Differentially expressed aqueous soluble proteins between Mycobacterium tuberculosis H37Ra and H37Rv were identified. The protein extracts were separated by two-dimensional gel electrophoresis followed by tandem mass spectrometric analysis. Twelve proteins were detected to be differentially expressed significantly between virulent strain H37Rv and attenuated strain H37Ra. The differentially expression of these proteins was validated by a recently isolated clinical virulent strains of M. tuberculosis, TB138. Out of the 12 proteins identified, which consisted of ten upregulated and two downregulated proteins, nine were belonged to intermediate metabolism and respiration protein group, two were in lipid metabolism, and one protein was involved in information pathways and virulence. Among these proteins, two of the upregulated proteins, namely, mmsA and pntAa, showed a consistent expression pattern in both virulent mycobacterium strains. These proteins can serve as potential biomarkers for the intervention treatment of TB.
An early detection of Mycobacterium tuberculosis is very important to reduce the number of fatal cases and allow for fast recovery. However, the interpretation of the result from smear microscopy requires skilled personnel due to the propensity of the method to produce false-negative results. In this work, a portable, rapid, and simple sandwich-type immunosensor reader has been developed that is able to detect the presence of M. tuberculosis in sputum samples. By using sandwich-type immunosensor, an anti-CFP10-ESAT6 antibody was immobilized onto the graphene/polyaniline (GP/PANI)-modified gold screen-printed electrode. After incubation with the target CFP10-ESAT6 antigen, the iron/gold magnetic nanoparticles (Fe3O4/Au MNPs) conjugated with anti-CFP10-ESAT6 antibody were used to complete the sandwich format. Differential pulse voltammetry (DPV) technique was used to detect the CFP10-ESAT6 antigen at the potential range of 0.0-1.0 V. The detection time is less than 2 h. Under optimal condition, CFP10-ESAT6 antigen was detected in a linear range from 10 to 500 ng mL-1 with a limit of detection at 1.5 ng mL-1. The method developed from this process was then integrated into a portable reader. The performance of the sensor was investigated and compared with the standard methods (culture and smear microscopy). It provides a good correlation (100% sensitivity and 91.7% specificity) with both methods of detection for M. tuberculosis in sputum samples henceforth, demonstrating the potential of the device as a more practical screening tool.Graphical abstract.
A MALDI TOF MS based minisequencing method has been developed and applied for the analysis of rifampin (RIF)- and isoniazid (INH)-resistant M. tuberculosis strains. Eight genetic markers of RIF resistance-nucleotide polymorphisms located in RRDR of rpoB gene, and three of INH resistance including codon 315 of katG gene and -8 and -15 positions of the promoter region of fabG1-inhA operon were worked out. Based on the analysis of 100 M. tuberculosis strains collected from the Moscow region in 1997-2005 we deduced that 91% of RIF-resistant and 94% of INH-resistant strains can be identified using the technique suggested. The approach is rapid, reliable and allows to reveal the drug resistance of M. tuberculosis strains within 12 h after sample isolation.