scholarly journals Genotypic Determination of Mycobacterium tuberculosisAntibiotic Resistance Using a Novel Mutation Detection Method, the Branch Migration Inhibition M. tuberculosis Antibiotic Resistance Test

2000 ◽  
Vol 38 (10) ◽  
pp. 3656-3662 ◽  
Author(s):  
Y. P. Liu ◽  
M. A. Behr ◽  
P. M. Small ◽  
N. Kurn
Keyword(s):  
Nucleic Acids ◽  
Genomic Dna ◽  
Mutation Detection ◽  
Detection Method ◽  
Clinical Isolates ◽  
Base Substitution ◽  
Enzyme Linked Immunosorbent Assay ◽  
Branch Migration ◽  
Migration Inhibition

A novel method for the detection of any alteration within a defined sequence has recently been demonstrated (A. Lishanski, N. Kurn, and E. F. Ullman, Nucleic Acids Res. 28:E42, 2000; A. Lishanski, Clin. Chem. 46:9, 2000). Essential to this method are the generation of partial duplexes that are capable of forming four-stranded structures and the ability to detect inhibition of branch migration in these structures (I. G. Panyutin and P. Hsieh, J. Mol. Biol. 230:413–424, 1993). Inhibition of branch migration indicates the presence of sequence alteration. This mutation detection method, termed branch migration inhibition (BMI), is suitable for the detection of drug resistance in M. tuberculosis, which is frequently associated with multiple mutations within known genes. We describe the genotypic determination of the rifampin (RMP) and pyrazinamide (PZA) susceptibilities of M. tuberculosis isolates, using BMI coupled with the luminescence oxygen channeling immunoassay (LOCI) (E. F. Ullman et al., Proc. Natl. Acad. Sci. USA 91:5426–5430, 1994). RMP and PZA resistances are associated with multiple mutations within the rpoB and pncA genes, respectively.M. tuberculosis genomic DNA samples prepared from 46 clinical isolates were used for genotypic determination of RMP resistance in a “blind study.” Similarly, PZA resistance was determined using genomic DNA samples prepared from 37 clinical isolates. Full agreement of the genotypic and phenotypic determinations of drug susceptibility was demonstrated. RMP susceptibility determination directly from cells of 10 clinical isolates grown in culture was also demonstrated. The genotypic result of only 1 out of 10 isolates did not agree with the phenotypic susceptibility testing result. Sequence analysis of the rpoB gene of this clinical isolate revealed a single base substitution, most likely a silent point mutation. The new BMI-LOCI mutation detection method is a rapid and accurate procedure for the genotypic determination of the RMP and PZA susceptibilities of M. tuberculosis clinical isolates. BMI can also be detected by using commercially available automated enzyme-linked immunosorbent assay plate formats (Lishanski et al., Nucleic Acids Res. 28:E42, 2000).

scholarly journals Branch migration inhibition in PCR-amplified DNA: homogeneous mutation detection

2000 ◽  
Vol 28 (9) ◽  
pp. e42-e42 ◽  
Author(s):  
A. Lishanski ◽  
N. Kurn ◽  
E. F. Ullman
Keyword(s):  
Mutation Detection ◽  
Branch Migration ◽  
Migration Inhibition

scholarly journals Reversion of argE3 to Arg(+) in Escherichia coli AB1157 -an informative bacterial system for mutation detection.

2010 ◽  
Vol 57 (4) ◽  
Author(s):  
Anna Sikora ◽  
Elżbieta Grzesiuk
Keyword(s):  
Dna Damage ◽  
Mutation Detection ◽  
Detection System ◽  
Base Substitution ◽  
E Coli ◽  
Level Ii ◽  
Nonsense Codon ◽  
Amber Suppressor ◽  
Detection Of Mutations

This review concerns reversion of the argE3 (ochre) nonsense mutation to prototrophy in E. coli AB1157 strain as an informative system for mutation detection. Strain AB1157 bears the argE3 (ochre), hisG4 (ochre) and thr-1 (amber) mutations, and the supE44 amber suppressor on its chromosome. The Arg(+) phenotype can be restored by (i) any base substitution at the argE3 site that changes the nonsense UAA codon to any sense nucleotide triplet or to UAG recognized by the supE44 amber suppressor, or (ii) suppressor mutations enabling the reading of the UAA nonsense codon. The argE3 → Arg(+) reversion-based system enables (i) determination of the spontaneous or induced mutation level; (ii) determination of base substitutions (suppressor analysis); (iii) examination of transcription-coupled repair (TCR) since targets for DNA damage are situated on the transcribed or coding strand of DNA; (iv) detection of mutations resulting from single stranded DNA damage. This review focuses on studies carried out since the early 1990s till now with the application of the AB1157-based mutation detection system. Recently, the system has been used to obtain new data on the processes of methyl methanesulfonate-induced mutagenesis and DNA repair in E. coli alkB⁻ mutants.

scholarly journals Direct PCR-free electrochemical biosensing of plant-food derived nucleic acids in genomic DNA extracts. Application to the determination of the key allergen Sola l 7 in tomato seeds

2019 ◽  
Vol 137 ◽  
pp. 171-177 ◽  
Author(s):  
Magda A. Pereira-Barros ◽  
M. Fátima Barroso ◽  
Laura Martín-Pedraza ◽  
Eva Vargas ◽  
Sara Benedé ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Genomic Dna ◽  
Plant Food ◽  
Direct Pcr ◽  
Electrochemical Biosensing ◽  
Tomato Seeds

An Enzyme Linked Immunosorbent Assay for Determination of Tissue Plasminogen Activator Applied to Patients with Thromboembolic Disease

1983 ◽  
Vol 50 (03) ◽  
pp. 740-744 ◽  
Author(s):  
Nils Bergsdorf ◽  
Torbjörn Nilsson ◽  
Per Wallén
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Specific Activity ◽  
Thromboembolic Disease ◽  
Tissue Type ◽  
Enzyme Linked Immunosorbent Assay ◽  
Type Plasminogen Activator ◽  
Tissue Plasminogen ◽  
Normal Human

SummaryUtilizing the immunoglobulin fraction from a goat antiserum against human uterine tissue plasminogen activator, an enzyme- linked immunoassay for tissue-type plasminogen activator in human plasma has been developed. With the new method, the concentration of t-PA in normal human acidified plasma is found to be 4.0 ± 1.8 (SD) ng/ml. It increases to 12 ng/ml after a tomiquet test, and to 14 ng/ml after strenous physical exercise. In a group of patients with idiopathic thromboembolic disease, the resting t-PA concentration was 5 ng/ml and the post-occlusion value 16 ng/ml. Furthermore, the patients also exhibited a normal post-occlusion rise in the concentration of plasmin-α2-antiplasmin complex. However, in 37% of the post-occlusion patient plasmas, virtually no increase in t-PA could be detected by a specific activity assay. The results indicate that the reason for a defective post-occlusion fibrinolytic activity in a majority of cases may be the presence of increased concentrations of a fast-acting specific t-PA inhibitor.

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