Detection of Mycobacterium tuberculosis based on H37R(v) binding peptides using surface functionalized magnetic...

Despite suffering from the major disadvantage of low sensitivity, microscopy of direct smear with the Ziehl-Neelsen stain is still broadly used for detection of acid-fast bacilli and diagnosis of tuberculosis. Here, we present a unique detection method of Mycobacterium tuberculosis (MTB) using surface functionalized magnetic microspheres (MMSs) coupled with quantum dots (QDs), conjugated with various antibodies and phage display-derived peptides. The principle is based upon the conformation of the sandwich complex composed of bacterial cells, MMSs, and QDs. The complex system is tagged with QDs for providing the fluorescent signal as part of the detection while magnetic separation is achieved by MMSs. The peptide ligand H8 derived from the phage display library Ph.D.-7 is developed for MTB cells. Using the combinations of MMS-polyclonal antibody+QD-H8 and MMS-H8+QD-H8, a strong signal of 10(3) colony forming units (CFU)/mL H37R(v) was obtained with improved specificity. MS-H8+QD-H8 combination was further optimized by adjusting the concentrations of MMSs, QDs, and incubation time for the maximum detection signal. The limit of detection for MTB was found to reach 10(3) CFU/mL even for the sputum matrices. Positive sputum samples could be distinguished from control. Thus, this novel method is shown to improve the detection limit and specificity of MTB from the sputum samples, and to reduce the testing time for accurate diagnosis of tuberculosis, which needs further confirmation of more clinical samples.