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.

设计了一种简便的分子诊断方法，用于高效检测泌尿科样本中低丰度的革兰氏阴性细菌病原体，其中含有多种核酸扩增抑制因子。我们开发了一种具有N，N -Dimethyl-4,4'-azodianiline的磁性纳米颗粒，用于细菌病原体样品制备，并结合了用于细菌病原体检测和鉴定的核酸扩增测试。我们雇用了N，N-Dimethyl-4,4'-azodianiline官能化的纳米颗粒可实现在单个试管中整合的双重策略：（i）通过疏水作用富集细菌，以及（ii）在pH控制下通过静电相互作用进行DNA提取和分离。它提供了更高的产量和更高质量的DNA，从而大大提高了定量PCR（qPCR）的荧光信号。我们使用的革兰氏阴性细菌包括评估我们提出的方法肠道沙门氏菌serover鼠伤寒沙门氏菌（S.鼠伤寒沙门氏菌），大肠埃希氏菌（大肠杆菌），肺炎克雷伯氏菌（K.肺炎）和布鲁氏羊（B.绵羊），存在于人类尿液样本中。我们提出的方法导致了5菌落形成单位（CFU）的检测极限/ 10 mL的S.鼠伤寒沙门氏菌和10 CFU / 10mL的对大肠杆菌，并导致在检测B.绵羊和肺炎克雷伯菌。这种新颖的方法可对革兰氏阴性细菌种类进行简单而灵敏的检测，可用于诊断尿路感染。