In multiplex pathogen detection, optimizing sensitivity is a core objective for improving clinical diagnostic accuracy. This process requires collaborative breakthroughs across multiple dimensions, including technical principles, reaction system design, hardware innovation, and clinical validation, to address issues such as cross-interference and uneven amplification efficiency that commonly occur in traditional multiplex detection methods.
The sensitivity bottleneck in multiplex pathogen detection essentially stems from competitive inhibition between primers in multiplex PCR reactions. When multiple primer pairs exist within the same reaction system, the amplification efficiency of different targets can fluctuate significantly due to primer dimer formation, differences in template binding ability, or enzyme activity bias. For example, some pathogens, due to excessively high GC content in their genome or complex template secondary structures, may experience primer extension obstruction, ultimately resulting in false-negative results. Optimization requires global screening of primer sequences using bioinformatics tools to ensure a high degree of matching between target primers in terms of annealing temperature, GC ratio, and product length. Simultaneously, locking nucleotide (LNA) or peptide nucleic acid (PNA) modification techniques should be introduced to enhance the specific binding ability of primers to the template.
Reaction system optimization is a crucial step in improving sensitivity. Traditional multiplex PCR relies on a single buffer formulation, making it difficult to meet the amplification requirements of different pathogens. Modern molecular diagnostic platforms utilize microfluidics to achieve precise control of reaction conditions. For example, nucleic acid extraction, amplification, and detection modules are integrated into a single chip. Electrowetting drives droplet splitting and merging, allowing each reaction unit to independently adjust Mg²⁺ concentration, dNTP ratio, and enzyme dosage. This "one tube, multiple detection" mode not only reduces sample transfer loss but also avoids primer cross-interference through spatial isolation, significantly improving the detection rate of low-abundance pathogens.
Hardware innovation provides the physical basis for breakthroughs in sensitivity. Taking digital microfluidics (DMF) technology as an example, it controls the movement path of nano-liter droplets through an electrode array, achieving a fully enclosed operation for nucleic acid extraction and amplification. In a fully automated molecular diagnosis platform, the magnetic bead-based nucleic acid extraction module and the electrowetting-driven PCR reaction chamber are connected by an "L"-shaped dispensing structure, ensuring that each reaction well obtains an equal amount of template nucleic acid. Combined with a four-channel fluorescence detection system, this platform can simultaneously detect multiple respiratory pathogens in a single run, with significantly improved sensitivity compared to traditional methods. The introduction of signal amplification technology has further broken through the detection limit. CRISPR-Cas-based diagnostic platforms achieve signal cascade amplification through bypass cleavage activity. For example, by combining the Cas13 enzyme with a luciferase reporter system, when the target RNA is recognized and cleaved, the released HiBiT peptide complements the LgBiT subunit to form an active luciferase. Bioluminescent readout significantly improves detection sensitivity. This technology is particularly suitable for detecting low viral load samples in resource-constrained environments.
Clinical sample validation is the ultimate test for sensitivity optimization. In studies targeting multiple respiratory pathogens, the research team collected various types of clinical samples for testing. The results showed that, using qPCR as a reference standard, the system's sensitivity and specificity met clinical requirements. This real-world validation ensures that the optimized technology not only performs excellently under laboratory conditions but also adapts to interference from complex clinical samples.
From a technological development perspective, the sensitivity optimization of molecular diagnosis platforms is evolving towards greater accuracy, speed, and integration. By integrating isothermal amplification technology, microsphere coding technology, and AI-assisted primer design algorithms, the next-generation platform is expected to achieve "one tube, one hundred tests" or even higher throughput detection capabilities, while significantly reducing testing time. These innovations will enable molecular diagnostics to move from central laboratories to primary healthcare and even home self-testing scenarios.
