Methodological Validation and Clinical Application Value of Microfluidic Multiplex PCR for Rapid Detection of Community-Acquired Respiratory Pathogens

Abstract

Objective : To evaluate the detection performance of microfluidic chip-based multiplex PCR technology for rapid detection of community-acquired respiratory pathogens, complete a systematic methodological validation, conduct a comprehensive comparison with traditional singleplex PCR and conventional bacterial culture, clarify the clinical application value of this technology in primary healthcare institutions, and provide a scientific basis for precise diagnosis and treatment of community-acquired respiratory infections as well as rational antibiotic stewardship strategies. Methods : A total of consecutive 50 patients with suspected community-acquired respiratory infections who attended the outpatient and inpatient departments of Guangxi—ASEAN Economic and Technological Development Zone People’s Hospital from March 1, 2025, to February 28, 2026, were enrolled as study subjects. The clinical diagnostic criteria for community-acquired respiratory infections followed the Guidelines for Diagnosis and Treatment of Community-Acquired Pneumonia (2023 edition) issued by the Chinese Thoracic Society 22. All samples were tested in parallel using three methods: microfluidic chip-based multiplex PCR, traditional singleplex PCR, and conventional bacterial culture. Complete methodological validation of the multiplex PCR technology was performed, including nucleic acid extraction quality, internal control assessment, accuracy, intra-assay precision, and inter-assay precision. The positive detection rate, negative detection rate, co-infection detection rate, turnaround time, and per-sample testing cost were statistically analyzed and compared among the three methods. The local pathogen spectrum distribution characteristics of community-acquired respiratory infections were analyzed. Discordant results were re-tested, analyzed, and arbitrated. Discordant samples were arbitrated using a composite reference standard based on repeat testing combined with clinical or microbiological evidence (including clinical symptoms, inflammatory markers, and imaging findings), without using multiplex PCR results as the sole basis for confirming its own results. SPSS 26.0 software was used for statistical analysis. Count data were expressed as numbers and percentages, and the McNemar test was used for comparison of positive rates between groups, with P < 0.05 considered statistically significant. Results : Among the 50 samples, microfluidic multiplex PCR yielded 39 positive results, with a positive rate of 78%; traditional singleplex PCR yielded 26 positive results, with a positive rate of 52%; and conventional bacterial culture yielded 12 positive results, with a positive rate of 24%. The positive rate of multiplex PCR was significantly higher than those of the two traditional methods (McNemar test, P < 0.01). The turnaround time of multiplex PCR was 2.5 ± 0.2 hours, much shorter than that of traditional singleplex PCR (7.0 ± 0.5 hours) and conventional bacterial culture (72.0 ± 2.0 hours). Multiplex PCR detected 8 cases of co-infection, accounting for 16%, while neither traditional method detected any co-infection. Methodological validation showed that the internal control positivity rate across 5 batches was 100%, with a mean Ct value of 24.7 ± 0.9; the intra-assay precision CV was 2.45%, and the inter-assay precision CV was 3.07%, both below 5%, indicating that the accuracy, repeatability, and stability all met clinical testing requirements. After arbitration of the 6 discordant samples, multiplex PCR was confirmed to accurately detect bacterial pathogens and co-infections, increasing the positive rate by 26 percentage points compared with traditional singleplex PCR. A secondary analysis limited to the 8 pathogen targets shared by multiplex PCR and traditional singleplex PCR (influenza A, influenza B, respiratory syncytial virus, parainfluenza virus, adenovirus, rhinovirus, Mycoplasma pneumoniae , Chlamydia pneumoniae ) showed that the positive detection rates were 78% (39/50) and 52% (26/50), respectively, still favoring multiplex PCR, suggesting that the performance improvement was not solely due to broader panel coverage but also related to higher analytical sensitivity. Conclusion : Microfluidic chip-based multiplex PCR technology offers advantages including rapid detection, comprehensive pathogen coverage, and stable results. Under the conditions of this study, it demonstrated a high detection rate and good precision. It can significantly improve the detection rate of community-acquired respiratory pathogens, effectively identify co-infections, and demonstrate stable and reliable methodological performance, making it suitable for the existing testing conditions in primary healthcare institutions. This technology can rapidly differentiate among viral, bacterial, and atypical pathogen infections, providing key evidence for early precise clinical diagnosis and treatment. It holds significant value for promoting the reduction of inappropriate antibiotic use and decreasing the risk of bacterial resistance.