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

Shijun Li,Yue Wang,Qing Ma,Ying Liu,Chunting Liu,Yi Wang

doi:10.1186/s12866-020-01945-x

Abstract

BackgroundBordetella 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.ResultsA 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.ConclusionsThe isothermal amplification based MCDA-LFB established in the present study is a specific, sensitive, rapid and economical technique for the detection of B. pertussis.

Highlights

Bordetella pertussis is the causative agent of pertussis, a respiratory tract infectious disease
Pertussis, referred whooping cough or named as “cough of 100 days”, is a respiratory tract infectious disease primarily leaded by the causative agent Bordetella pertussis [1], a gram negative bacterium which can attach to the ciliated cells and colonize the human upper respiratory tract [2]
Confirming the effectiveness of B. pertussis-Multiple cross displacement amplification (MCDA) primer set To confirm the effectiveness of B. pertussis-MCDA primer set targeting on the pertussis toxin (PT) promoter region (Fig. 1, Table 1), the Deoxyribo Nucleic Acid (DNA) extracted from B. pertussis strain was Optimizing the reaction temperature of B. pertussisMCDA-LFB To optimize amplification temperature, genomic DNA templates from B. pertussis The Global Bioresource Center (ATCC)-9340 were applied as the positive control with 10 pg DNA for each reaction

Summary

Introduction

Bordetella pertussis is the causative agent of pertussis, a respiratory tract infectious disease. Referred whooping cough or named as “cough of 100 days”, is a respiratory tract infectious disease primarily leaded by the causative agent Bordetella pertussis [1], a gram negative bacterium which can attach to the ciliated cells and colonize the human upper respiratory tract [2]. It can damage the respiratory epithelium by producing multiple toxins and leads to systemic effects such as the lymphocytosis [3], local inflammatory changes in the mucosal lining of the respiratory tract [1]. According to the estimation of WHO in 2013, B. pertussis infection caused about 60,257 deaths in children younger than 5 years [5]

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