scholarly journals Detection of Viable Mycobacterium tuberculosis by Reverse Transcriptase-Strand Displacement Amplification of mRNA

1999 ◽  
Vol 37 (3) ◽  
pp. 518-523 ◽  
Author(s):  
T. J. Hellyer ◽  
L. E. DesJardin ◽  
L. Teixeira ◽  
M. D. Perkins ◽  
M. D. Cave ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Reverse Transcriptase ◽  
Therapeutic Efficacy ◽  
Sputum Sample ◽  
Drug Susceptibility ◽  
Strand Displacement ◽  
Strand Displacement Amplification ◽  
Amplification System

Numerous assays have been described for the detection of DNA and rRNA sequences that are specific for the Mycobacterium tuberculosis complex. Although beneficial to initial diagnosis, such assays have proven unsuitable for monitoring therapeutic efficacy owing to the persistence of these nucleic acid targets long after conversion of smears and cultures to negative. However, prokaryotic mRNA has a typical half-life of only a few minutes and we have previously shown that the presence of mRNA is a good indicator of bacterial viability. The purpose of the present study was to develop a novel reverse transcriptase-strand displacement amplification system for the detection of M. tuberculosis α-antigen (85B protein) mRNA and to demonstrate the use of this assay in assessing chemotherapeutic efficacy in patients with pulmonary tuberculosis. The assay was applied to sequential, noninduced sputum specimens collected from four patients: 10 of 11 samples (91%) collected prior to the start of therapy were positive for alpha-antigen mRNA, compared with 1 of 8 (13%), 2 of 8 (25%), 2 of 8 (25%), and 0 of 8 collected on days 2, 4, 7, and 14 of treatment, respectively. In contrast, 39 of 44 samples (89%) collected on or before day 14 were positive for α-antigen DNA. The loss of detectable mRNA corresponded to a rapid drop over the first 4 days of treatment in the number of viable organisms present in each sputum sample, equivalent to a mean fall of 0.43 log10 CFU/ml/day. Analysis of mRNA is a potentially useful method for monitoring therapeutic efficacy and for rapid in vitro determination of drug susceptibility.

scholarly journals Detection of Mycobacterium tuberculosis DNA with thermophilic strand displacement amplification and fluorescence polarization

1996 ◽  
Vol 42 (10) ◽  
pp. 1604-1608 ◽  
Author(s):  
G T Walker ◽  
C P Linn
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Restriction Enzyme ◽  
Fluorescence Polarization ◽  
Target Sequence ◽  
Strand Displacement ◽  
Insertion Element ◽  
Strand Displacement Amplification ◽  
Simple Protocol ◽  
Polymerase Binding

Abstract Strand displacement amplification (SDA) is an isothermal, in vitro method for diagnostics that amplifies a target DNA sequence by using a restriction enzyme and DNA polymerase. We have combined a new thermophilic form of SDA that involves restriction enzyme BsoBI and polymerase exo-Bca with fluorescence polarization for detection of Mycobacterium tuberculosis DNA by using the IS6110 insertion element as the target sequence. A 5'-fluorescein-labeled oligodeoxynucleotide detector probe hybridizes to the amplified product as it rises in concentration during SDA, and the single- to double-stranded conversion is monitored through an increase in fluorescence polarization. The associated change in polarization upon amplification of the target sequence is enhanced by specific polymerase binding to the double-stranded detector probe. Fewer than 10 M. tuberculosis genomes can be amplified and detected with an extremely simple protocol that takes only 20 min and uses relatively simple instrumentation and reagents, all of which can be purchased off-the-shelf.

scholarly journals Quantitative Analysis of mRNA as a Marker for Viability of Mycobacterium tuberculosis

1999 ◽  
Vol 37 (2) ◽  
pp. 290-295 ◽  
Author(s):  
T. J. Hellyer ◽  
L. E. DesJardin ◽  
G. L. Hehman ◽  
M. D. Cave ◽  
K. D. Eisenach
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Quantitative Analysis ◽  
16S Rrna ◽  
Therapeutic Efficacy ◽  
Rapid Determination ◽  
Drug Susceptibility ◽  
Resistant Strains ◽  
Time Required ◽  
Rrna Sequences

Numerous assays which use conserved DNA or rRNA sequences as targets for amplification have been described for the diagnosis of tuberculosis. However, these techniques have not been applied successfully to the monitoring of therapeutic efficacy owing to the persistence of amplifiable nucleic acid beyond the point at which smears and cultures become negative. Semiquantitative analysis of rRNA has been used to reduce the time required for antimicrobial susceptibility testing of Mycobacterium tuberculosis, although growth for up to 5 days in the presence of some drugs is still required to discriminate resistant strains. The purpose of the present study was to determine whether quantitative analysis of M. tuberculosis mRNA could be used to assess bacterial viability and to illustrate the application of this technique to rapid determination of drug susceptibility. Levels of mRNA encoding the 85B protein (α-antigen), IS6110 DNA, and 16S rRNA were compared in parallel cultures of M. tuberculosis that were treated with either no drug, 0.2 μg of isoniazid per ml, or 1 μg of rifampin per ml. Exposure of sensitive strains to isoniazid or rifampin for 24 h reduced the levels of 85B mRNA to <4 and <0.01%, respectively, of those present in control cultures without drug. In contrast, the levels of IS6110 DNA and 16S rRNA did not diminish over the same period. Strains which were resistant to either isoniazid or rifampin demonstrated no reduction in 85B mRNA in the presence of the drug to which they were nonresponsive. Quantitative analysis of 85B mRNA offers a potentially useful tool for the rapid determination of M. tuberculosis drug susceptibility and for the monitoring of therapeutic efficacy.

scholarly journals Strand displacement amplification (SDA) and transient-state fluorescence polarization detection of Mycobacterium tuberculosis DNA

1996 ◽  
Vol 42 (1) ◽  
pp. 9-13 ◽  
Author(s):  
G T Walker ◽  
J G Nadeau ◽  
C P Linn ◽  
R F Devlin ◽  
W B Dandliker
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Dna Sequence ◽  
Fluorescence Polarization ◽  
Near Infrared ◽  
Detection System ◽  
Transient State ◽  
Strand Displacement ◽  
Strand Displacement Amplification ◽  
Polarization Detection

Abstract Strand displacement amplification (SDA) is an isothermal, in vitro method of amplifying a DNA sequence for diagnostic purposes. We have combined SDA with fluorescence polarization detection in a closed, homogeneous format. A fluorescently labeled oligodeoxynucleotide detector probe hybridizes to the amplification product that increases in concentration during SDA. The single- to double-stranded conversion of the probe is accompanied by an increase in fluorescence polarization values, which can be measured in real-time without physical manipulation of the sample. The probe was labeled with the near-infrared dye La Jolla Blue, and fluorescence polarization was measured on a transient-state fluorometer. We have applied this homogeneous SDA/detection system to a target DNA sequence specific for Mycobacterium tuberculosis DNA.

Fluorometric determination of microRNA based on strand displacement amplification and rolling circle amplification

2017 ◽  
Vol 184 (11) ◽  
pp. 4359-4365 ◽  
Author(s):  
Yunlei Zhou ◽  
Bingchen Li ◽  
Minghui Wang ◽  
Jun Wang ◽  
Huanshun Yin ◽  
...  
Keyword(s):  
Rolling Circle Amplification ◽  
Strand Displacement ◽  
Fluorometric Determination ◽  
Strand Displacement Amplification ◽  
Rolling Circle

scholarly journals Microscopic Observation Drug Susceptibility (MODS) Assay: A Convenient Method for Determining Antibiogram of Clinical Isolates of Mycobacterium tuberculosis in Ghana

2020 ◽  
Vol 8 (1) ◽  
pp. 5
Author(s):  
Enid Owusu ◽  
Mercy Jemima Newman
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Rural Areas ◽  
Microscopic Observation ◽  
Drug Susceptibility ◽  
Ease Of Use ◽  
Health Centers ◽  
Culture Methods ◽  
Susceptibility Tests ◽  
User Friendly

(1) Background: Present methods for drug susceptibility tests (DST) rely on culture methods that are sophisticated and relatively faster, or a slow and cheaper option. These methods frustrate disease control; therefore, there is a need for methods that incorporate key functions of microscopy and culture, with reduced cost burden and sophistry. Thus, the purpose of this study was to identify which, among the most commonly used (in Ghana) methods, can conveniently be used at health centers located in rural areas for effective DST determination of Mycobacterium tuberculosis (MTB). (2) Methods: Mycobacterium tuberculosis isolates were tested for their susceptibility to streptomycin, isoniazid, rifampicin, ethambutol (SIRE), and pyrazinamide by microscopic observation drug susceptibility (MODS) and BACTEC MGIT 960 methods. Evaluations were based on shorter turnaround periods, rapidity, ease of use, cost, etc. A comparative analysis was statistically expressed as kappa values. (3) Results: Endpoints for drug susceptibilities by MODS averaged 13 days (7–32), whilst that for BACTEC MGIT 960 was 10 days with a further 12 days to detect resistance. Therefore, a turnaround period of 22 days was needed for DST by BACTEC MGIT 960, compared to 13 days for MODS. There were differences in correlation levels between the two methods, as determined by their kappa values. (4) Conclusion: The MODS assay was found to be less costly, more user-friendly, and still able to be conveniently used at health centers located in rural areas known to be endemic for TB, particularly in Ghana.

scholarly journals In VitroandIn VivoActivities of Three Oxazolidinones against Nonreplicating Mycobacterium tuberculosis

2014 ◽  
Vol 58 (6) ◽  
pp. 3217-3223 ◽  
Author(s):  
Ming Zhang ◽  
Claudia Sala ◽  
Neeraj Dhar ◽  
Anthony Vocat ◽  
Vasan K Sambandamurthy ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Latent Tuberculosis ◽  
Drug Susceptibility ◽  
New Combination ◽  
Drug Candidate ◽  
Bacteriostatic Effect ◽  
Content Type ◽  
New Class ◽  
Modest Activity

ABSTRACTOxazolidinones represent a new class of antituberculosis drugs that exert their function by inhibiting protein synthesis. Here, we compared the activities of three oxazolidinones, linezolid, PNU-100480, and AZD5847, against latent tuberculosis using a simple model employing the streptomycin-starvedMycobacterium tuberculosisstrain 18b. Thein vitrodrug susceptibility results showed that the three oxazolidinones had a bacteriostatic effect against actively growing bacilli but potent bactericidal activity against nonreplicating cells. In the murine model of latent infection withM. tuberculosis18b, the efficacy of the three compounds varied greatly. Indeed, AZD5847 or its prodrug exhibited no activity or only modest activity, respectively, after 2 months of treatment, whereas both linezolid and PNU-100480 were effective against latent bacilli in mice and showed promising outcomes in combination therapy with rifampin. Moreover, the potency of PNU-100480 was significantly greater than that of linezolid, making it an attractive drug candidate in the development of new combination therapies for latent tuberculosis.

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