Rapid detection and identification of clinically important bacteria by High- Resolution Melting anly

Improved strategies for rapid diagnosis of bacterial infections are critical step to control clinically important infections. Therefore, in clinical application, the use of real-time PCR for wide- spectrum amplification of bacterial DNA has additional advantages such as minimal labor, lower running cost, rapid turnaround time, specificity and sensitivity, and decreased risk of PCR carryover contamination.

Amplification of the gene that encodes 16s rRNA using universal primers are used for identification of bacteria at the genus and species levels after their isolation from BCO lesions. variable region 2 (V2) of the 16s rDNA was found to be more specific compared to V3, and the combination of V2F and V3R in species identification. Also, using of LC green and Eva green both were found to be more specific than previously used SYBR green (more distinct and sharp peaks)

HRM assay optimization required a DNA of good quality, target size, primer selection, dye selection, mg cl2 concentration, and HRM analysis software.

First step includes amplification of the region of interest using standard PCR technique in the presence of inter-chelate , double stranded DNA binding dye which is highly fluorescent when it bind to dsDNA but poorly fluorescent in the unbound state. this change allows monitoring the DNA amplification during PCR. In the next step, the amplified target is gradually denatured by increasing the temperature, in small increments to produce characteristic melting profile.