Advanced Search Abstract A rapid, high-resolution melting HRM analysis protocol was developed to detect sequence variations associated with resistance to the QoIs, benzimidazoles and dicarboximides in Botrytis cinerea airborne inoculum. Three and five different genotypes were detected and classified according to their melting profiles in BenA and bos1 genes associated with resistance to benzimidazoles and dicarboximides, respectively. The sensitivity of the methodology was evident in the case of the QoIs, where genotypes varying either by a single nucleotide polymorphism or an additional bp intron were separated accurately with a single pair of primers. The developed two-step protocol was completed in 82 min and showed reduced variation in the melting curves' formation.
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This article has been cited by other articles in PMC. Abstract Quantitative polymerase chain reaction-high-resolution melting qPCR-HRM analysis was used to screen for mutations related to drug resistance in Mycobacterium tuberculosis.
Plasmids can be handled outside of biosafety level 3 facilities, reducing the risk of contamination and the cost of the assay. Plasmids have a high stability, are normally maintained in Escherichia coli and can be extracted in large amounts.
The rapid detection of Mycobacterium tuberculosis reduces treatment failure, the rate of transmission and the period of infectiousness Martin et al. High-resolution melting HRM analysis can be used to distinguish DNA sequences with slight differences in the melting curve profile.
HRM analysis has been used in screens for gene mutations prior to DNA sequencing, in methylation studies and for genotyping, with high throughput, reliability, rapidity, simplicity and cost efficiency Ereqat et al.
A number of DNA deletions, insertions and single-nucleotide polymorphisms in the M. HRM analysis of these mutations are useful for the rapid prediction of the drug sensitivity profile and, consequently, allow for the adjustment of treatment regimens Ramirez et al.
Rifampicin is considered a good marker for multidrug resistance.
Mutations in the rpoB gene are associated with rifampicin resistance and are usually located within an 81 bp hypervariable region of the rpoB gene known as the rifampicin resistance-determining region RRDR Khadka et al. To improve the specificity of HRM analysis, genomic DNA samples with specific mutations are used as positive or negative controls Choi et al.
However, the production of large amounts of genomic DNA carrying resistance-associated mutations must be performed in a biosafety level 3 laboratory. Cloning strategies that allow for the large-scale production of recombinant DNA in Escherichia coli or other less pathogenic bacteria may provide a useful method to obtain M.
A total of 54 M. Rifampicin susceptibility was determined by the resazurin microtitre assay REMA plate method according to Palomino et al. The sequence was analysed with BioEdit software v7. Recombinant plasmids and M. The cycling parameters were as follows: This software was used to optimise the assay and group the samples.
Similar results were obtained by Ramirez et al. PCR products that were directly amplified from M.Coeus HRM is a web based human resource management solution for businesses of all sizes. It offers such capabilities as document repository, learning management, expense management, and others.
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