关键词: 聚合酶链反应, 分枝杆菌, 结核, 微生物敏感性试验, 研究

Abstract:

Objective: This study aimed to systematically compare the performance differences of three digital PCR (dPCR) platforms on detection of Mycobacterium tuberculosis (MTB) nucleic acid, and to provide a scientific basis for platform selection in different application scenarios. Methods: Genomic DNA of the MTB standard strain H37Rv was extracted and serially diluted two-fold to prepare 22 samples with different concentration gradients. Three commercial dPCR platforms, namely TargetingOne D20 (Platform A), Snjper DQ24 (Platform B), and Maccura D610 (Platform C), along with their matched MTB detection kits, were used for testing. Evaluation metrics included detection sensitivity (the highest dilution at which a positive signal could be detected) and quantitative linear range (the span of consecutive concentration gradients meeting a coefficient of determination, R²≥0.99). A comprehensive analysis integrated core instrumental parameters (e.g., partition number, detection principle) and reagent kit characteristics (target genes, cost) was performed. Results: Regarding detection sensitivity, Platforms A, B, and C could detect positive signals at the highest dilution of 2²¹, 2⁹, and 2¹¹, respectively. Regarding the quantitative linear range (R²≥0.99), Platform A covered 11 consecutive concentration gradients, outperforming Platform B (5 gradients) and Platform C (4 gradients). Technical correlation analysis indicated that the high droplet count (100000) and the laser-photomultiplier tube (PMT) detection system of Platform A were the basis for its high sensitivity and wide linear range. The vibrating droplet and complementary metal oxide semiconductor (CMOS) imaging design adopted by Platforms B and C offered relative advantages in terms of cost and operational speed. For the accompanying reagent kits, both kits for Platform A and B contained target IS1081 and IS6110, enabling both qualitative and quantitative detection, while the kit for Platform B additionally integrated probes for drug resistance mutations rpoB and gyrB. The kit for Platform C contained only a single target IS6110, resulting in relatively limited functionality. Conclusion: Platforms following different technical pathways showed distinct differences in performance, functionality, and cost. Appropriate selection can be made according to specific needs, including scientific research monitoring, routine clinical diagnosis, and large-scale screening with emphasis on cost-effectiveness.

Key words: Polymerase chain reaction, Mycobacterium tuberculosis, Microbial sensitivity tests, Research