Ultrasensitive Detection of Multidrug-Resistant Mycobacterium tuberculosis Using SuperSelective Primer-Based Real-Time PCR Assays

Abstract

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.

Analysis of three cases with false positive PCR results of non tuberculosis mycobacterium

“…Clinical examiners encounter this situation and cannot proceed directly to negative or false-positive results. Further measures should be taken to sequence and analyze the amplified products and compare them with relevant databases, or using MALDI-TOF mass spectrometry, which has become a standard routine identification tool for bacteria, which is very helpful for infections of clear pathogens, especially rare [ 18 ].…”

Exploring Diagnostic Methods for Drug-Resistant Tuberculosis: A Comprehensive Overview

“…In 23 clinical MTB isolates (including 11 MDR and 6 INHmonoresistant isolates) the assay detected HR populations with a detection limit of 0.01%. The assay is ultrasensitive, but not all isolates were compared with WGS and or with pDST [122]. Tamilzhalagan and co-authors retrospectively performed WGS on MTB isolates from India to detect mutations that might have been undetected by the MGIT.…”

Optimization of DNA amplification temperature in quantitative polymerase chain reaction for Identification of isoniazid-resistant Mycobacterium tuberculosis

“…Suhu denaturasi dan ekstensi pada proses qPCR yang akan dioptimasi diatur dengan menggunakan komputer yang terhubung dengan qPCR (Gentier 96, Tianlong,China). Ct ideal yang digunakan ialah 19-25 (Narang et al, 2022).…”