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

Abstract

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.

A Lab-on-a-Chip Approach for the Detection of the Quarantine Potato Cyst Nematode Globodera pallida

“…), even so lower than 1%. These data are in line with previous studies, whose reports show a positive detection signal of 1.8 ± 0.7% and a negative control of 0.4 ± 0.3% [ 20 ].…”

“…Prior to probe immobilization, biochips underwent a cleaning procedure described in Viveiros et al (2020) [ 20 ].…”

Biosensors for European Zoonotic Agents: A Current Portuguese Perspective

“…In addition to their application in the context of food safety, potential biosensors for E. coli could also represent a significant advance for disease prevention in companion animals and animal production. For example, untrained personnel could use biosensors at the point-of-use to prevent or detect mastitis in cattle [ 29 ].…”

“…An MR biochip was recently used to genetically analyse the 16S rRNA gene in the multiplex detection of five important mastitis-causing bacteria, including E. coli , Klebsiella sp., S. aureus, Streptococcus uberis , and Streptococcus agalactiae [ 29 ]. These MR biochips consisted of an array of 30 magnetic field sensors (spin valves) modified with a series of oligonucleotide probes representing the most common mastitis-causing bacteria.…”

“…These MR biochips consisted of an array of 30 magnetic field sensors (spin valves) modified with a series of oligonucleotide probes representing the most common mastitis-causing bacteria. This was followed by amplification of the five other bacterial targets by asymmetric Polymerase Chain Reaction (PCR), which allowed successful detection of the targets without cross-reactivity and an LOD of 10 3 cell/mL [ 29 ].…”