Frontiers in cellular and infection microbiology · 2026
Rapid and sensitive detection of Pseudomonas aeruginosa in traditional Chinese medicine herbal pieces using loop-mediated isothermal amplification combined with fluorescent microsphere-based lateral flow strip.
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Tian Wenlin, Yang Chao, Chen Zhilan, Wen Jingtao, Zhong Yujie, Guo Yongcan
Аннотация
OBJECTIVE: Pseudomonas aeruginosa (P. aeruginosa) is a common contaminant in traditional Chinese medicine (TCM) herbal pieces, and its detection is often hindered by polysaccharides, polyphenols, and other inhibitory compounds in the herbal matrix. This study aimed to develop a rapid, sensitive, and matrix-tolerant method for detecting P. aeruginosa in TCM herbal pieces for routine quality control.
METHODS: A loop-mediated isothermal amplification (LAMP) assay targeting the species-specific ecfX gene was developed and integrated with a fluorescent microsphere-based lateral flow strip (LFS) for visual result interpretation. LAMP primers were dual labeled with digoxigenin and biotin to enable specific amplicon to capture at the test and control lines of the LFS. Reaction conditions for both LAMP reaction and LFS were optimized using genomic DNA extracted from P. aeruginosa-spiked powdered herbal samples. Analytical performance, including specificity, sensitivity, reproducibility, and limit of detection (LOD), was rigorously evaluated. The validated method was then applied to six commonly used TCM herbal pieces, including Astragali radix and Angelicae sinensis radix. Additionally, the assay performance was validated using six representative TCM herbal pieces and 90 clinical-relevant herbal samples in parallel with qPCR.
RESULTS: The LAMP-LFS assay demonstrated strict specificity for P. aeruginosa, with no cross-reactivity observed against any non-target bacterial strains tested. Optimal LAMP conditions were determined as 65 °C for 60 min in the presence of 6 mM Mg2+ and 1.4 mM dNTPs. The entire detection workflow, including DNA extraction, amplification, and strip readout, was completed within approximately 70 min. The assay achieved a limit of detection of 1 × 10² CFU/250 mL in spiked herbal matrices, with high reproducibility (CV < 5%). Consistent detection was observed across six different TCM herbal species, indicating strong tolerance to complex phytochemical backgrounds. In parallel validation with qPCR, the assay demonstrated a sensitivity of 94.0%, specificity of 97.5%, and overall accuracy of 95.6%, with a Cohen's kappa coefficient of 0.92.
CONCLUSION: The developed fluorescent microsphere-based LAMP-LFS assay provides a rapid, sensitive, and reproducible platform for detecting P. aeruginosa in TCM herbal pieces. Compared with conventional PCR-based approaches, this method demonstrates improved tolerance to inhibitory herbal matrices and enables reliable on-site applicability with minimal instrumentation requirements. The assay shows potential for routine microbiological quality control and field-based monitoring of TCM herbal materials.
METHODS: A loop-mediated isothermal amplification (LAMP) assay targeting the species-specific ecfX gene was developed and integrated with a fluorescent microsphere-based lateral flow strip (LFS) for visual result interpretation. LAMP primers were dual labeled with digoxigenin and biotin to enable specific amplicon to capture at the test and control lines of the LFS. Reaction conditions for both LAMP reaction and LFS were optimized using genomic DNA extracted from P. aeruginosa-spiked powdered herbal samples. Analytical performance, including specificity, sensitivity, reproducibility, and limit of detection (LOD), was rigorously evaluated. The validated method was then applied to six commonly used TCM herbal pieces, including Astragali radix and Angelicae sinensis radix. Additionally, the assay performance was validated using six representative TCM herbal pieces and 90 clinical-relevant herbal samples in parallel with qPCR.
RESULTS: The LAMP-LFS assay demonstrated strict specificity for P. aeruginosa, with no cross-reactivity observed against any non-target bacterial strains tested. Optimal LAMP conditions were determined as 65 °C for 60 min in the presence of 6 mM Mg2+ and 1.4 mM dNTPs. The entire detection workflow, including DNA extraction, amplification, and strip readout, was completed within approximately 70 min. The assay achieved a limit of detection of 1 × 10² CFU/250 mL in spiked herbal matrices, with high reproducibility (CV < 5%). Consistent detection was observed across six different TCM herbal species, indicating strong tolerance to complex phytochemical backgrounds. In parallel validation with qPCR, the assay demonstrated a sensitivity of 94.0%, specificity of 97.5%, and overall accuracy of 95.6%, with a Cohen's kappa coefficient of 0.92.
CONCLUSION: The developed fluorescent microsphere-based LAMP-LFS assay provides a rapid, sensitive, and reproducible platform for detecting P. aeruginosa in TCM herbal pieces. Compared with conventional PCR-based approaches, this method demonstrates improved tolerance to inhibitory herbal matrices and enables reliable on-site applicability with minimal instrumentation requirements. The assay shows potential for routine microbiological quality control and field-based monitoring of TCM herbal materials.