 30 (R1) or from Day 31 to 100 of gestation (R2) or a 100% diet throughout gestation (Control). After parturition lambs were fed to appetite. At 10 months of age offspring were euthanised and livers were removed. Maternal undernutrition did not affect offspring bodyweight at birth or at 10 months of age. However, liver weight of males of the R2 group was lower (P &lt; 0.05) in relation to other groups. A significant (P &lt; 0.05) hypomethylation of the hepatic GR promoter was revealed in males of the R2 group and a tendency towards the same in the R1 group, along with increased (P &lt; 0.001) GR gene expression in both restricted groups. A significant increase (P &lt; 0.05) in hepatic phosphoenolpyruvate carboxykinase (PEPCK) gene expression was found in male lambs of both undernourished groups, accompanied by increased (P &lt; 0.01) protein levels, while no differences were detected for glucose-6-phosphatase (G6Pase) mRNA abundance and protein levels. In female lambs, no differences between groups were observed for any parameter studied. These data represent potential mechanisms by which insults in early life may lead to persistent physiological changes in the offspring.</jats:p

CERTIFICATION REPORT: The Certification of the PFGE fragment sizes of Listeria monocytogenes (strain H2446) DNA in agarose plugs: ERM®-AD624

Summary This report describes the production of ERM®-AD624, a Listeria monocytogenes DNA material certified for the size of the DNA fragments obtained by enzymatic restriction digestion and Pulsed Field Gel Electrophoresis (PFGE). This material was produced following ISO Guide 34:2009 [ ] and is certified in accordance with ISO Guide 35:2006 [ ]. The CRM was produced from a culture of Listeria monocytogenes strain H2446 and processed into agarose plugs suitable for PFGE. The bacteria were lysed to release the DNA within the plugs. Between unit-homogeneity and stability during dispatch and storage were assessed in accordance with ISO Guide 35:2006. The material was characterised by an interlaboratory comparison of laboratories of demonstrated competence and adhering to ISO/IEC 17025. Technically invalid results were removed but no outliers were eliminated on statistical grounds only. Uncertainties of the certified values were calculated in accordance with the Guide to the Expression of Uncertainty in Measurement (GUM). The material is intended for quality control and assessment of method performance. As with any reference material, it can be used for establishing control charts or validation studies. The CRM is available in plastic screw cap vials containing one plug suspended in TE solution. The minimum amount of sample to be used is the whole CRM.JRC.F.6-Reference Material

Direct detection of unamplified DNA from pathogenic mycobacteria using DNA-derivatized gold nanoparticles

Mycobacterial infections have a high economic, human and animal health impact. Herein, we present the development of a colorimetric method that relies on the use of gold nanoparticles for fast and specific detection of Mycobacterium spp. dispensing with the need for DNA amplification. The result can be recorded by visual and/or spectrophotometric comparison of solutions before and after acid induced AuNP-probe aggregation. The presence of a complementary target prevents aggregation and the solution remains pink, whereas in the opposite event it turns to purple. The application of the proposed method on isolated bacteria produced positive results with the mycobacterial isolates and negative with the controls. The minimum detection limit of the assay was defined at 18.75 ng of mycobacterial DNA diluted in a sample-volume of 10 μl. In order to obtain an indication of the method's performance on clinical samples we applied the optimized assay to the detection of Mycobacterium avium subsp. paratuberculosis DNA in faeces, in comparison with real-time PCR. The concordance of the two methods with connection to real-time PCR positive and negative sample was defined respectively as 87.5% and 100%. The proposed method could be used as a highly specific and sensitive screening tool for the detection of mycobacteria directly from clinical samples in a very simple manner, without the need of high-cost dedicated equipment. The technology described here, may develop into a platform that could accommodate detection of many bacterial species and could be easily adapted for high throughput and expedite screening of samples

Specific detection of unamplified mycobacterial DNA using fluorescent semiconductor quantum dots and magnetic beads

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. Cadmim selenite QDs conjugated with streptavidin and species specific probes were used to produce 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 on isolated bacteria produced the expected result in all cases. The minimum detection limit of the assay was defined at 12.5 ng of DNA diluted in a sample volume of 20 μl. In order to obtain an indication of the method's performance on clinical samples we applied the optimized assay to the detection of Mycobacterium tuberculosis in DNA isolated from bronchoalveolar lavage of patients with tuberculosis, and Mycobacterium avium subsp. paratuberculosis in faeces and paraffin embedded tissues, in comparison with culture, Ziehl-Neelsen stain and Real Time PCR. The concordance of these methods compared to the proposed with connection to positive and negative samples varied between 53.84% - 87.23% and 84.61%-100% respectively. The overall accuracy of the QD assay compared to Real Time PCR was 70-90% 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

Bacterial cells (a) are separated from the matrix using MBs coupled with genus specific polyclonal antibodies (b) and a magnetic device (c).

<p>These complexes are then tagged with anti-HBHA (d) and anti-mouse biotynylated antibody and streptavidin-conjugated QDs (e) which lead to the detection of a fluorescent signal (f).</p

MBs/bacteria/QDs complexes visualized by fluorescent microscopy.

<p>Magnetic beads (arrows) are surrounded by a fluorescent halo with shape and dimensions compatible with mycobacteria. 1000X.</p