Table 1.
Primer sequences for PCR and DNA sequencing.
Table 2.
RPA primer sequences and optimum conditions used in this study.
Table 3.
Phenotypic drug susceptibility testing.
Table 4.
Frequency of amino acid substitutions within 81-base-pair hotspot region of the rpoB gene of 141 M. tuberculosis.
Table 5.
Frequency of amino acid substitutions within the katG gene of 141 M. tuberculosis.
Fig 1.
Results of the AS-RPA/SYBR assay validated with the M. tuberculosis wild-type strain.
(A) RPA amplicons were detected via agarose gel electrophoresis. The product sizes from each pair of primers were 173 bp, 363 bp, 213 bp, 182 bp, 250 bp, 276 bp and 152 bp for the IS1081, rpoB, rpoB516, rpoB526, rpoB531, katG and katG315 primers, respectively. Lane L: 100-bp DNA ladder, Lane N: no template control; Lane 1: IS1081 primer; Lane 2: rpoB primer; Lane 3: rpoB516 primer; Lane 4: rpoB526 primer; Lane 5: rpoB531 primer; Lane 6: katG primer; Lane 7: katG315 primer. (B) The naked-eye endpoint detection method was performed by adding SYBR Green I directly to the reaction tubes. Tube N is a no-template control; tubes 1 to 7 contained the IS1081, rpoB, rpoB516, rpoB526, rpoB531, katG and katG315 primers, respectively, showing a green colour, which implied a positive amplification result and no mutation at those allele-specific sites.
Fig 2.
Results of the AS-RPA/SYBR assay when validated with M. tuberculosis strains with known mutations within the rpoB and katG genes.
This figure represents the results after testing with M. tuberculosis DNA containing the rpoB531 and katG315 mutations. (A) RPA amplicons from different primers were detected via agarose gel electrophoresis. The product sizes from each pair of primers were 173 bp, 363 bp, 213 bp, 182 bp and 276 bp for the IS1081, rpoB, rpoB516, rpoB526 and katG primers, respectively. Reactions with the rpoB531 and katG315 primers showed no visible target band. Lane N: no-template control; Lane 1: IS1081 primer; Lane 2: rpoB primer; Lane 3: rpoB516 primer; Lane 4: rpoB526 primer; Lane 5: rpoB531 primer; Lane 6: katG primer; Lane 7: katG315 primer; Lane L: 100-bp DNA ladder. (B) The naked-eye endpoint detection method was performed by adding SYBR Green I directly to the reaction tubes. Tube N was a no-template control; tubes 5 and 7 were tested for rpoB531 and katG315, respectively, showing an orange colour, which implied a negative amplification result. For allele-specific primers, these results also indicated that the DNA template carried mutations at a particular site. Tubes 1–4 and 6 contained the IS1081, rpoB, rpoB516, rpoB526 and katG primers, respectively, showing a green colour, which implied a positive amplification result and no mutation at those allele-specific sites.
Table 6.
Validation of AS-RPA/SYBR by a DNA sequencing analysis (N = 141).
Table 7.
Validation of AS-RPA/SYBR by a phenotypic drug susceptibility test (N = 141).
Fig 3.
(A) Agarose gel electrophoresis showed the expected rpoB526 RPA product when testing with wild-type M. tuberculosis H37Rv DNA only (lane 10). Lane L is a 100-bp DNA ladder, lane N is a no-template control, and lanes 1–9 are DNA extracted from A. baumannii, H. influenzae, K. pneumoniae, M. catarrhalis, M. intracellulare, S. pyogenes, S. pneumoniae, M. avium, and P. aeruginosa, respectively. (B) RpoB526 RPA products were observed for the SYBR Green I colour change by the naked eye. Tube N is a no-template control, and tubes 1–9 are DNA extracted from A. baumannii, H. influenzae, K. pneumoniae, M. catarrhalis, M. intracellulare, S. pyogenes, S. pneumoniae, M. avium, and P. aeruginosa, respectively, showing an orange colour, which indicates no amplification. Tube 10 is M. tuberculosis H37Rv DNA, showing a green colour, which implies a positive amplification result.