A fully automated microfluidic PCR-array system for rapid detection of multiple respiratory tract infection pathogens

Abstract

Rapid and accurate identification of respiratory tract infection pathogens is of utmost importance for clinical diagnosis and treatment, as well as prevention of pathogen transmission. To meet this demand, a microfluidic chip-based PCR-array system, Onestart, was developed. The Onestart system uses a microfluidic chip packaged with all the reagents required, and the waste liquid is also collected and stored on the chip. This ready-to-use system can complete the detection of 21 pathogens in a fully integrated manner, with sample lysis, nucleic acid extraction/purification, and real-time PCR sequentially implemented on the same chip. The entire analysis process is completed within 1.5 h, and the system automatically generates a test report. The lower limit-of-detection (LOD) of the Onestart assay was determined to be 1.0 × 10³ copies·mL^-1. The inter-batch variation of cycle threshold (Ct) values ranged from 0.08% to 0.69%, and the intra-batch variation ranged from 0.9% to 2.66%. Analytical results of the reference sample mix showed a 100% specificity of the Onestart assay. The analysis of batched clinical samples showed consistency of the Onestart assay with real-time PCR. With its ability to provide rapid, sensitive, and specific detection of respiratory tract infection pathogens, application of the Onestart system will facilitate timely clinical management of respiratory tract infections and effective prevention of pathogen transmission. Onestart, a ready-to-use system, can detect 21 pathogens in a fully integrated manner on a microchip within 1.5 h.

Keywords: Automation; Microfluidic; Multiplex detection; Point-of-care testing; Rapid diagnosis; Respiratory tract infection.

Onestart, a ready-to-use system, can detect 21 pathogens in a fully integrated manner on a microchip within 1.5 h.

Fig. 1

Schematic illustration of the microfluidic chip. The microfluidic chip contains a sample reservoir, three reagent reservoirs, an SPE chamber, six diaphragm valves, a lyophilized powder tube, and an amplification module with 32 PCR chambers

Fig. 2

a The Onestart microchip analyzer. b The composite structure diagram of the Onestart microchip analyzer

Fig. 3

Operation procedure of the Onestart system

Fig. 4

Variations in Ct value in the analysis of the positive references using the Onestart system

Fig. 5

Ct values of sample diluents with target concentration around LOD₉₅