The detection and identification of microbial pathogens in meat and fresh produce play an essential role in food safety for reducing foodborne illnesses every year. A new approach based on targeting a specific sequence of the 16S rRNA region for each bacterium is proposed and validated. The probe complex consists of a C60, a conjugated RNA detector which targets a specific 16S rRNA sequence, and a complementary fluorescent reporter. The RNA detectors were designed by integrating NIH nucleotide and Vienna RNA Webservice databases, and their specificities were validated by the RDP database. Probe complexes were synthesized for identifying E. coli K12, E. coli O157: H7, S. enterica, Y. enterocolitica, C. perfringens, and L. monocytogenes. First, under controlled conditions of known bacterial mixtures, the efficiency and crosstalk for identifying the foodborne bacteria were quantified to be above 94% and below 5%, respectively. Second, experiments were designed by inoculating meat products by known numbers of bacteria and measuring the limit of detection. In one experiment, 225 g of autoclaved ground chicken was inoculated with 9 E. coli O157:H7, where 6.8 ± 1.2 bacteria with 95% confidence interval were recovered. Third, by positionally printing probe complexes in microwells, specific microorganisms were identified with only one fluorophore. The proposed protocol is a cell-based system, can identify live bacteria in 15 min, requires no amplification, and has the potential to open new surveillance opportunities.

Key points

• The identification of foodborne bacteria is enabled in live-cell assays.

• The limit of detection for 100 g of fresh chicken breast inoculated with 4 bacteria is 2.7 ± 1.4 with 95% confidence interval.

• The identification of five bacteria in a coded microwell chip is enabled with only one fluorophore.

肉类和新鲜农产品中微生物病原体的检测和鉴定在每年减少食物传播疾病的食品安全中起着至关重要的作用。提出并验证了一种基于针对每种细菌的16S rRNA区特定序列的新方法。该探针复合物由C60，其靶向特定的16S rRNA序列的缀合的RNA检测器，和互补荧光报道的。通过整合NIH核苷酸和Vienna RNA Webservice数据库设计了RNA检测器，并通过RDP数据库验证了其特异性。合成探针复合物以鉴定大肠杆菌K12，大肠杆菌O157：H7，肠炎链球菌，肠结肠炎耶尔森氏菌，产气荚膜梭菌和单核细胞增生李斯特菌。首先，在已知细菌混合物的受控条件下，鉴定食源性细菌的效率和串扰分别被量化为高于94％和低于5％。其次，通过在肉制品中接种已知数量的细菌并测量检出限来设计实验。在一项实验中，将225克高压灭菌地面鸡接种了9株大肠杆菌O157：H7，回收了具有95％置信区间的6.8±1.2个细菌。第三，通过在微孔中位置印刷探针复合物，仅使用一种荧光团即可鉴定出特定的微生物。拟议的协议是一个基于细胞的系统，可以在15分钟内识别出活细菌，无需扩增，并且有可能带来新的监视机会。

关键点

•在活细胞分析中可以鉴定食源性细菌。

• 100 g新鲜鸡胸肉接种4种细菌的检出限为2.7±1.4，置信区间为95％。

•仅用一种荧光团就能识别编码微孔芯片中的5种细菌。