Multiple Bacteria Identification in the Point-of-Care: An Old Method Serving a New Approach

Sensors

Mónica Rodrigues¹  Sofia A. M. Martins²  Verónica C. Martins²  Susana Cardoso³  Sara Viveiros³  Débora Albuquerque³ 

[1] Centre for Ecology, Evolution and Environmental Changes, Faculdade de Ciências, Universidade de Lisboa, 1749-016 Lisboa, Portugal;INESC-MN- Microsystems and Nanotechnologies, 1000-029 Lisboa, Portugal;Instituto Superior Técnico, University of Lisbon, 1049-001 Lisboa, Portugal;

关键词: magnetoresistance;    spin-valve;    asymmetric PCR;    biosensor;    bacterial pathogens;   

DOI  :  10.3390/s20123351

来源: DOAJ

【 摘 要 】

The accurate diagnosis of bacterial infections is of critical importance for effective treatment decisions. Due to the multietiologic nature of most infectious diseases, multiplex assays are essential for diagnostics. However, multiplexability in nucleic acid amplification-based methods commonly resorts to multiple primers and/or multiple reaction chambers, which increases analysis cost and complexity. Herein, a polymerase chain reaction (PCR) offer method based on a universal pair of primers and an array of specific oligonucleotide probes was developed through the analysis of the bacterial 16S ribosomal RNA gene. The detection system consisted of DNA hybridization over an array of magnetoresistive sensors in a microfabricated biochip coupled to an electronic reader. Immobilized probes interrogated single-stranded biotinylated amplicons and were obtained using asymmetric PCR. Moreover, they were magnetically labelled with streptavidin-coated superparamagnetic nanoparticles. The benchmarking of the system was demonstrated to detect five major bovine mastitis-causing pathogens: Escherichia coli, Klebsiella sp., Staphylococcus aureus, Streptococcus uberis, and Streptococcus agalactiae. All selected probes proved to specifically detect their respective amplicon without significant cross reactivity. A calibration curve was performed for S. agalactiae, which demonstrates demonstrating a limit of detection below 30 fg/µL. Thus, a sensitive and specific multiplex detection assay was established, demonstrating its potential as a bioanalytical device for point-of-care applications.

【 授权许可】

Unknown