Content area
Full text
During the last 2decades, an unparalleled interest has been stimulated to design new tuberculosis (TB) diagnostic tests but still a dependable test for rapid diagnosis of various clinical forms of TB is lacking. Immuno-PCR (I-PCR) assay comprises the versatility of ELISA and the enormous amplification power of PCR (1,2), which has been utilized for the detection of an array of Mycobacterium tuberculosis biomarkers, in other words, CFP-10, ESAT-6 (Rv3875), Ag85B (Rv1886c), lipoarabinomannan, and so on, in TB patients that revealed superiority over ELISA (2,3). We recently developed real-time I-PCR (RT-I-PCR) for the quantitative detection of M. tuberculosis MPT64 (Rv1980c) and PstS1 (Rv0934) secreted proteins in TB patients (4,5). Coupling of detection antibodies to reporter DNA is the most critical step of I-PCR/RT-I-PCR assay. Various strategies are employed for this coupling, in other words, streptavidin-protein A, streptavidin-biotin conjugate or succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate/N-succinimidyl S-acetylthioacetate (SATA) system by a covalent binding (1,6). Moreover, high accuracy of I-PCR/RT-I-PCR is limited due to complicated preparation of antibody-DNA conjugates and the low ratio of DNA to detection antibody (∼1:1) (7,8). Owing to small size, unusual target-binding properties and high surface-to-volume ratio, nanoparticles (NPs) have also been used as signal amplification tools (9). Among these, gold nanoparticles (GNPs), magnetic beads (MBs), carbon nanotubes and quantum dots are the major nanomaterials employed in I-PCR/RT-I-PCR assays (6). Notably, GNPs provide various chemistries to couple with antibodies and DNA and the large ratio of DNA to antibody (∼100-300:1) in those functionalized GNPs may further improve the PCR amplification capacity of GNP-based I-PCR (GNP-I-PCR) (6,9).
Indeed, GNP-I-PCR/GNP-RT-I-PCR is a modified biobarcode amplification assay where PCR/real-time PCR is being employed for the detection of short oligonucleotides (as surrogate targets) instead of scanometric detection (6). GNP-I-PCR/GNP-RT-I-PCR has been utilized for the detection of several pathogens, for example, HIV, Hantaan virus, foot and mouth disease virus (FMDV) and Giardia lamblia (10-13) with promising results, however, no report is available to detect M. tuberculosis biomarkers in clinical samples by this assay. Strikingly, M. tuberculosis CFP-10 secretion has been shown to elicit immunopathologic host response, while its deficiency attenuates the virulence of organism (14), thus implying CFP-10 to be an attractive biomarker for active TB disease (3,15). To further improve the technology and diagnostic efficacy of RT-I-PCR,...