Optimization of the real-time PCR platform method using high-resolution melting analysis and comparison with...

Area of Science:

• Medical diagnostics
• Molecular biology
• Parasitology

Background:

• Malaria diagnosis is critical for public health in developing nations.
• Accurate identification of Plasmodium species is essential for effective treatment and infection control.
• Current diagnostic methods face challenges, with molecular techniques offering higher accuracy than microscopy.

Purpose of the Study:

• To optimize the real-time PCR platform with high-resolution melting (HRM) analysis for malaria diagnosis.
• To compare the efficacy of HRM analysis with sequencing and phylogenetic analyses.
• To develop an optimal malaria diagnostic method using HRM technology.

Main Methods:

• Collected 300 blood samples from individuals with suspected malaria in southeastern Iran.
• Performed microscopic examination and PCR-HRM analysis using the Light Cycler 96 Instrument.
• Conducted sequencing and phylogenetic analysis for further validation.

Main Results:

• PCR identified 3% Plasmodium falciparum and 6.66% Plasmodium vivax.
• HRM detected 5% Plasmodium falciparum and 4.66% Plasmodium vivax.
• Sequencing revealed 4.33% Plasmodium falciparum and 5.33% Plasmodium vivax; HRM showed complete agreement with sequencing.

Conclusions:

• The HRM technique, with optimized primer design, is a reliable and optimal method for malaria species diagnosis.
• HRM analysis demonstrated high sensitivity and specificity in identifying Plasmodium species.
• HRM offers a valuable molecular tool for accurate malaria diagnostics in clinical settings.