Microfluidic genotyping by rapid serial PCR and high-speed melting analysis

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Clinical molecular testing often relies on sample batching or multiplex assays for cost-efficiency.
• Analyzing multiple genetic variants necessitates efficient workflows for rapid, multi-locus analysis.

Purpose of the Study:

• To evaluate a novel microfluidic platform for rapid genotyping of single nucleotide variants.
• To assess the accuracy and speed of a system combining rapid serial PCR and high-speed melting (HSM).

Main Methods:

• A microfluidic platform was utilized for serial PCR and high-speed melting (HSM).
• Four single nucleotide variants (F2, F5, and two MTHFR variants) were genotyped in 100 samples per site.
• Automated liquid handling facilitated PCR and HSM, with internal temperature controls enhancing precision.

Main Results:

• Genotyping was completed in 12.5 minutes using PCR and HSM at 0.5 °C/s.
• Homozygous variants were distinguished by melting temperature, and heterozygotes by curve shape.
• The platform achieved high accuracy, with only 1.38% of assays requiring follow-up testing.

Conclusions:

• A novel microfluidic platform demonstrates accurate genotyping using rapid serial PCR and HSM.
• The platform enables short turnaround times for multiple genetic variants in up to 8 samples.
• The system supports automated reflexive or repeat testing, offering a flexible genetic workflow.