N,N-Dimethyl-4,4′-azodianiline functionalized magnetic nanoparticles for enhanced sensitivity of nucleic acid amplification tests

Abstract

A facile molecular diagnostic method was devised for highly efficient detection of gram-negative bacterial pathogens with low abundance in urological samples, which contain various inhibitory factors of nucleic acid amplification. We developed a magnetic nanoparticle with N,N-Dimethyl-4,4′-azodianiline for bacterial pathogen sample preparation combining nucleic acid amplification test for bacterial pathogen detection and identification. We employed N,N-Dimethyl-4,4′-azodianiline functionalized nanoparticles to enable a dual strategy integrated within a single tube: (i) bacteria enrichment via the hydrophobic effect and (ii) DNA extraction and isolation via electrostatic interaction under pH control. It provided more yield and higher-quality DNA, resulting in a significant increase in fluorescent signal of quantitative PCR (qPCR). We evaluated our proposed method using gram-negative bacteria including Salmonella enterica serover Typhimurium (S. Typhimurium), Escherichia coli (E. coli), Klebsiella pneumonia (K. pneumonia) and Brucella ovis (B. ovis), found in human urine samples. Our proposed method resulted in a detection limit of five colony-forming units (CFU)/10 mL for S. Typhimurium and 10 CFU/10 mL for E. coli, and resulted in the detection of B. ovis and K. pneumonia. This novel method provides simple and sensitive detection of gram-negative bacteria species and can be used for diagnosis of urinary tract infections.