Establishment and application of a multiple cross displacement amplification combined with nanoparticles-based biosensor method for the detection of Bordetella pertussis

摘要

Bordetella pertussis is the causative agent of pertussis, a respiratory tract infectious disease. Efficient techniques for detection of B. pertussis isolates are important for clinical diagnosis. Multiple cross displacement amplification (MCDA), a novel isothermal amplification based molecular detection method, has been developed to overcome the technical drawback of the current methods in recent years. This aim of this study is to develop a MCDA with Nanoparticles-based Lateral Flow Biosensor (MCDA-LFB) for the detection of B. pertussis. A set of 10 primers based on the pertussis toxin (PT) promoter region sequence of B. pertussis was designed. The B. pertussis-MCDA-LFB assay was successfully established and optimized at 64?°C for reaction of 40?min. The detection limit was determined as 10?fg/reaction of pure DNA, and no cross-reactions to non-B. pertussis strains were observed, based on the specificity validation. The whole operation, ranging from template preparation to result reporting, could be completed within 70?min without requirement of costly equipment. The B. pertussis-MCDA-LFB in clinic sample detection yielded identical positive rates with traditional culture and showed higher sensitivity than conventional PCR. The results of MCDA-LFB are easier to read due to the usage of LFB. The isothermal amplification based MCDA-LFB established in the present study is a specific, sensitive, rapid and economical technique for the detection of B. pertussis.