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Diagnosis of Mycobacterium tuberculosis from Sputum DNA Using Multiplex PCR
Published in P. Mereena Luke, K. R. Dhanya, Didier Rouxel, Nandakumar Kalarikkal, Sabu Thomas, Advanced Studies in Experimental and Clinical Medicine, 2021
R. Gopinath, Suthandira Munisamy, M. Jeyadevasena, J. M. Jeffrey, Elanchezhiyan Manickan
The MPCR products were then analyzed by electrophoresis at 100 V on 2% agarose gels stained with Ethidium bromide (0.5 μg/ml) and visualized under Gel documentation system (GELSTAN, India). The PCR products were compared with H37Rv positive control and PCR grade water and patient negative sample as negative control.
Application of Next-Generation Plant-Derived Nanobiofabricated Drugs for the Management of Tuberculosis
Published in Richard L. K. Glover, Daniel Nyanganyura, Rofhiwa Bridget Mulaudzi, Maluta Steven Mufamadi, Green Synthesis in Nanomedicine and Human Health, 2021
Charles Oluwaseun Adetunji, Olugbenga Samuel Michael, Muhammad Akram, Kadiri Oseni, Ajayi Kolawole Temidayo, Osikemekha Anthony Anani, Akinola Samson Olayinka, Olerimi Samson E, Wilson Nwankwo, Iram Ghaffar, Juliana Bunmi Adetunji
The completion and first release of the complete nucleotide sequence of the circular chromosome M. tuberculosis strain H37Rv occurred in 1988 (Cole et al., 1998) and has immensely improved the understanding of Mtb biology in its ramifications. The M. tuberculosis H37Rv genome consists of 4.4 × 106 bp with a relatively high G-C content (65%) and contains about 4,000 genes. (Glickman and Jacobs, 2001; Smith 2003; Cerezo, 2015) Other unique features of the genome include its content of the two related protein families of genes named PE (derived from proline-glutamate) and PPE (derived from proline–proline glutamate), which makes up about 9% of the genome and could be implicated in the antigenic variation of Mtb during infection. About 200 genes were also annotated as encoding enzymes for the metabolism of fatty acids, comprising 6% of the total, and the genome also contains homologs of the fatty acid β-oxidation system having 36 FadD and 36 FadE homologs, which is suggestive of Mtb’s involvement in lipid synthesis and assimilation as carbon sources (Glickman and Jacobs, 2001). Since the release of this complete sequence in 1988, its functional annotation has grown, giving much information to explore Mtb and its infection control (Cerezo, 2015).
Mycobacterium tuberculosis
Published in Lloyd N. Friedman, Martin Dedicoat, Peter D. O. Davies, Clinical Tuberculosis, 2020
A landmark in TB research occurred with the publication of the genome sequence of M. tuberculosis in 199822 on a widely used reference strain, H37Rv. No extrachromosomal genetic elements were detected. The genome of M. bovis has also been sequenced and shows more than 99.95% similarity with that of M. tuberculosis although it is very slightly smaller, with 4,345,492 base pairs.23 In contrast, the genome of M. leprae,24 with 3.27 million base pairs, is considerably smaller than that of M. tuberculosis, and it differs from the latter in that many of its genes, around a half, are defective and non-functional, explaining why this organism has never been cultivated in vitro and is an obligate intracellular pathogen.
Effect of mycobacterial proteins that target mitochondria on the alveolar macrophages activation during Mycobacterium tuberculosis infection
Published in Experimental Lung Research, 2022
Iris Selene Paredes-González, Omar Emiliano Aparicio-Trejo, Octavio Ramos-Espinosa, Manuel Othoniel López-Torres, Milena Maya-Hoyos, Monserrat Mendoza-Trujillo, Alejandra Barrera-Rosales, Dulce Mata-Espinosa, Juan Carlos León-Contreras, José Pedraza-Chaverri, Clara Espitia, Rogelio Hernández-Pando
Cells were seeded into a 75 cm2 culture flask (SC-200263, Santa Cruz Biotechnology Inc., Dallas, Texas, USA) at a density of 2.5 × 106 cells per flask and incubated overnight at 5% CO2, 37 °C. MQs were pretreated with P27 or PE_PGRS33 Mtb recombinant proteins (2 µg/mL) or left untreated for 4 h. Subsequently, cells were infected for 3 h with Mtb H37Rv (MOI 1:5). Cells were fixed with Karnovsky solution (4% paraformaldehyde and 1.5% glutaraldehyde in 0.2 M Sörensen Buffer, pH 7.2) for 1 min, collected, pelleted, and re-suspended in the same fixer solution. MQs were post-fixed with 2% OsO4 buffer, dehydrated in graded ethyl alcohol solutions, and embedded in Low Viscosity Resin Spurr (Electron Microscopy Sciences, Industry Road Hatfield PA, USA). Thin sections of 70–90 nm were placed on copper grids, contrasted with uranyl acetate and lead citrate Reynol’s for examination with FEI Tecnai G2 Spirit transmission electron microscope (Hillsboro, OR., USA). Images of mitochondria were analyzed with the ImageJ software (National Institutes of Health, USA) to quantify area and perimeters to determine interconnectivity and elongation with the following formulas27:
An evaluation of liposome-based diagnostics of pulmonary and extrapulmonary tuberculosis
Published in Expert Review of Molecular Diagnostics, 2020
Nikunj Tandel, Anish Z Joseph, Aishwarya Joshi, Priya Shrama, Ravi PN Mishra, Rajeev K. Tyagi, Prakash S Bisen
A simple and cost-effective diagnostic tool (TB Screen Test) for the screening of patients with pulmonary and extrapulmonary tuberculosis has been developed by a research group which further allows to distinguish among TB and other infectious diseases [47] (Figure 1). The serological responses of purified mycobacterial glycolipid antigens were examined by a liposome agglutination assay. The assay was able to detect very low antiglycolipid antibody concentrations in the infected individuals with 94% sensitivity and 98.3% specificity. H37Rv antigens were isolated, purified, and characterized for this assay. These purified antigens were intercalated with liposome particles and were observed to specifically bind to the antiglycolipid antibodies present in the sera of patients with tuberculosis, resulting in the formation of a blue agglutination. This protocol clearly differentiates healthy controls and M. bovis BCG-vaccinated subjects from those with active tuberculosis. The resultant diagnostic tool, the TB Screen Test, is more economical and rapid (4 min) than other currently available products and can be used for the mass screening of a heavily afflicted population [47].
Molecular identification of mutations conferring resistance to rifampin, isoniazid and pyrazinamide among Mycobacterium tuberculosis isolates from Iran
Published in Journal of Chemotherapy, 2020
Ahad Ali Haratiasl, Gholamreza Hamzelou, Sirus Amini, Jalil Kardan-Yamchi, Mehri Haeili, Fereshteh Heidari, Mohammad Mehdi Feizabadi
Isolates showing resistance to rifampin, isoniazid, and pyrazinamide were selected and subjected to PCR amplification of different genes accounting for resistance. Primers described in Table 1 were used to amplify different fragments including, 570 bp for the rpoB, 209 bp and 704 bp for katG, 455 bp for inhA promoter region, 860 bp for ahpC coding region, 456 bp for oxyR-ahpC intergenic region, and 954 bp for pncA genes. Amplification was performed using Applied Biosystems Vertical 96 well under the following conditions: 1 cycle of 95 °C for 10 min, followed by 30/35 cycles of 95 °C for 40 s, 59 °C (rpoB), 56 °C (ahpc), 63 °C (oxyR-ahpC intergenic region, katG and inhA), 57 °C (pncA) for 30 s, 72 °C for 30 s, with a final extension at 72 °C for 10 min. The PCR products were analyzed on 1.5% agarose gel and purified for sequencing. PCR products were sent to the MACROGENE Corporation (South Korea) and the MICROSYNTH Company (Switzerland) for Sanger sequencing. Sequencing results were analyzed using Chromaspro v2.6.6 software and compared to the genome of H37Rv (NC-000962.3) strain as a reference. The chromatogram of sequencing results can be seen in figure S1 in the supplementary material.