EXTRACTING TB DNA FROM MONOCYTES

4 posts / 0 new
Last post
920201687
920201687's picture
EXTRACTING TB DNA FROM MONOCYTES

HI TO ALL,

I HAVE INFECTED SOME MONOCYTES WITH TB(in vitro assay). I now want to confirm that i've infected these cells using REAL TIME PCR.

Does any one have protocols as to the best DNA extraction method (keeping in mind that lysing of the bacterial wall is more difficult versus host cell membranes).
Once the DNA has been extracted, how can i separate the host's DNA from that of the bacteria's???

NOTE: need these results ASAP, all suggestions welcome.

Regards
Aurelia

Tony Rook
Tony Rook's picture
I performed a Solutions

I performed a Solutions Search! on this site (see search tool above) and found the results listed below.

Check it out and try your own Customized Life Science Search.

Takahashi T, Nakayama T. Novel technique of quantitative nested real-time PCR assay for Mycobacterium tuberculosis DNA. J Clin Microbiol. 2006 Mar;44(3):1029-39.

Abstract:
The diagnosis of tuberculous meningitis (TBM) remains a complex issue because the most widely used conventional diagnostic tools, such as culture and PCR assay for cerebrospinal fluid (CSF) samples, are unable to rapidly detect Mycobacterium tuberculosis with sufficient sensitivity in the acute phase of TBM. Based on TaqMan PCR, we designed a novel technique consisting of an internally controlled quantitative nested real-time (QNRT) PCR assay that provided a marked improvement in detection sensitivity and quantification. We applied this novel technique to quantitatively detect M. tuberculosis DNA in CSF samples from patients with suspected TBM. For use as the internal control in the measurement of the M. tuberculosis DNA copy numbers in the QNRT-PCR assay, the original mutation (M) plasmid, which included an artificial random 22-nucleotide sequence within an inserted DNA fragment of the MPB64 gene of M. tuberculosis, was prepared. The QNRT-PCR assay showed high sensitivity and specificity that were approximately equivalent to those of the conventional nested PCR assay. Moreover, the QNRT-PCR assay made it possible to precisely and quantitatively detect the initial copy number of M. tuberculosis DNA in CSF samples. Therefore, compared to the conventional PCR assay, the QNRT-PCR assay can be considered a more useful and advanced technique for the rapid and accurate diagnosis of TBM. To establish the superiority of this novel technique in TBM diagnosis, it will be necessary to accumulate data from a larger number of patients with suspected TBM.

________________________________________________________

Nabin K. Shrestha, Marion J. Tuohy, Gerri S. Hall, Udo Reischl, Steven M. Gordon, and Gary W. Procop. Detection and Differentiation of Mycobacterium tuberculosis andNontuberculous Mycobacterial Isolates by Real-Time PCR. JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 2003, p. 51215126 Vol. 41, No. 11
DOI: 10.1128/JCM.41.11.51215126.2003

Abstract:
Mycobacteria cause a variety of illnesses that differ in severity and public health implications. The differentiation of Mycobacterium tuberculosis from nontuberculous mycobacteria (NTM) is of primary importance for infection control and choice of antimicrobial therapy. Despite advances in molecular diagnostics, the ability to rapidly diagnose M. tuberculosis infections by PCR is still inadequate, largely because of the possibility of false-negative reactions. We designed and validated a real-time PCR for mycobacteria by using the LightCycler system with 18 reference strains and 168 clinical mycobacterial isolates. All clinically significant mycobacteria
were detected; the mean melting temperatures (with 99.9% confidence intervals [99.9% CI] in parentheses) for the different mycobacteria were as follows: M. tuberculosis, 64.35°C (63.27 to 65.42°C); M. kansasii, 59.20°C (58.07 to 60.33°C); M. avium, 57.82°C (57.05 to 58.60°C); M. intracellulare, 54.46°C (53.69 to 55.23°C); M. marinum, 58.91°C (58.28 to 59.55°C); rapidly growing mycobacteria, 53.09°C (50.97 to 55.20°C) or 43.19°C
(42.19 to 44.49°C). This real-time PCR assay with melting curve analysis consistently accurately detected and differentiated M. tuberculosis from NTM. Detection of an NTM helps ensure that the negative result for M. tuberculosis is a true negative. The specific melting temperature also provides a suggestion of the identity of the NTM present, when the most commonly encountered mycobacterial species are considered. In a parallel
comparison, both the LightCycler assay and the COBAS Amplicor M. tuberculosis assay correctly categorized 48 of 50 specimens that were proven by culture to contain M. tuberculosis, and the LightCycler assay correctly characterized 3 of 3 specimens that contained NTM.

________________________________________________________

Volpe E, Cappelli G, Grassi M, Martino A, Serafino A, Colizzi V, Sanarico N, Mariani F. Gene expression profiling of human macrophages at late time of infection with Mycobacterium tuberculosis. Immunology. 2006 Aug;118(4):449-60.

Abstract:
Macrophages play an essential role in the immune response to Mycobacterium tuberculosis (Mtb). Previous transcriptome surveys, by means of micro- and macroarrays, investigated the cellular gene expression profile during the early phases of infection (within 48 hr). However, Mtb remains within the host macrophages for a longer period, continuing to influence the macrophage gene expression and, consequently, the environment in which it persists. Therefore, we studied the transcription patterns of human macrophages for up to 7 days after infection with Mtb. We used a macroarray approach to study 858 human genes involved in immunoregulation, and we confirmed by quantitative real-time reverse transcriptase polymerase chain reaction (q-rt RT-PCR) and by enzyme-linked immunosorbent assay the most relevant modulations. We constantly observed the up-regulation in infected macrophages versus uninfected, of the following genes: interleukin-1 beta and interleukin-8, macrophage inflammatory protein-1 alpha, growth-related oncogene-beta, epithelial cell-derived neutrophil-activating peptide-78, macrophage-derived chemokine, and matrix metalloproteinase-7; whereas macrophage colony-stimulating factor-receptor and CD4 were down-regulated in infected macrophages. Mtb is able to withstand this intense cytokine microenvironment and to survive inside the human macrophage. Therefore we simultaneously investigated by q-rt RT-PCR the modulation of five mycobacterial genes: the alternative sigma factors sigA, sigE and sigG, the alpha-crystallin (acr) and the superoxide dismutase C (sodC) involved in survival mechanisms. The identified host and mycobacterial genes that were expressed until 7 days after infection, could have a role in the interplay between the host immune defences and the bacterial escape mechanisms.

_________________________________________________________

Hillemann D, Galle J, Vollmer E, Richter E. Real-time PCR assay for improved detection of Mycobacterium tuberculosis complex in paraffin-embedded tissues. Int J Tuberc Lung Dis. 2006 Mar;10(3):340-2.

Abstract:
OBJECTIVE: To test the usefulness of a commercially available real-time polymerase chain reaction (PCR) kit for the detection of Mycobacterium tuberculosis complex (MTBC) in formalin-fixed, paraffin-embedded tissues. RESULTS: The examination of 24 specimens of patients with a final diagnosis of TB shows that the real-time PCR assay exhibits a higher sensitivity (66.7%) for the detection of MTBC DNA than an alternative in-house IS6110 PCR (33.3%), whereas staining detected acid-fast bacilli in only two cases (8.3%). CONCLUSION: The real-time PCR assay provides a highly sensitive and specific means for the detection of MTBC DNA in histopathological specimens.

_________________________________________________________

Bernal S., Freyre C., Torres M.J., Jesus de la Calle I., Rodríguez-Iglesias J.M., Palomares J.C., Martín-Mazuelos E. Comparison of a LightCycler real-time PCR method and the BDProbeTec ET system for direct detection of Mycobacterium tuberculosis complex in respiratory specimens. 15th European Congress of Clinical Microbiology and Infectious Diseases
Copenhagen / Denmark, April 2-5, 2005. Abstract number: 1134_01_144.

Objectives:
Detection of micobacteria by culture takes between one week and two months.To reduce the delay in tuberculosis diagnosis, we compared a home-made LightCycler real-time PCR method to the BDProbeTec ET system (Becton Dickinson, Sparks, Md.), for direct detection of Mycobacterium tuberculosis in 60 clinical samples and to the conventional mycobacterial culture.
Methods:
The real-time assay amplifies a region from the IS6110 sequence and the BDProbe Tec amplifies a region of the mycobacterial 16S rDNA. Conventional mycobacterial culture was performed with BACTEC MGIT 960 and Lowestein-Jensen medium. Sixty respiratory specimens were used (36 sputa, 14 pleural fluid and 10 BAL) from patient with negative smear microscopy and a high probability of tuberculosis were assessed by the three methods.
Results:
Of 60 specimens, 10 grew MTBC (and in one culture grew one M. avium-intracellulare). BDProbeTec ET detected 9 of the 10 MTBC culture-positive specimens and gave a positive result in one of the 49 negative cultures. LC real-time PCR detected the same 9 MTBC culture-positive specimens and gave a positive result in another but different from that of the BDProbeTec ET of the 49 negative cultures. Thus, the sensitivities of the real-time PCR method and SDA were similar for respiratory specimens.
Conclusion:
The data demonstrate that both LC real-time PCR and BDProbeTec assays are highly sensitive and specific techniques for the rapid detection of M. tuberculosis comples in respiratory smear negative specimens.

_______________________________________________________

Hopefully one or more of these links will point you in the right direction.

Try your own Solution Search today!

This search engine was built on the Google search engine technology but searches only the selected life science websites which the expert moderators at Scientist Solutions chose. This Customized Life Science Search Engine is continually maintained and update with new relevant life science websites.

Tony Rook
Tony Rook's picture
These references may be of

These references may be of some help too....

Genome Technology's Clinical qPCR Tech Guide - Volume 4

________________________________________________________

Sarah M. Fortune, Alejandra Solache, Alejandra Jaeger, Preston J. Hill, John T. Belisle, Barry R. Bloom, Eric J. Rubin and Joel D. Ernst. Mycobacterium tuberculosis Inhibits Macrophage Responses to IFN-{gamma} through Myeloid Differentiation Factor 88-Dependent and -Independent Mechanisms. he Journal of Immunology, 2004, 172: 6272-6280.

Abstract:
Mycobacterium tuberculosis overcomes macrophage bactericidal activities and persists intracellularly. One mechanism by which M. tuberculosis avoids macrophage killing might be through inhibition of IFN-{gamma}-mediated signaling. In this study we provide evidence that at least two distinct components of M. tuberculosis, the 19-kDa lipoprotein and cell wall peptidoglycan (contained in the mycolylarabinogalactan peptidoglycan (mAGP) complex), inhibit macrophage responses to IFN-{gamma} at a transcriptional level. Moreover, these components engage distinct proximal signaling pathways to inhibit responses to IFN-{gamma}: the 19-kDa lipoprotein inhibits IFN-{gamma} signaling in a Toll-like receptor (TLR)2-dependent and myeloid differentiation factor 88-dependent fashion whereas mAGP inhibits independently of TLR2, TLR4, and myeloid differentiation factor 88. In addition to inhibiting the induction of specific IFN-{gamma} responsive genes, the 19-kDa lipoprotein and mAGP inhibit the ability of IFN-{gamma} to activate murine macrophages to kill virulent M. tuberculosis without inhibiting production of NO. These results imply that inhibition of macrophage responses to IFN-{gamma} may contribute to the inability of an apparently effective immune response to eradicate M. tuberculosis.

_______________________________________________________

Holly M. Scott Algood, Philana Ling Lin, David Yankura, Alvin Jones, John Chan and JoAnne L. Flynn. TNF Influences Chemokine Expression of Macrophages In Vitro and That of CD11b+ Cells In Vivo during Mycobacterium tuberculosis Infection. The Journal of Immunology, 2004, 172: 6846-6857.

Abstract:
Granulomas, focal accumulations of immune cells, form in the lung during Mycobacterium tuberculosis infection. Chemokines, chemotactic cytokines, are logical candidates for inducing migration of T lymphocytes and monocytes to and within the lung. TNF influences chemokine expression in some models. TNF-deficient mice infected with M. tuberculosis are highly susceptible to disease, and granuloma formation is inhibited. Through in vitro assays, we demonstrate that neutralization of TNF in M. tuberculosis-infected macrophages led to a reduction in many inflammatory chemokines, such as C-C chemokine ligand 5, CXC ligand 9 (CXCL9), and CXCL10. In TNF-deficient mice, immune cells migrated to the lungs early after infection, but did not organize to form granulomas within the lung. Although chemokine expression, as measured in whole lung tissue, was not different, the expression of chemokines in the CD11b+ subset of cells isolated ex vivo from the lungs of TNF-deficient mice had reduced expression of C-C chemokine ligand 5, CXCL9, and CXCL10 at early time points after TNF neutralization. Local expression of CXCR3-binding chemokines within the lungs, as determined by in situ hybridization, was also affected by TNF. Therefore, TNF affects the expression of chemokines by macrophages in vitro and CD11b+ cells in vivo, which probably influences the local chemokine gradients and granuloma formation.

______________________________________________________

Robert J. Wilkinson et al. An increase in expression of a Mycobacterium tuberculosis mycolyl transferase gene (fbpB) occurs early after infection of human monocytes. Molecular Microbiology Volume 39 Issue 3 Page 813-821, February 2001

Abstract:
Changes in the mRNA levels of two Mycobacterium tuberculosis genes (fbpB known as antigen 85B, and hspX known as Acr) were studied in infected human monocytes. Antigen 85B is an enzyme involved in cell wall biosynthesis and is also a major target of the immune response. Acr is a stress protein believed to be involved in the bacillary response to adverse conditions and in non-replicating persistence. During the first 24 h of intracellular infection, the intramonocyte 85B mRNA level increased 54-fold (P = 0.00001) and 14.6 times in comparison with the 16S ribosomal rRNA. In contrast, the Acr mRNA fell 14.3 times. Although monocyte cytokine production was very variable, the 24 h secretion of tumour necrosis factor (TNF)-α correlated with the 85B−16S RNA ratio at 24 h (r = 0.77, Pcorr < 0.01). Furthermore, the addition of exogenous TNF-α to cultures was associated with a twofold increase in the 85B−16S ratio and, conversely, neutralization of endogenous TNF-α reduced the ratio. As antigen 85B also induces TNF-α, the positive feedback implied by our findings suggests a previously unsuspected role for this protein in the immunopathogenesis of tuberculosis.

________________________________________________________

B J Johnson and D N McMurray Cytokine gene expression by cultures of human lymphocytes with autologous Mycobacterium tuberculosis-infected monocytes. Infect Immun. 1994 April; 62(4): 14441450.

_______________________________________________________

Michael Weiden, et al. Differentiation of Monocytes to Macrophages Switches the Mycobacterium tuberculosis Effect on HIV-1 Replication from Stimulation to Inhibition: Modulation of Interferon Response and CCAAT/Enhancer Binding Protein Expression The Journal of Immunology, 00, 165: 2028-2039.

Abstract:
HIV-1 replication is inhibited in uninflamed lung macrophages and is stimulated during tuberculosis. Attempts to recapitulate activation of HIV-1 replication in primary monocytes and macrophages ex vivo and in the untreated and PMA-treated THP-1 cell line model in vitro have produced opposite results depending on the state of differentiation of the cells. After infection with Mycobacterium tuberculosis, monocytes enhanced HIV-1 replication and produced a stimulatory 37-kDa CCAAT/enhancer binding protein (C/EBP) transcription factor, whereas macrophages suppressed HIV-1 replication and produced an inhibitory 16-kDa C/EBP transcription factor. IFN- induced inhibitory 16-kDa C/EBP in macrophages, but had no effect on C/EBP expression in monocytes. Macrophages, but not monocytes, were able to activate IFN-stimulated gene factor-3 (ISGF-3), a transcription factor composed of STAT-1, STAT-2, and IFN regulatory factor (IRF)-9, after infection with M. tuberculosis or stimulation with type I IFN. Macrophages expressed IRF-9 DNA-binding activity, but monocytes did not, and addition of the IRF-9 component reconstituted ISGF-3 in extracts of IFN-treated monocytes. Modulation of IFN responsiveness upon differentiation occurred at least in part through a post-transcriptionally regulated increase in IRF-9 expression. Both monocytes and macrophages maintained IFN responsiveness, activating STAT-1 homodimer formation and transcription of the STAT-1 gene after IFN stimulation. In addition, both monocytes and macrophages were able to activate NF-{kappa}B upon infection with M. tuberculosis. These results show that induction of ISGF-3, expression of the inhibitory 16-kDa C/EBP, and suppression of HIV-1 replication via a transcriptional mechanism are macrophage-specific responses to infection with M. tuberculosis.

_______________________________________________________

Good luck!

920201687
920201687's picture
Thank you for your prompt

Thank you for your prompt response. The articles are of great use.
Regards
Aurelia