Evaluation of the RNA simultaneous amplification and testing for detection of rifampin resistance in Mycobacterium tuberculosis

Abstract

To establish and evaluate a method for detection of rifampin resistance in Mycobacterium tuberculosis (M. tuberculosis) by the RNA simultaneous amplification and testing (SAT). RNA probe and primer of reverse transcription with T7 promoter targets pre-16S rRNA were designed, and the isothermal RNA amplification at 42 °C was performed for real-time detection of the levels of pre-16S rRNA in drug exposed MTB. Twenty clinical isolates were detected by the SAT after treatment with rifampicin 1, 2 and 3 d in order to determine the best drug effect time. Fifty clinical isolates with known drug susceptibility results were used to determine the best cutoff value of rifampicin drug susceptibility by SAT. In total, 128 clinical isolates were detected by SAT to evaluate the accuracy compared with the Bactec MGIT 960. The best drug effect time and cutoff value for the detection of rifampin resistance by SAT were 2 d and 2.95. With the result of Bactec MGIT 960 as the reference, the sensitivity and the specificity of the assay was 100% (51/51) and 97.4% (75/77), respectively. The isothermal RNA amplification assay is a highly sensitive and specific tool for the detection of rifampicin resistance in M. tuberculosis, and therefore, it may be a new method to detect rifampicin resistance in M. tuberculosis.