Rapid detection of multidrug-resistant tuberculosis based on allele-specific recombinase polymerase amplification and colorimetric detection

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.

From saliva to SNP: non-invasive, point-of-care genotyping for precision medicine applications using recombinase polymerase amplification and giant magnetoresistive nanosensors

“…Neither study integrated sample preparation into their POC protocols, suggesting the development of straightforward extraction methods going forward. 25,28 Though impressive, these publications ultimately affirm the paucity of multiplexed RPA SNP detection technologies that are readily translatable to the POC.…”

Recent advances in recombinase polymerase amplification: Principle, advantages, disadvantages and applications

“…Singpanomchai et al established an allele-specific RPA-SYBR amplification system to detect multidrug-resistant tuberculosis, this method designed specific primers for the alleles of four major mutations rpoB516, rpoB526, rpoB531, and katG315. The experimental results showed that RPA-SYBR had 100% sensitivity and specificity compared with DNA sequencing, and its detection limit for these special mutation sites was 5 ng (Singpanomchai et al, 2021). Wang et al developed RPA-LFS based on the four most common carbapenemase genes: bla KPC , bla NDM , bla OXA-48like , and bla IMP for rapid on-site detection of carbapenemaseproducing Enterobacterales.…”

Evolution of tuberculosis diagnostics: From molecular strategies to nanodiagnostics

“…The hybridized probes form a spot and give signals on the immobilized microarray chip. If no spots are obtained then the mutation is confirmed [ 95 ]. DNA microarray chips are rapid, accurate, and highly efficient and can address the shortcomings of traditional methods of nucleic acid amplification like low automation and low detection efficiency.…”