Identification of S315T mutation in katG gene using probe-free exclusive mismatch primers for a rapid diagnosis of isoniazid-resistant Mycobacterium tuberculosis by real-time loop-mediated isothermal amplification

Abstract

We developed a LAMP-based technique that employs primers that carry base mismatches to specifically detect the katG (S315T) SNP associated with isoniazid resistance in tuberculosis (TB). Antibiotic resistance in TB is still predominantly determined by the standard drug susceptibility testing that lacks sensitivity, and often requires a weeks-long effort of manual labor and extensive laboratory resources. Advanced real-time PCR techniques are highly efficient for detecting these drug-resistant SNPs, yet their accessibility is limited in TB-endemic regions due to prohibitive instrumental costs and a lack of highly-trained personnel. The isothermal assay described herein was optimized for fluorescence detection on the real-time PCR system. Its ability to discriminate the S315T mutant from wild-type katG without using the melting curve analysis has granted its adaptation into an end-point detection format performed on a hand-held potentiostat device through rapid electrochemical detection. Our technique exhibited the statistical sensitivity and specificity of 100% (95% CI: 98% − 100%) and 96% (95% CI: 87% − 99%), respectively, based on the validation with 218 tuberculosis isolates collected from patients performed under real-time detection mode. The versatility of analysis of this assay has unprecedently allowed the determination of a key INH-resistant associated SNP to be diagnosed without relying on detection probes or the melting curve analysis as often required by standard real-time PCR.