Establishment and Application of a Multiple Cross Displacement Amplification Coupled With Nanoparticle-Based Lateral Flow Biosensor Assay for Detection of Mycoplasma pneumoniae.

Yacui Wang,Adong Shen,Yonghong Wang,Xue Qi,Xingyun Wang,Lin Sun,Jieqiong Li,Shuting Quan,Weiwei Jiao,Yi Wang

doi:10.3389/fcimb.2019.00325

Abstract

Mycoplasma pneumoniae (M. pneumoniae) is responsible for pneumonia, and is a causative agent of other respiratory tract infections (e.g., bronchiolitis and tracheobronchitis). Herein, we established and applied a multiple cross displacement amplification (MCDA) coupled with a nanoparticle-based lateral flow biosensor (LFB) assay (MCDA–LFB) for rapid, simple, and reliable detection of target pathogen. A set of 10 primers was designed based on M. pneumoniae-specific P1 gene, and optimal reaction conditions were found to be 30 min at 65°C. The detection results were visually reported using a biosensor within 2 min. The M. pneumoniae–MCDA–LFB method specifically detected only M. pneumoniae templates, and no cross-reactivity was generated from non-M. pneumoniae isolates. The analytical sensitivity for this assay was 50 fg of genomic templates in the pure cultures, as obtained from colorimetric indicator and real-time turbidimeter analysis. The assay was applied to 197 oropharyngeal swab samples collected from children highly suspected of M. pneumoniae infection, and compared to culture-based method and real-time PCR assay. The detection rates of M. pneumoniae using a culture-based method, real-time PCR assay, and MCDA–LFB assay were 8.1%, 33.0%, and 52.3%, respectively, which indicated that the MCDA–LFB assay was superior to the culture-based method and real-time PCR method for detection of target agent. Using this protocol, 25 min for rapid template extraction followed by MCDA reaction (30 min) combined with LFB detection (2 min) resulted in a total assay time of ~60 min. In conclusion, the MCDA–LFB assay established in this report was a simple, rapid, sensitive, and reliable assay to detect M. pneumoniae strains, and can be used as a potential diagnostic tool for M. pneumoniae in basic and clinical laboratories.

Highlights

Mycoplasma pneumoniae (M. pneumoniae) is one of the most common pathogens causing community-acquired pneumonia (CAP) in children and adolescents (Waites and Talkington, 2004; Atkinson et al, 2008)
The results of the multiple cross displacement amplification (MCDA)–lateral flow biosensor (LFB) assay for M. pneumoniae detection were compared with that of the culturebased method and real-time PCR assay for the identical samples
Using an LFB, two visible red lines (Test line, TL; Control line, CL) were seen in MCDA-positive products; only one red line (CL) was observed in negative and blank controls (Figure 2B). These results suggested that the MCDA primer set designed here was a good candidate for development of MCDAbased assay for M. pneumoniae detection

Summary

Introduction

Mycoplasma pneumoniae (M. pneumoniae) is one of the most common pathogens causing community-acquired pneumonia (CAP) in children and adolescents (Waites and Talkington, 2004; Atkinson et al, 2008). During epidemics, this causative agent can cause 40% and 70% of CAP in populations with general and high population density, respectively (Loens et al, 2010; Jacobs et al, 2015). Owing to lack of cell walls, M. pneumoniae is not sensitive to β-lactam antibiotics, which are selected first for treatment of common pathogens. Developing a simpler, faster, and more accurate method for M. pneumoniae detection is imperative to treat timely with effective antibiotics

Methods

Results

Conclusion