HIGH RESOLUTION MELTING ANALYSIS (HRM) FOR LEISHMANIA SPECIES - - PowerPoint PPT Presentation

HIGH RESOLUTION MELTING ANALYSIS (HRM) FOR LEISHMANIA SPECIES IDENTIFICATION

Group: Bayan, Ali and Dima meBOP, 29-07-16

High Resolution Melting (HRM) analysis

• New post-PCR analysis method used to identify variations in

nucleic acid sequences.

• Detection of small differences in PCR melting curves.
• It is enabled by improved dsDNA-binding dyes used in

conjunction with real-time PCR instrumentation.

5 10 15 20 25 30 35 40 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5

qPCR Results for DNA amplification of Blood samples

Real-time LightCycler PCR assay for 20 samples using primers JW13 and JW14 amplifying a DNA fragment from the kinetoplast DNA Cycle Fluorescence Control + Samples Control -

(Nicolas et al. 2002)

Cycle 5 10 15 20 25 30 35 40 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5

qPCR Results for DNA amplification of Blood samples

Cycle Fluorescence

Control +

deg. 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 100 90 80 70 60 50 40 30 20 10

Data acquisition

Temperature Fluorescence

deg. 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 dF/dT 1.2 1.0 0.8 0.6 0.4 0.2 0.0

Melting curve analysis for species identification

Real-time LightCycler PCR assay using primers JW13 and JW14 amplifying a DNA fragment from the kinetoplast DNA

• L. donovani
• L. tropica
• L. major
• L. major:83.86
• L. donovani: 87.12
• L. tropica:

88.89

• L. infantum:

89.21 – 89.47

Advantages of HRM

• Low reagent consumption
• (requires only PCR reaction volume (20 µL) for analysis of each

sample).

• Simple, fast workflow:
• no additional instrumentation is required after PCR amplification.
• Low sample consumption:
• Following HRM analysis, the PCR amplicon can be used directly in

a Sanger sequencing reaction

Disadvantages of HRM

• Limited sensitivity.
• Limited to genes characterized by low rate of

mutations

• Returning to others methods in case of co-infection.