A Fully Automated Microfluidic PCR-array System For Rapid Detection Of Multiple Respiratory Tract Infection Pathogens

Objective:An integrated microfluidic chip PCR-array system is developed to detect multiple respiratory tract infection pathogens in resource-limited settings.Methods:A PCR-array system including instrument and microfluidic chip was designed and manufactured,which integrated modules of sample lysis,nucleic acid（NA）extraction/purification,and NA amplification/detection.Six diaphragm valves control the opening and closing of fluid paths to ensure that fluid flows programmatically through the chip,so a series of analysis steps are completed in sequence.PCR is completed in 32 array chambers with preloaded primers and probes for different pathogens,allowing simultaneous detection of 21 respiratory tract infection pathogens and internal quality control（RNase P gene）in a single test.The fluorescence signal is collected by the instrument in real-time,and the detection results are determined according to the Ct value.In this study,the limit of detection（LOD）,reproducibility,and specificity of the microchip method were determined by analyzing the standard samples.The clinical application value of the chip method was investigated by detecting batch clinical samples.Results:The microfluidic chip system can detect multiple respiratory pathogens in a fully integrated manner within 1.5 hours.The LOD of the microchip method was confirmed by testing a series of diluted positive controls,and the LODs of each target were calculated at 1.0×10³ copies·m L^-1 based on Probit regression analysis（P>0.05）.Positive control substances of each target were repeatedly tested,and the experimental results showed that the inter-batch variation of Ct values ranged from0.08%to 0.69%,and the intra-batch variation ranged from 0.9%to 2.66%.The reference samples mixed with multiple pathogens were analyzed,and the results showed that there was no cross-influence among the pathogens,and the detection specificity of the chip method was 100%.Through the analysis of batch clinical samples,it can be seen that the detection results of the chip method and real-time PCR are consistent,which verifies the clinical practical value of the microchip method.Conclusion:The microfluidic-based multiple respiratory tract infection pathogens detection method has the advantages of simple operation,suitable throughput,and rapid detection,and thus is a powerful tool for rapid detection of pathogens in resource-limited settings.