Table 1.
Bacteria and plasmid; source and function in template preparation.
Table 2.
Primers used in PCR amplification for the detection of INH and RIF resistance.
Fig 1.
(A) Multiplex PCR amplification products for the rpoB assay and (B) Semi-nested PCR amplification products for the katG assay, on agarose gel electrophoresis.
Fig 2.
Schematic illustration of NALF design.
The NALF device functions to indicate the presence of dual labeled amplicons in PCR products. At the conjugate pad, AuNP-anti-biotin binds to biotin on one end of the amplicon, and the complex flows towards the test-lines, T1 and T2. T1 is lined with anti-FITC mAb to capture biotin-FITC labeled amplicons, indicative of RIF resistance for the rpoB assay or INH resistance for the katG assay. T2 is lined with anti-DIG mAb to capture biotin-DIG labeled amplicons for Mtb DNA control. The excess AuNP-anti-biotin is captured at the control line (C) by anti-mouse IgG.
Fig 3.
Example of possible NALF outcomes.
Fig 4.
Distribution of MDR-TB determining mutations.
A broad compilation of MDR-TB global epidemiological statistics represents the estimated percentage distribution of MDR-TB determining mutations. The compilation demonstrates that 85–95% of RIF resistance is accompanied by INH resistance to determine MDR-TB (purple). Five to fifteen percent of RIF resistance (brown crest) and 5–25% of INH resistance (blue crest) are monoresistance occurrences. [9–11,15,17,21–37,39–52].
Fig 5.
NALF and agarose gel electrophoresis results for the katG assay.
NALF; the appearance of T1 and T2 for katG MT template indicates a positive detection of INH resistant Mtb isolate, whereas the appearance of T2 (only) for WT template indicates a positive detection of an INH susceptible Mtb isolate. Correspondingly, for gel electrophoresis, two bands are generated for MT template (630 bp, equivalent to NALF T2, and 335 bp to NALF T1), and a single band for WT template (630 bp, equivalent to NALF T2).
Fig 6.
RpoB primer designs for codon 526 (mutations Y and R) have been demonstrated above as examples. SM (single mutation) primers are designed with a single base mismatch at the 3’-terminus to complement MT templates. DM (double mutation) primers, which have also been designed to complement MT templates, carry an added mismatch at the third position from the 3’-terminus. The red colored bases indicate an intentional mismatch and the blue colored bases indicate a complementary base to the SM and DM primers.
Fig 7.
NALF and agarose gel electrophoresis results for the rpoB assay.
(A) The use of SM primers demonstrate an effective template distinction with the appearance of NALF T1 and T2 for all MT templates to indicate RIF resistant Mtb isolates, which relates to the inner and outer DNA bands on agarose gel for the MT templates. As for WT templates, the assays correctly resulted in the appearance of NALF T2 only, to indicate RIF susceptible Mtb isolates, which corresponds to PCR product size 314 bp on agarose gel.
Fig 8.
NALF results for multiplex PCR presented with the correct appearance of T1 and T2 for MT templates to register RIF resistance Mtb isolates, and the appearance of just T2 for WT template to register RIF susceptible Mtb isolates.
Fig 9.
The least limit of detection (LOD);
For both the rpoB and the katG assays, the NALF results present the LOD as 104 DNA copies (0.1 ng) per PCR reaction, whereas the gel electrophoresis results present the LOD as105 DNA copies (1 ng) per PCR reaction. Below these DNA concentrations, the NALF test lines or the agarose gel DNA bands are not observable. The LOD results for rpoB codon 531 have been shown above to represent the results for other rpoB codons.