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

2003 ◽  
pp. 141-155
Author(s):  
Tobin J. Hellyer
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Reverse Transcriptase ◽  
Strand Displacement ◽  
Strand Displacement Amplification

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 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.

scholarly journals Defective Strand-Displacement DNA Synthesis Due to Accumulation of Thymidine Analogue Resistance Mutations in HIV-2 Reverse Transcriptase

2020 ◽  
Vol 6 (5) ◽  
pp. 1140-1153 ◽  
Author(s):  
Samara Martín-Alonso ◽  
Mar Álvarez ◽  
María Nevot ◽  
Miguel Á. Martínez ◽  
Luis Menéndez-Arias
Keyword(s):  
Reverse Transcriptase ◽  
Dna Synthesis ◽  
Strand Displacement ◽  
Resistance Mutations ◽  
Thymidine Analogue
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