High-Resolution DNA Melt Curve Analysis of the Clustered, Regularly Interspaced Short-Palindromic-Repeat Locus of Campylobacter jejuni

Classification of “same” and “different” genotypes using HRM analysis of the Campylobacter jejuni CRISPR locus. Data that illustrate the limits of the technology were deliberately chosen: i.e., the alleles shown here were especially refractory to HRM discrimination. (Top) Normalized high-resolution melt curve. (Middle) Derivative of fluorescence with respect to temperature (dF/dT) dissociation curve. (Bottom) Difference graph. The left panel shows an example of two genotypes classed as “different” from a control sample (isolate F470, shown by the black lines) due to the erratic pattern in the difference graph, despite falling within the cutoff (≤±5 in the difference graph). The normalized and dF/dT melt curves confirmed that these genotypes are different from F470. The right panel shows genotypes classed as “same” (in black lines) compared with a control sample (isolate QHSS2). The difference graph, normalized, and dF/dT melt curves confirmed that these genotypes are indistinguishable from QHSS2. A “different” genotype (gray lines) is shown for comparison.