Fig 1.
Schematic diagram of dual probe based mutation detection.
(A-B) Hybridization of probes to the amplicon or intermediary amplification product obtained using isothermal amplification method like LAMP. The signals from the probes can deviate depending on the presence or absence of mutation or template concentration. Mutation is determined by deviation of calibrated signals from sample compared to that from wild type. The raw data signal is related to the amount of amplified product and hybridization efficiency between target and probe. Lower panel: an example by real time detection. WT: Wild type; Probe I: “Indicator” probe; Probe C: “Calibrator” probe. (C) A plot of dual probe detection using real-time readout in isothermal amplification. X-axis is time; Y-axis is signal; Tb: baseline or background signal time point; Te: plateau phase signal end time point. Right panel: Equations for signal calibration. Z-score is used to determine presence or absence of mutation.
Fig 2.
RIF-drug resistance related mutation sites and their detection by the dual probe method.
(A) Sequences of plasmid inserts used for the study. The mutation sites considered in the study are highlighted in blue and the mutations are denoted in small alphabet in red. (B) Probe C and Probe I binding region RIF drug resistance detection using dual probe based strategy in LAMP. (C) Mutation detection at 526 and 531 sites using dual probe method. Fluorescence signals from the calibrator (Probe C) and indicator (Probe I) probes in presence of plasmid samples: (i) Wild type plasmid (His-526, Ser-531). (ii) P2 plasmid (Leu-526, Ser-531). (iii) P3 plasmid (Tyr-526, Ser-531). (iv) P4 plasmid (His-526, Trp-531). (D) Mutation detection at site 516 using dual probe method. Fluorescence signals from the calibrator (Probe C) and indicator (Probe I) probes in presence of plasmid samples: (i) Wild type plasmid (Asp-516). (ii) P5 plasmid (Val-516). (iii) P6 plasmid (Tyr-516). (iv) P7 plasmid (Tyr-516, Leu-526, Trp-531). X-axis is time (min), Y-axis is fluorescence signal (RFU). Z-score is labeled in each sample’s dual probe detection plot.
Fig 3.
Dual probe based mutation detection performance on contrived samples.
(A) Scatter plot of Z-score of 40 valid contrived samples of different genotypes at different concentrations for detection of mutations at sites 526 and 531 using the 526, 531 primer set. Initial sample concentration in reaction is labeled using different shapes. Dashed line Z-score = 5.5 is a threshold. The 2X2 table showing 100% accuracy in determining the sample type. (B) Scatter plot of Z-score of 80 valid contrived samples of different genotypes at different concentrations for detection of mutations at site 516 using the 516 primer set. Dashed line Z-score = 2 is a threshold. The 2X2 table shows 100% accuracy for wild type detection and 95% accuracy for mutation detection.
Fig 4.
Mutation detection in MTB verification panel.
(A) Fluorescence signals from the calibrator (Probe C, blue) and indicator (Probe I, red) probes (designed to detect mutations at sites 526 and 531). (B) Fluorescence signals from the calibrator (Probe C, blue) and indicator (Probe I, red) probes (designed to detect mutations at site 516). (i) Zeptometrix Wild type strain, (ii) Zeptometrix RIF-resistance strain carrying Ser531Leu change, (iii) Wild type strain (ATCC-25177DQ), (iv) Wild type strain (ATCC-BAA-2337D-2), (v) Wild type strain (ATCC-BAA-2336D-2), (vi) Wild type strain (BEI-NR-44096), (vii) Mutant type strain (ATCC-35838D-2) carrying single mutation at codon 531 (TCG to TTG). X-axis is time (min), Y-axis is fluorescence signal (RFU). Z-score is labeled in each sample’s dual-probe detection plot. Clinical strains ID are labeled below each plot.