The multi-channel detection capability of the fluorescence quantitative PCR instrument significantly improves the accuracy of multiplexed target analysis through the dual mechanisms of physical isolation and signal-specific recognition. The core of this feature lies in the independent optical pathways of each detection channel, allowing only fluorescence signals within specific wavelengths to pass through. For example, the FAM and HEX channels can operate simultaneously without signal crosstalk. This design ensures that probes labeled with different fluorophores can be independently detected in the same reaction system, avoiding the signal interference caused by overlapping fluorescence wavelengths in traditional single-channel instruments. For example, in pathogen detection, different channels can be used to simultaneously detect SARS-CoV-2 RNA and influenza virus nucleic acids, achieving a highly efficient "one-tube, multiple-test" model while eliminating the risk of contamination from repeated openings of the instrument's lid.
The accuracy of multi-channel detection is also enhanced by its dynamic calibration capability. The fluorescence quantitative PCR instrument's built-in calibration module monitors the baseline level, fluorescence gain, and signal drift of each channel in real time, automatically correcting for detection deviations caused by light source attenuation or ambient temperature fluctuations. For example, some high-end models utilize fiber-optic single-well scanning technology, which uses independent optical fibers to scan each reaction well, channel by channel. Combined with the high sensitivity of CMOS imaging sensors, this technology can detect nucleic acid templates as low as a single copy, while minimizing signal variability between channels.
This technology not only supports higher-multiplicity target analysis (e.g., detecting multiple pathogens in a single tube), but also reduces detection time to one-third of traditional methods through optimized optical system design, providing a more efficient and accurate tool for clinical diagnosis and scientific research. Regarding experimental design, multi-channel detection capabilities provide researchers with greater flexibility in probe selection. Different fluorophores (such as FAM, VIC, ROX, and CY5) have unique emission spectral characteristics, and multi-channel systems can simultaneously capture these signals, enabling simultaneous detection of multiple targets in the same reaction system. For example, in tumor gene testing, mutation probes for genes such as EGFR, KRAS, and BRAF can be labeled with different channels, enabling simultaneous analysis of multiple gene mutations. This design not only shortens detection cycles but also improves the reliability of results by eliminating operational errors between reactions. In addition, some instruments support melting curve analysis, which can further verify amplification specificity. For example, by analyzing the melting temperature of SYBR Green dye-labeled products, specific products can be distinguished from nonspecific dimers, ensuring the accuracy of each target in multiplexed assays.
From an application validation perspective, multichannel detection has been tested in a variety of complex experimental scenarios. For example, in the differential diagnosis of SARS-CoV-2 and influenza virus co-infection, a multichannel fluorescence quantitative PCR instrument can simultaneously detect the ORF1ab gene and nucleoprotein gene (N gene) of the novel coronavirus, as well as the M gene and NS1 gene of the influenza virus, achieving over 98% sensitivity and specificity. This performance far exceeds that of traditional single-channel instruments, thanks to the multichannel system's precise differentiation and dynamic calibration of fluorescence signals. Furthermore, in gene expression studies, multichannel detection can simultaneously analyze the expression levels of reference genes and target genes, eliminating inter-sample variability through relative quantification methods (such as the ΔΔCt method), thereby improving data comparability.
With the continuous improvement of technology, the new generation of fluorescence quantitative PCR instruments is further expanding the application boundaries of multiplexed detection by integrating more detection channels (such as 6-channel and 8-channel) and compatibility with new fluorescent groups (such as LSS long Stokes shift dyes). These improvements not only support the analysis of higher-multiplicity targets, but also increase detection throughput to thousands of samples per hour through optimization of the optical system and temperature control module, providing technical support for large-scale screening and precision medicine.
