Feng Xiaoyan, Zhou Donggen, Xie Guoyang, Liu Ju, Xiong Qin, Xu Hengyi
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047, P.R. China.
Ningbo International Travel Healthcare Center (Ningbo Customs Port Outpatient Department), Ningbo, 315010, P.R. China.
J Dairy Sci. 2022 Jun;105(6):4895-4902. doi: 10.3168/jds.2022-21900. Epub 2022 Apr 18.
In addition to Cronobacter spp., Klebsiella pneumoniae is another opportunistic bacterial pathogen present in powdered infant formula (PIF) that can cause pneumonia, septicemia, and other diseases. In this study, a rapid and specific method based on a fluorescence probe was developed for detecting viable K. pneumoniae in PIF samples via the combination of recombinase-aided amplification (RAA) with thiazole orange monoazide (TOMA) dye (the TOMA-RAA assay hereafter). As a novel photosensitive DNA-intercalating dye, TOMA was used to penetrate bacterial cells, including both dead and viable cells, as verified by confocal laser scanning microscopy and fluorescent emission spectrometry. Importantly, the RAA assay exhibited good performance in detecting K. pneumoniae within 40 min at 39°C. Under optimal conditions, the TOMA-RAA assay can detect as low as 2.6 × 10 cfu/mL of K. pneumoniae in pure culture and 2.3 × 10 cfu/g of K. pneumoniae in spiked PIF sample. After 3 h of pre-enrichment, 3 × 10 cfu/g of K. pneumoniae can be detected. Furthermore, the TOMA-RAA assay displayed an excellent anti-interference ability to nontarget bacteria. In short, the proposed method has great potential application for the rapid and accurate detection of viable K. pneumoniae in PIF.
除了阪崎肠杆菌属外，肺炎克雷伯菌是存在于婴儿配方奶粉（PIF）中的另一种机会性细菌病原体，可导致肺炎、败血症和其他疾病。在本研究中，基于荧光探针开发了一种快速、特异的方法，通过重组酶辅助扩增（RAA）与噻唑橙单叠氮化物（TOMA）染料相结合（以下简称TOMA-RAA检测法）来检测PIF样品中的活肺炎克雷伯菌。作为一种新型的光敏DNA嵌入染料，通过共聚焦激光扫描显微镜和荧光发射光谱验证，TOMA可穿透包括死细胞和活细胞在内的细菌细胞。重要的是，RAA检测法在39℃下40分钟内检测肺炎克雷伯菌表现出良好的性能。在最佳条件下，TOMA-RAA检测法在纯培养物中可检测低至2.6×10 cfu/mL的肺炎克雷伯菌，在加标PIF样品中可检测2.3×10 cfu/g的肺炎克雷伯菌。经过3小时的预富集后，可检测到3×10 cfu/g的肺炎克雷伯菌。此外，TOMA-RAA检测法对非靶标细菌表现出优异的抗干扰能力。简而言之，所提出的方法在快速、准确检测PIF中活肺炎克雷伯菌方面具有巨大的潜在应用价值。
