A rapid, sensitive, naked eye RPA assay can be integrated into point-of-care diagnosis for Mtb detection, especially in remote areas where laboratory instrument resources are limited.

Multidrug-resistant tuberculosis (MDR-TB) poses a serious threat to TB control. Early diagnosis and proper treatment are essential factors to limit the spread of the disease. The existing molecular tests for MDR-TB usually require specific instruments, steady power supply, and routine maintenance, which might be obstacles for low-resource settings. This study aimed to develop allele-specific isothermal recombinase polymerase amplification (allele-specific RPA) to simultaneously detect the most common mutations in the rpoB gene at codons 516, 526, and 531, which are associated with rifampicin resistance, and in the katG gene at codon 315, which is related to isoniazid resistance. Allele-specific primers targeting four major mutations, rpoB516, rpoB526, rpoB531, and katG315, were constructed and used in individual RPA reactions. The RPA amplicons were endpoints detected by the naked eye immediately after applying SYBR Green I. The optimised RPA assay was evaluated with the Mycobacterium tuberculosis wild-type strain H37Rv and 141 clinical M. tuberculosis isolates. The results revealed that allele-specific RPA combined with SYBR Green I detection (AS-RPA/SYBR) detected these four major mutations with 100% sensitivity and specificity relative to DNA sequencing. The limits of detection for these particular mutations with AS-RPA/SYBR were 5 ng. As a result of the outstanding performance of AS-RPA/SYBR, including its easy setup, speed, lack of a specific instrument requirement, and lack of cross-reaction with other bacteria, this technique may be integrated for the molecular diagnosis of MDR-TB, especially in low-resource settings.