Abstract
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.
Highlights
Tuberculosis (TB) remains a significant health problem worldwide
Over 95% of rifampicinresistant isolates possess single point mutation clustering in the 81-base-pair hotspot region of the rpoB gene known as a rifampicin-resistant determining region (RRDR) that encodes codons 507 to 533
A previous study from Thailand showed that all 143 Multidrug-resistant tuberculosis (MDR-TB) isolates had mutations at rpoB, with 98% of strains mutated in the hot-spot RRDR and the remaining isolates mutated in the area of the N-terminus
Summary
Tuberculosis (TB) remains a significant health problem worldwide. In 2019, the World Health Organization (WHO) estimated that approximately 10 million people developed TB disease and that approximately 1.4 million affected people died [1]. Multidrug-resistant tuberculosis (MDR-TB), defined as Mycobacterium tuberculosis that resists at least two of the most potent medications against TB infection, rifampicin and isoniazid, is a challenge for TB control. In 2019, the incidence of multidrug-resistant/rifampicin-resistant tuberculosis (MDR/RR-TB) was estimated to be approximately 465,000 cases, while 206,030 cases were noted with a 57% success rate for treatment outcomes [1]. Over 95% of rifampicinresistant isolates possess single point mutation clustering in the 81-base-pair hotspot region of the rpoB gene known as a rifampicin-resistant determining region (RRDR) that encodes codons 507 to 533. Mutations in several genes can lead to isoniazid resistance. The majority (60–80%) of isoniazid resistance in M. tuberculosis is due to a single point mutation in the katG gene at codon 315 (50–93% of katG mutations) [2, 5]
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