[Methodology research and preliminary assessment of Mycobacterium tuberculosis detection by immunomagnetic beads combined with functionalized fluorescent quantum dots].

OBJECTIVE

To establish a detection method for Mycobacterium tuberculosis (MTB) by immunomagnetic beads combined with functionalized fluorescent quantum dots technology, and to investigate the optimal test condition and the diagnostic value of this method.

METHODS

MTB standard strain H37Rv was used as detection object. Nanobeads and quantum dots were prepared by using wet chemical method, and conjugated separately with MTB binding peptide H8 to obtain immunomagnetic beads and functionalized fluorescent quantum dots, which could react with H37Rv simultaneously and form a ternary complex structure. Based on measurement of the fluorescence value and observation under fluorescence microscopy to determine if MTB existed in the sample, a new detection method of MTB using nanotechnology was established. The optimal detection concentration and reaction time of immunomagnetic beads and quantum dots were investigated, and the detection limit and specificity of this detection method were evaluated by using bacterial suspension and simulation sputum samples.

RESULTS

By fluorescence microscopy examination, it was found that conjugated immunomagnetic beads and functionalized fluorescent quantum dots both bound with H37Rv and formed the ternary complex structure. The fluorescent value ratio of the experimental group and the control group could be 4:1. The best detection concentration of immunomagnetic beads and functionalized fluorescent quantum dots was 100 mg/L and the optimal incubation time was 2 h. The detection limit of H37Rv bacterial suspension and simulation sputum sample were both 10(3) CFU/ml. The detection results for 3 non-mycobacteria were all negative, while for the 12 types of NTM, only Mycobacterium parafortuitum, Mycobacterium aurum, Mycobacterium smegmatis and Mycobacterium fortuitum were positive, and others were all negative.

CONCLUSION

The detection method of immunomagnetic beads combined with fluorescence quantum dots can be a new detection method for MTB, but the clinical value needs to be evaluated further.