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Drug-resistant Mycobacterium tuberculosis continues to be a global public health threat despite the development of new diagnostics and antitubercular drugs. [1] Shockingly, the World Health Organization (WHO) has estimated that for more than 10 years, the proportion of people diagnosed with tuberculosis (TB) for the first time who had multidrug-resistant (MDR)/rifampin-resistant (RR)-TB has remained unexpectedly as high as about 3%-4% and for those previously treated for TB has remained about 18%-21%. [2] Hence, identifying patients with drugresistant TB as rapidly and accurately as possible is crucial for effective treatment and appropriate infection control. [3] Existing methods for diagnosing drug-resistant TB mainly include phenotypic and genotypic drug susceptibility testing (DST). [4] Phenotypic DST, the gold standard for identifying drug-resistant M. tuberculosis, can detect as low as 1% of mutants, making it the most sensitive method available so far. [5] However, phenotypic DST is limited in terms of turnaround time, availability, and accuracy. [6] Genotypic DST has been wildly used recently by rapidly providing definitive results and availability. [7][8][9][10][11][12] Nevertheless, most current genotypic DST assays face great challenges in achieving the same sensitivity as phenotypic DST, which may cause false diagnosis, inappropriate treatment, and further deterioration of resistance. For example, Xpert MTB/ RIF is a highly respected genotypic DST diagnostic tool for RR-TB endorsed by the WHO in 2008 [13] due to the convenience of operation. However, this assay only identifies rifampin resistance when the mixture contains at least 65.6% of mutant DNA, [14] which may result in misdiagnosis of some patients with drug-resistant TB. Another XDR-TB detection assay based on the multiplex melting curve analysis could identify DNA with a mutation ratio of 20%-30%. [15] However, it still could not meet the needs in clinical practice. Besides, Folkvardsen et al. [16] also concluded that line probe assays, such as GenoType Establishing simple, rapid, and highly sensitive molecular assays is crucial for timely diagnosis and effective treatment of drug-resistant tuberculosis. However, current genotypic drug susceptibility testing (DST) still encounters enormous challenges including lower sensitivity than phenotypic DST and insufficient accuracy. Herein, a simple, low-cost, multiplex real-time polymerase chain reaction-based assay is established to achieve highly sensitive detection of low-abundant mutants through competitive wild-type blocking (COWTB). Analytical performance of the COWTB assay can achieve 1% or even 0.1% mutants under background of 10 000 wild-type genomes/test. Furthermore, clinical practice feasibility is evaluated to identify resistance to rifampicin (RIF), isoniazid (INH), and streptomycin (SM) on 92 actual clinical samples, its sensitivity is 93.8% for RIF and 100% for INH and SM, and specificity is 100% each for RIF, INH, and SM when using DNA sequencing as the reference standard. In compari...

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