Analysis of the Technical Principle and Detection Advantages of a Fluorescent PCR Melting Curve-Based MSI Kit

Technical principle: How melt curve analysis achieves MSI classification

MSI (microsatellite instability) is an important molecular marker for solid tumors such as colorectal cancer and endometrial cancer. The fluorescent PCR melt curve method MSI kit uses multiplex fluorescent PCR technology to simultaneously amplify multiple microsatellite marker loci (such as common loci including BAT-25, BAT-26, NR-21, NR-24, MONO-27, etc.). The amplification products are subjected to melt curve analysis on a real-time fluorescent PCR instrument using labeled probes: the difference in melting temperature (Tm) between the test sample and the normal control can reflect changes in repeat sequence length. When the Tm shift at a marker locus exceeds a preset threshold, the locus is determined to be unstable. This method does not require post-processing steps such as electrophoresis or capillary electrophoresis, and closed-tube operation can reduce the risk of contamination.

Workflow and key control points

A typical workflow includes: DNA extraction from formalin-fixed paraffin-embedded tissue (FFPE) or fresh tissue samples, multiplex PCR amplification, melt curve analysis, and automatic interpretation by software. Kits generally require an initial DNA amount in the range of 10~50 ng, and internal reference quality control is needed when amplification efficiency is affected by inhibitors. The key control points are: (1) paired analysis of normal controls and tumor samples to exclude interference from germline polymorphisms; (2) consistency of melt curve peak shape to avoid false positives caused by nonspecific amplification; (3) the Tm threshold set by the kit must be validated with clinical samples. Operators should strictly follow the instructions to set up positive and negative quality control materials, and the effectiveness of amplification must be verified in each experiment.

Routine performance characteristics and precautions

The routine characteristics of the fluorescent PCR melt curve method MSI kit include: the detection cycle is about 3~4 hours, 5~7 single nucleotide repeat markers can be analyzed simultaneously, and the sensitivity generally can reach a mutation frequency of 1~5% (varying somewhat with different kit lots). Compared with capillary electrophoresis, this method has strong instrument versatility (commonly used real-time fluorescent PCR instruments), and result interpretation relies more on software algorithms, so attention should be paid to calibration of Tm baseline values across different platforms. Kit storage usually requires protection from light at -20°C, and the number of freeze-thaw cycles should not exceed 5. In clinical promotion, it is recommended that laboratories regularly participate in external quality assessment to maintain interpretation consistency.