Identification of Novel Cytotoxic T Lymphocyte Epitopes of Drug- Resistance Related Protein InhA from Mycobacterium tuberculosis

2020 ◽  
Vol 27 (11) ◽  
pp. 1141-1150
Author(s):  
Dezhi Li ◽  
Zelong Dou ◽  
Yahong Wu ◽  
Yuanming Qi ◽  
Junhui Chen ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Mycobacterium Tuberculosis ◽  
Solid Phase ◽  
Immune Defense ◽  
Phase Method ◽  
Enzyme Linked Immunosorbent Assay ◽  
Drug Resistant ◽  
Ctl Epitopes ◽  
Ldh Assay ◽  
Ifn Γ

Background: Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB), especially the drug-resistant MTB, poses serious challenges to human healthcare worldwide. Cytotoxic T lymphocytes (CTLs) play a vital role in immune defense against MTB. Objective: To identify novel CTL epitopes that could induce cellular immunity against MTB infections. Methods: The HLA-A*0201 restricted CTL epitopes of the drug-resistant protein InhA from MTB were predicted by online algorisms and synthesized by the Fmoc solid phase method. The candidate peptides were used to induce CTLs from human peripheral blood mononuclear cells (PBMCs) of HLA-A*0201 healthy donors and the HLA-2.1/Kb mice. IFN-γ productions of CTLs were detected by enzyme linked immunospot assay (ELISPOT), flow cytometry and enzyme-linked immunosorbent assay (ELISA), and cytotoxicity was analyzed by lactate dehydrogenase (LDH) assay. Results: A group of 4 epitopes were screened out with high affinities to HLA-A*0201. ELISPOT and flow cytometry analysis indicated these peptides significantly induced that IFN-γ release of CTLs from the HLA-A*0201+/PPD+ donors, as the mutant analogues had more potent stimulation effects. LDH assay showed that CTLs from PPD+ donors and the immunized mice exhibited significant cytotoxicity and low cross-reactivity. ELISA analysis revealed comparative levels of IFN-γ were released by CTLs isolated from the mice spleen. Conclusion: Our study has identified 4 novel CTL epitopes of InhA that could elicit potent CTL immunity, establishing a foundation for the development of multivalent peptide vaccines against the drug-resistant MTB.

scholarly journals PERCENTAGE OF CD3+ T LYMPHOCYTES EXPRESSING IFN-γ AFTER CFP-10 STIMULATION (Persentase Limfosit T-CD3+ yang Mengekspresikan Interferon Gamma Setelah Stimulasi Antigen CFP-10)

2018 ◽  
Vol 23 (1) ◽  
pp. 31
Author(s):  
Yulia Nadar Indrasari ◽  
Betty Agustina Tambunan ◽  
Jusak Nugraha ◽  
Fransiska Sri Oetami
Keyword(s):  
Flow Cytometry ◽  
Mycobacterium Tuberculosis ◽  
T Lymphocytes ◽  
Interferon Gamma ◽  
Tuberculosis Antigen ◽  
Mycobacterium Tuberculosis Antigen ◽  
Ifn Γ

Tuberkulosis (TB) merupakan penyakit infeksi menular, disebabkan oleh Mycobacterium tuberculosis. Respons imun adaptif yangdiperantarai oleh limfosit T berperan sangat penting dalam menyingkirkan bakteri intraseluler. Hasilan sitokin IFN-γ merupakanmekanisme efektor utama dari limfosit T. Pengembangan vaksin yang efektif dalam melawan infeksi TB mempertimbangkan faktor yangmengatur hasilan IFN-γ. CFP-10 merupakan antigen yang disekresikan oleh Mycobacterium tuberculosis. Antigen ini dikenal sebagaikomponen vaksin potensial untuk TB. Tujuan penelitian ini adalah membandingkan respons imun seluler yaitu persentase limfosit T-CD3+yang mengekspresikan IFN-γ setelah dirangsang antigen CFP-10 di pasien TB paru kasus baru, TB laten dan orang sehat. Penelitianini menggunakan desain eksperimen murni di laboratorium secara in vitro pada kultur PBMC pasien TB paru kasus baru, TB latendan orang sehat. Subjek penelitian adalah 8 pasien TB paru kasus baru, 7 TB laten dan 7 orang sehat di RS Khusus Paru Surabaya.Pemeriksaan persentase limfosit T-CD3+ yang mengekspresikan IFN-γ dengan metode Flow cytometry (BD FACSCalibur). Hasil dianalisisdengan Kruskal-Wallis atau ANOVA satu arah. Rerata persentase limfosit T-CD3+ yang mengekspresikan IFN-γ di TB paru kasus barusetelah stimulasi antigen CFP-10 (4,36%) lebih tinggi daripada sebelum stimulasi (3,50%) (nilai P=0,015). Rerata persentase limfositT-CD3+ yang mengekspresikan IFN-γ di TB laten setelah stimulasi antigen CFP-10 (3,96%) lebih tinggi dibandingkan sebelum stimulasi(2,50%) tetapi tidak bermakna (nilai P=0,367). Rerata persentase limfosit T- CD3+ yang mengekspresikan IFN-γ di orang sehat setelahstimulasi (1,66%) lebih rendah daripada sebelum stimulasi (2,89%) tetapi tidak bermakna (nilai P=0,199). Perubahan persentaselimfosit T-CD3+ yang mengekspresikan IFN-γ setelah stimulasi antigen CFP-10 antarkelompok tidak berbeda bermakna (nilai P=0,143).Berdasarkan hasil telitian ini dapat disimpulkan bahwa terdapat peningkatan persentase limfosit T-CD3+ yang mengekspresikan IFN-γdi TB paru kasus baru setelah stimulasi antigen CFP-10. Hal ini menunjukkan limfosit T-CD3+ yang mengekspresikan IFN-γ berperandalam perlindungan terhadap infeksi TB paru.

scholarly journals Evaluation of a lateral flow assay for rapid and simple detection of IFN-γ for the diagnosis of Mycobacterium tuberculosis infection

2020 ◽  
Author(s):  
Jeong-Ran Kim ◽  
Hae Yeong Kang ◽  
Su-Bin Seong ◽  
Nari Kim ◽  
Tae Sun Shim ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Lateral Flow ◽  
Lateral Flow Assay ◽  
Tuberculosis Infection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mycobacterium Tuberculosis Infection ◽  
Blood Samples ◽  
Simple Detection ◽  
Laboratory Workers ◽  
Ifn Γ

Abstract Background: Interferon-gamma (IFN-γ) release assays (IGRAs) are useful for the diagnosis of Mycobacterium tuberculosis infection. Current IGRAs use either enzyme-linked immunosorbent assay (ELISA) or enzyme-linked immunospot assay, which require complex procedures and techniques to determine IFN-γ secretion. We aimed to compare the usefulness of the easy-to-use lateral flow assay (LFA) with that of the QuantiFERON-TB Gold In-Tube (QFT-GIT) or QuantiFERON-TB Gold Plus (QFT-plus) ELISAs for detecting IFN-γ, produced by the blood T cells stimulated by tuberculosis (TB) antigen. Methods: Following informed consent, 176 participants, including health care workers such as TB laboratory workers and radiologists, were enrolled for the study from June 2017 to June 2018. Blood samples were collected and tested using QFT-GIT and QFT-plus. The secreted IFN-γ was quantified by LFA, which took approximately 15 min, and ELISA, which took approximately 3 h. Results: A total of 176 blood samples were screened. The positive rates of QFT-GIT and QFT-plus were 34.1% and 37.5%, respectively. Overall agreement between QFT-GIT and QFT-plus was 93.1% ( κ = 0.86). The positive rates of LFA with QFT-GIT tube and QFT-plus tube were 25.6% and 31.3%, respectively, overall agreement of LFA being 90.3% ( κ = 0.78) and 89.2% ( κ = 0.77), respectively, compared to the QFT-GIT and QFT-plus ELISA. Conclusion: The ability of LFA to measure IFN-γ was similar to that of ELISA. The current findings suggested that the new LFA could be more conveniently utilized for diagnosing TB infection.

Catgut Implantation at Acupoints has an Immunosuppressive Effect on Autoimmune Uveitis by Regulating Th1/Th17 Lymphocytes

2021 ◽  
Author(s):  
Feifei Chen ◽  
Jianying Zhao ◽  
Shuyang Zhong ◽  
Fengming Zheng ◽  
Xiaobo Hao
Keyword(s):  
Flow Cytometry ◽  
Rat Model ◽  
Inhibitory Effect ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mrna Levels ◽  
Autoimmune Uveitis ◽  
Post Immunization ◽  
Th17 Lymphocytes ◽  
Catgut Implantation At Acupoints ◽  
Ifn Γ

Catgut implantation at acupoints (CIA) has a long history as a medical treatment for a wide variety of diseases, including autoimmune diseases. However, the effect and mechanism of this therapy in autoimmune uveitis is still largely unknown. The aim of this study was to explore the immunity-inhibitory effect of CIA in an experimental autoimmune uveitis (EAU) rat model. EAU was established in Lewis rats by the injection of IRBP1177–1191 peptide. The rats were randomly divided into control and CIA groups. Phenotypic and histological assessments were performed days 9, 13, 18, 23 post-immunization. The percentage of Th1 and Th17 lymphocytes isolated from lymph nodes were determined by flow cytometry. The expression of IL-17 and IFN-γ was detected by real-time quantitative PCR and enzyme-linked immunosorbent assay (ELISA). In the CIA group, delayed mild inflammation was observed. Pathological investigation found alleviated infiltration of lymphocytes and ocular damage. Flow cytometry showed significantly decreased Th17 lymphocytes at day 9, 13, and 18 post-immunization (P<0.05) and no significant changes at day 23 post-immunization (P=0.868) after CIA. The Th1 lymphocytes were significantly decreased at day 13 and 18 post-immunization (P<0.05) and comparable at day 9 (P=0.111) and 23 (P=0.551) post-immunization in the CIA group. IL-17 and IFN-γ mRNA levels were notably decreased at day 9, 13 and 18 post-immunization (P<0.05) and showed a downward trend at day 23 post-immunization, although with no significance (P=0.080 and P=0.137, respectively) after CIA. Serum IL-17 and IFN-γ levels in the CIA group were significantly decreased at day 9, 13 and 18 post-immunization (P<0.05) and were comparable at day 23 post-immunization (P=0.078 and P=0.979, respectively). Ocular inflammation was markedly inhibited after catgut implantation at Pishu (BL20) and Shenshu (BL23) acupoints in an EAU rat model. Moreover, CIA reduced Th1 and Th17 lymphocytes and the expression IFN-γ and IL-17.

scholarly journals Role of Interferon-Gamma (Ifn-γ) in Immune Response Regulation in Hiv-1 and Hiv-1 + Mycobacterium Tuberculosis (Tb) Infected Patients / Interferona-gamma (Ifn-γ) Loma Imūnās Atbildes Regulēšanā Pacientiem Ar HIV-1 un HIV-1 + mycobacterium Tuberculosis

2016 ◽  
Vol 70 (4) ◽  
pp. 211-214
Author(s):  
Inga Januškevica ◽  
Baiba Rozentāle ◽  
Elvīra Hagina ◽  
Jeīena Eglīte ◽  
Tatjana Kolupajeva ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Viral Load ◽  
University Hospital ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cell Counts ◽  
Cd4 Cell Counts ◽  
Ifn Γ ◽  
Cd4 Cell ◽  
Hiv 1

Abstract The aim of this research was to investigate the role of IFN-γ in interaction between IL-10, IL-18, IL-1b, CD4 cell counts and HIV-1 RNA viral load in the development of HIV-1 in patients co-infected with Mycobacterium tuberculosis (TB). The study was conducted by Rīga East Clinical University Hospital with data from the HIV-1 register, in collaboration with the RSU Joint Laboratory of Clinical Immunology and Immunogenetics. 200 HIV-1 infected patients and 184 HIV-1 with TB co-infection patients divided in four groups were included in the study. IFN-γ, IL-10, IL-18, IL-1b levels were measured in serum with commercially enzyme-linked immunosorbent assay (ELISA Vector-Best Corporation, Novosibirsk, Russia). CD4 cell counts were measured by flow Partec IVD cytometry (USA). HIV-1 RNA quantification was performed using the COBAS AmpliPrep/COBAS Taqman HIV-1 Test (Germany). All groups were compared with each another. IFN-γ production was significantly lower, and IL-10 and CD4 cell counts were significantly higher, in HIV-1 patients without TB compared with the other groups. The group with HIV-1 and TB had significantly elevated IL-18 production. HIV patients with primary TB had significantly elevated IFN-γ production and HIV-1 RNA viral load and significantly lower IL-10 production.

scholarly journals Influence of Immunotherapy with Autologous Dendritic Cells on Innate and Adaptive Immune Response in Cancer

2013 ◽  
Vol 7 ◽  
pp. CMO.S12268 ◽  
Author(s):  
Bruna F. Matias ◽  
Tânia M. De Oliveira ◽  
Cláudia M. Rodrigues ◽  
Douglas R. Abdalla ◽  
Letícia Montes ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Immune Response ◽  
Dendritic Cell ◽  
B Lymphocytes ◽  
Dendritic Cell Vaccine ◽  
Cell Vaccine ◽  
Enzyme Linked Immunosorbent Assay ◽  
Autologous Dendritic Cell ◽  
Tnf Α ◽  
Ifn Γ

The objective of this study was to evaluate some of the mechanisms involved in the activation of the immune system in patients with advanced-stage cancer (n = 7) who received an autologous dendritic cell vaccine. We examined the immune response mediated by macrophages (CD14+), natural killer cells (CD56+), and B lymphocytes (CD19+) by flow cytometry and assessed the expression of Th1 (IFN-γ, TNF-α, IL-2, and IL-12), Th2 (IL-4), and Treg (TGF-β) cytokines by flow cytometry and an enzyme-linked immunosorbent assay. The CD14+ TNF-α+ population was significantly increased ( P < 0.04) when patients received the vaccine; IL-2 expression in both NK cells and in B lymphocytes was increased after a transient initial increase showed a nearly significant decrease ( P < 0.07 and P < 0.06 respectively), whereas the CD19+ and CD56+ populations did not show significant changes. Dendritic cell-based immunotherapy led to increased secretion of IFN-γ and IL-12 and reduced secretion of TGF-β. In conclusion, it is likely that the autologous dendritic cell vaccine stimulated the immune cells from the peripheral blood of patients with cancer and generally increased the production of Th1 cytokines, which are related to immunomodulatory responses against cancer.

scholarly journals DOP68 Thiomyristoyl ameliorates colitis by blocking the differentiation of Th17 cells via STAT3/IL-6 signal pathway

2020 ◽  
Vol 14 (Supplement_1) ◽  
pp. S105-S106
Author(s):  
Y XU ◽  
M zhang ◽  
G Xu ◽  
X Zou
Keyword(s):  
Flow Cytometry ◽  
Western Blotting ◽  
Th17 Cells ◽  
Culture Supernatant ◽  
Specific Inhibitor ◽  
Signal Pathway ◽  
T Cell Subsets ◽  
Enzyme Linked Immunosorbent Assay ◽  
Control Group ◽  
Ifn Γ

Abstract Background The pathogenesis of inflammatory bowel disease (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), has not been fully elucidated, but a strong correlation between IBD and the immune dysregulation of the host has been persistently detected. Sirtuin 2 (SIRT2), a histone deacetylase, has recently been found to play an important role in inflammation, infection and immunomodulatory. As a potent SIRT2-specific inhibitor, thiomyristoyl (TM) has an extensive anticancer activity, but its anti-inflammatory property remains unclear. Methods Human peripheral blood mononuclear cells (PBMC) were differentiated into T helper 17 (Th17) cells in vitro and were treated with TM simultaneously. Interleukin (IL)-17A in the culture supernatant, the ratio of T cells, the levels of phosphorylated-signal transducer and activator of transcription (p-stat3) and phosphorylated-nuclear factor kappa-B (p-NF κB) were determined by enzyme-linked immunosorbent assay (ELISA), flow cytometry or western blotting, respectively. Then, colitis C57BL/6J mice induced by 2.5% dextran sulphate sodium salt (DSS) were treated with TM, and were evaluated by disease activity index (DAI) and haematoxylin-eosin (HE) staining. T-cell subsets in the mice spleen, IL-6, IL-10 and IL-17A in serum, related factors retinoic acid receptor-related orphan receptor-γ t (ROR-γt), forkhead box protein P3 (FOXP3), IL-17A, interferon γ γ (IFN-γ), IL-23, IL-10 and hypoxia-inducible factor (HIF)-1α in mice colon were measured by flow cytometry, ELISA, quantitative real-time polymerase chain reaction (Q-PCR) and western blotting, respectively. Results Compared with the positive control group, the levels of IL-17A in culture supernatant, the percentage of Th17, the levels of p-stat3 and p-NF kappa B in cell lysate were lower in the TM group. Compared with PBS-treated colitis mice, TM-treated colitis mice had longer colons, fewer weight-losses, lower DAI and histopathologic scores. Interestingly, although the expression of IFN-γ, IL-17A, and ROR-γt was inhibited in the colons of TM-treated mice, the level of FOXP3 did not change. Consistently, the percentage of spleen Th17 cells was decreased in the TM group while the percentage of Treg cells was not affected. In addition, the TM group had reduced levels of IL-23 and HIF-1α, and an increased level of IL-10 in the colon, compared with colitis group. Conclusion TM ameliorates DSS-induced experimental colitis by blocking the differentiation of Th17 cells, which may be associated with the STAT3/IL-6 signal pathway. SIRT2 may represent a potential target for the treatment of IBD.

scholarly journals Flow cytometry analysis of CD4+IFN-γ+ T-cells for the diagnosis of mycobacterium tuberculosis infection

2015 ◽  
Vol 90 (3) ◽  
pp. 303-311 ◽  
Author(s):  
Chrysovalantis V. Papageorgiou ◽  
Andreas Anastasopoulos ◽  
Maria Ploussi ◽  
Michail Leventopoulos ◽  
Simona Karabela ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Mycobacterium Tuberculosis ◽  
Tuberculosis Infection ◽  
Flow Cytometry Analysis ◽  
Mycobacterium Tuberculosis Infection ◽  
Ifn Γ

scholarly journals MyD88 Primes Macrophages for Full-Scale Activation by Interferon-γ yet Mediates Few Responses to Mycobacterium tuberculosis

2003 ◽  
Vol 198 (7) ◽  
pp. 987-997 ◽  
Author(s):  
Shuangping Shi ◽  
Carl Nathan ◽  
Dirk Schnappinger ◽  
Jörg Drenkow ◽  
Michele Fuortes ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Large Scale ◽  
Nuclear Factor Κb ◽  
Interferon Γ ◽  
Full Scale ◽  
Enzyme Linked Immunosorbent Assay ◽  
Ifn Γ ◽  
Microbial Products ◽  
Oxide Synthase

Macrophages are activated from a resting state by a combination of cytokines and microbial products. Microbes are often sensed through Toll-like receptors signaling through MyD88. We used large-scale microarrays in multiple replicate experiments followed by stringent statistical analysis to compare gene expression in wild-type (WT) and MyD88−/− macrophages. We confirmed key results by quantitative reverse transcription polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay. Surprisingly, many genes, such as inducible nitric oxide synthase, IRG-1, IP-10, MIG, RANTES, and interleukin 6 were induced by interferon (IFN)-γ from 5- to 100-fold less extensively in MyD88−/− macrophages than in WT macrophages. Thus, widespread, full-scale activation of macrophages by IFN-γ requires MyD88. Analysis of the mechanism revealed that MyD88 mediates a process of self-priming by which resting macrophages produce a low level of tumor necrosis factor. This and other factors lead to basal activation of nuclear factor κB, which synergizes with IFN-γ for gene induction. In contrast, infection by live, virulent Mycobacterium tuberculosis (Mtb) activated macrophages largely through MyD88-independent pathways, and macrophages did not need MyD88 to kill Mtb in vitro. Thus, MyD88 plays a dynamic role in resting macrophages that supports IFN-γ–dependent activation, whereas macrophages can respond to a complex microbial stimulus, the tubercle bacillus, chiefly by other routes.

scholarly journals Peptides-Based Vaccine MP3RT Induced Protective Immunity Against Mycobacterium Tuberculosis Infection in a Humanized Mouse Model

2021 ◽  
Vol 12 ◽  
Author(s):  
Wenping Gong ◽  
Yan Liang ◽  
Jie Mi ◽  
Zaixing Jia ◽  
Yong Xue ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
T Lymphocytes ◽  
Binding Capacity ◽  
Protective Efficacy ◽  
Humanized Mice ◽  
Enzyme Linked Immunosorbent Assay ◽  
Human Beings ◽  
Wild Type ◽  
New Findings ◽  
Ifn Γ

BackgroundTuberculosis (TB) is still a global infectious disease that seriously threatens human beings. The only licensed TB vaccine Bacille Calmette-Guérin (BCG)’s protective efficacy varies significantly among populations and regions. It is very urgent to develop more effective vaccines.MethodsIn this study, eleven candidate proteins of Mycobacterium tuberculosis were selected to predict peptides with high-affinity binding capacity for the HLA-DRB1*01:01 molecule. The immunodominant peptides were identified with the enzyme-linked immunospot assay (ELISPOT) and linked in silico to result in a novel polypeptide vaccine in Escherichia coli cells. The vaccine’s protective efficacy was evaluated in humanized and wild-type C57BL/6 mice. The potential immune protective mechanisms were explored with Enzyme-linked Immunosorbent Assay (ELISA), flow cytometry, and ELISPOT.ResultsSix immunodominant peptides screened from 50 predicted peptides were used to construct a new polypeptide vaccine named MP3RT. After challenge with M. tuberculosis, the colony-forming units (CFUs), lung lesion area, and the number of inflammatory cells in humanized mice rather than wild-type mice vaccinated with MP3RT were significantly lower than these in mice immunized with PBS. The humanized mice vaccinated with MP3RT revealed significant increases in IFN-γ cytokine production, IFN-γ+ T lymphocytes, CD3+IFN-γ+ T lymphocytes, and the MP3RT-specific IgG antibody.ConclusionsTaken together, MP3RT is a promising peptides-based TB vaccine characterized by inducing high levels of IFN-γ and CD3+IFN-γ+ T lymphocytes in humanized mice. These new findings will lay a foundation for the development of peptides-based vaccines against TB.

A PCR Referenced Comparative Study for Evaluation of Different Mycobacterium tuberculosis DNA Extraction Methods Directly from Sputum and from LJ Culture Isolates in Egypt

2021 ◽  
Vol 30 (2) ◽  
pp. 59-65
Author(s):  
Heba Elshahawy ◽  
Ehab B. Rakha ◽  
Mona B. Elhadidi ◽  
Sanaa S. Hamam
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Dna Extraction ◽  
Solid Phase ◽  
Laboratory Diagnosis ◽  
Extraction Methods ◽  
Phase Method ◽  
Conventional Pcr ◽  
Dna Yield ◽  
Microbiological Methods ◽  
Solid Phase Method

Background: Tuberculosis is a critical infectious disease primarily affecting the lungs and is more common in developing countries. In the 21st century, it forms a significant problem for world public health especially with the emergence and rising of drug resistant TB. Microbiological methods are the clue for the laboratory diagnosis. The ordinary methods for TB identification showed either weak sensitivity as in microscopy or lateness for many weeks as in culture. The evolution in molecular biology gives a chance for fast diagnosis of Mycobacterium tuberculosis helping start proper treatment early and holding its spread. The initial critical step in PCR is DNA extraction. Objective: The aim to evaluate different extraction methods of Mycobacterium tuberculosis retrieved directly from sputum samples and from LJ isolates from same patients and comparing DNA yield using conventional PCR. Methodology: DNA from 32 sputum samples from TB patients extracted by solid, digestion and phenol extraction methods, DNA from 40 LJ isolates extracted by solid, boiling and Cetyl trimethylammonium bromide methods. Extracted DNA was evaluated by conventional PCR. Results: Among 32 sputum samples, the extracted DNA by phenol method was 21/32 (65.62%) with highest DNA yield, digestion method 14/32 (43.75%) and solid phase method 1/32 (2.5%) with least DNA yield. From 40 MTB LJ culture isolates, the extracted DNA by boiling method was 28/40 (70%), CTAB method 18/40 (45%) and solid phase method 2/40 (5%). Conclusion: Phenol method was the best method (mean rank 2.34) for DNA extraction from sputum samples, while the easy and economic boiling method was the best method (mean rank 2.45) for DNA extraction from LJ culture isolates. The worst method of DNA extraction from both sputum and culture was phase solid method. A greater and easier yield of DNA was obtained from MTB LJ Culture than sputum.

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