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Ultrasensitive Detection of Multidrug-Resistant Mycobacterium tuberculosis Using SuperSelective Primer-Based Real-Time PCR Assays.
Narang, Anshika; Marras, Salvatore A E; Kurepina, Natalia; Chauhan, Varsha; Shashkina, Elena; Kreiswirth, Barry; Varma-Basil, Mandira; Vinnard, Christopher; Subbian, Selvakumar.
Affiliation
  • Narang A; Public Health Research Institute, New Jersey Medical School, Rutgers University, Newark, NJ 07103, USA.
  • Marras SAE; Public Health Research Institute, New Jersey Medical School, Rutgers University, Newark, NJ 07103, USA.
  • Kurepina N; Center for Discovery and Innovation, Nutley, NJ 07110, USA.
  • Chauhan V; Department of Microbiology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi 110021, India.
  • Shashkina E; Center for Discovery and Innovation, Nutley, NJ 07110, USA.
  • Kreiswirth B; Center for Discovery and Innovation, Nutley, NJ 07110, USA.
  • Varma-Basil M; Department of Microbiology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi 110021, India.
  • Vinnard C; New Jersey Medical School, Rutgers University, Newark, NJ 07103, USA.
  • Subbian S; Public Health Research Institute, New Jersey Medical School, Rutgers University, Newark, NJ 07103, USA.
Int J Mol Sci ; 23(24)2022 Dec 12.
Article in En | MEDLINE | ID: mdl-36555395
The emergence of drug-resistant tuberculosis is a significant global health issue. The presence of heteroresistant Mycobacterium tuberculosis is critical to developing fully drug-resistant tuberculosis cases. The currently available molecular techniques may detect one copy of mutant bacterial genomic DNA in the presence of about 1-1000 copies of wild-type M. tuberculosis DNA. To improve the limit of heteroresistance detection, we developed SuperSelective primer-based real-time PCR assays, which, by their unique assay design, enable selective and exponential amplification of selected point mutations in the presence of abundant wild-type DNA. We designed SuperSelective primers to detect genetic mutations associated with M. tuberculosis resistance to the anti-tuberculosis drugs isoniazid and rifampin. We evaluated the efficiency of our assay in detecting heteroresistant M. tuberculosis strains using genomic DNA isolated from laboratory strains and clinical isolates from the sputum of tuberculosis patients. Results show that our assays detected heteroresistant mutations with a specificity of 100% in a background of up to 104 copies of wild-type M. tuberculosis genomic DNA, corresponding to a detection limit of 0.01%. Therefore, the SuperSelective primer-based RT-PCR assay is an ultrasensitive tool that can efficiently diagnose heteroresistant tuberculosis in clinical specimens and contributes to understanding the drug resistance mechanisms. This approach can improve the management of antimicrobial resistance in tuberculosis and other infectious diseases.
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Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: Tuberculosis / Tuberculosis, Multidrug-Resistant / Mycobacterium tuberculosis Type of study: Diagnostic_studies Limits: Humans Language: En Journal: Int J Mol Sci Year: 2022 Document type: Article Affiliation country: United States Country of publication: Switzerland

Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: Tuberculosis / Tuberculosis, Multidrug-Resistant / Mycobacterium tuberculosis Type of study: Diagnostic_studies Limits: Humans Language: En Journal: Int J Mol Sci Year: 2022 Document type: Article Affiliation country: United States Country of publication: Switzerland