Multiplex Detection Design: Technical Features Analysis of Nucleic Acid Detection Kits for Aspergillus fumigatus, Aspergillus flavus, and Aspergillus niger (Fluorescence PCR Method)

Basic principle of fluorescent PCR

Fluorescent PCR (also known as real-time fluorescent quantitative PCR) uses the accumulation of fluorescent signals to monitor the nucleic acid amplification process. The reaction system contains specific primers, fluorescent probes (such as TaqMan probes), and a thermostable DNA polymerase. When template DNA is present, primer extension causes probe hydrolysis, separating the fluorescent group from the quencher group, and the fluorescence intensity is proportional to the amount of template. Qualitative or semi-quantitative analysis of the results is performed using the Cq value (or Ct value). For the detection of Aspergillus fumigatus, Aspergillus flavus, and Aspergillus niger, the ribosomal RNA gene (such as the ITS1-5.8S-ITS2 region) is usually selected as the target because it has sequence differences among species and a relatively high copy number, which helps improve sensitivity.

Design considerations for multiplex detection

Multiplex fluorescent PCR requires simultaneous amplification of multiple targets in the same reaction tube. The core challenges are avoiding cross-dimers between primers, nonspecific amplification, and crosstalk between fluorescence channels. When designing kits, different fluorescent groups are often selected (such as FAM labeling Aspergillus fumigatus, VIC labeling Aspergillus flavus, and ROX labeling Aspergillus niger) along with corresponding quencher groups, and fine adjustments are made at the 3′ end of the probe sequence (such as introducing MGB modification) to improve annealing specificity. At the same time, an internal control (such as the human GAPDH gene) must be added to monitor sample quality and nucleic acid extraction efficiency. Primer concentration, annealing temperature, and magnesium ion concentration all need to be optimized to ensure similar amplification efficiency for each target.

Comparison with culture methods

Fungal culture is a classic method for Aspergillus detection, but its limitations are obvious: some Aspergillus species grow slowly and may be masked by bacterial contamination; certain species (such as Aspergillus fumigatus) are inhibited from growing in specimens containing antifungal drugs. Serological tests (such as the GM test and G test) have false positives (such as in patients taking β-lactam antibiotics or undergoing hemodialysis) and cannot distinguish species-level identification. In contrast, fluorescent PCR directly detects nucleic acids, is not affected by phenotype, and can simultaneously identify to species level, providing clinical practice with a more precise etiological basis. However, it should be noted that a positive PCR result does not distinguish between viable and dead organisms and must be interpreted in combination with clinical manifestations.

Quality control and standards

When using this kit, the instructions must be strictly followed, including sufficient removal of inhibitors (such as polysaccharides and heme) during the DNA extraction step. Each test requires a positive control (plasmid or strain DNA containing the three targets), a negative control (no-template control), and an internal control channel. When interpreting results, the test result is valid only when the internal control Cq value is within the normal range and the controls meet expectations. Laboratories should participate in external quality assessment (EQA) to verify performance stability. In addition, the storage temperature of the kit (generally below -20°C) and the number of repeated freeze-thaw cycles must be strictly controlled to avoid performance degradation.