Plasmid-based high-resolution melting analysis for accurate detection of rpoB mutations in Mycobacterium tuberculosis isolates from Moroccan patients

BMC Infectious Diseases

Research Article

Ali K. Yetisen¹  Saaïd Amzazi²  Mohammed Abid³  My Driss El Messaoudi⁴  Malika Messaoudi⁴  Hassan Sefrioui⁵  Hassan Ait Benhassou⁵  El Mehdi Bentaleb⁶ 

[1] Harvard−MIT Division of Health Sciences and Technology, Harvard University and Massachusetts Institute of Technology, 65 Landsdowne Street, 02139, Cambridge, MA, USA;Laboratory of Biochemistry and Immunology, Faculty of Sciences, Mohammed V University, Rabat, Morocco;Laboratory of Mycobacteria Genetics, Pasteur Institute of Morocco, Tangier, Morocco;Laboratory of Tuberculosis, Pasteur Institute of Morocco, Casablanca, Morocco;Medical Biotechnology Center, Moroccan Foundation for Advanced Science, Innovation and Research (MAScIR), Rabat Design Center, Avenue Mohamed El Jazouli - Madinat Al Irfane, 10100, Rabat, Morocco;Medical Biotechnology Center, Moroccan Foundation for Advanced Science, Innovation and Research (MAScIR), Rabat Design Center, Avenue Mohamed El Jazouli - Madinat Al Irfane, 10100, Rabat, Morocco;Laboratory of Biochemistry and Immunology, Faculty of Sciences, Mohammed V University, Rabat, Morocco;

关键词: Tuberculosis;    Drug resistance;    qPCR-HRM;    Plasmid-based controls;    Mycobacterium tuberculosis;   

DOI  :  10.1186/s12879-017-2666-4

 received in 2017-04-25, accepted in 2017-08-02,  发布年份 2017

来源: Springer

【 摘 要 】

BackgroundRapid diagnosis of drug resistance in tuberculosis (TB) is pivotal for the timely initiation of effective antibiotic treatment to prevent the spread of drug-resistant strains. The development of low-cost, rapid and robust methods for drug-resistant TB detection is highly desirable for resource-limited settings.MethodsWe report the use of an in house plasmid-based quantitative polymerase chain reaction-high-resolution melting (qPCR-HRM) analysis for the detection of mutations related to rifampicin-resistant Mycobacterium tuberculosis (MTB) in clinical isolates from Moroccan patients. Five recombinant plasmids containing predominant mutations (S531L, S531W, H526Y and D516V) and the wild-type sequence of the Rifampicin Resistance-Determining Region (RRDR) have been used as controls to screen 45 rifampicin-resistant and 22 rifampicin-susceptible MTB isolates.ResultsThe sensitivity and the specificity of the qPCR-HRM analysis were 88.8% and 100% respectively as compared to rifampicin Drug Susceptibility Testing (DST). The results of qPCR-HRM and DNA sequencing had a concordance of 100%.ConclusionOur qPCR-HRM assay is a sensitive, accurate and cost-effective assay for the high-throughput screening of mutation-based drug resistance in TB reference laboratories.

【 授权许可】

CC BY   
© The Author(s). 2017