Zhang Ni, Hou Laiwang, Li Darong, Lan Weiqing, Zhao Yong, Sun Xiaohong
College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China.
Shanghai Engineering Research Center of Aquatic-Product Processing & Preservation, Shanghai 201306, China.
Foods. 2025 Feb 9;14(4):573. doi: 10.3390/foods14040573.
and are significant and prevalent pathogens associated with bovine mastitis on dairy farms worldwide, resulting in severe infections in both dairy cows and, subsequently, human beings. Fast and dependable pathogen diagnostics are essential to minimize the effects of cow mastitis and human infections. The aim of this research was to develop a duplex recombinase-aided amplification (RAA) combined with the lateral flow dipstick (LFD) method, which was used for rapid, simultaneous detection of and . The SKII culture medium for and cocultivation was developed in this study. By optimizing the duplex RAA-LFD reaction conditions in terms of primer concentration, amplification temperature, and reaction time, the duplex RAA-LFD assay could successfully detect and when the reaction was conducted at 39 °C for 20 min. The duplex RAA-LFD method demonstrated good specificity, exhibiting no cross-reactivity with other pathogens. In addition, the detection limit of the duplex RAA-LFD for and was 60 fg of genomic DNA and 1.78 × 10 and 2.46 × 10 CFU/mL of bacteria in pure culture. Moreover, the duplex RAA-LFD technique is capable of identifying and in artificially spiked milk samples even at very low initial concentrations of 1.78 × 10 and 2.46 × 10 CFU/mL, respectively, after 6 h of enrichment. The result of the actual samples showed that the total concordance rate of the duplex RAA-LFD method with the biochemical identification method and PCR method could reach 92.98~98.25% with high consistency. The results of this study indicated that the duplex RAA-LFD assay, which is a precise, sensitive, and simple field testing technique, can be used to identify and and is expected to be used for disease diagnosis.
[病原体名称1]和[病原体名称2]是全球奶牛场与牛乳腺炎相关的重要且普遍存在的病原体，会导致奶牛严重感染，随后还会感染人类。快速可靠的病原体诊断对于将奶牛乳腺炎和人类感染的影响降至最低至关重要。本研究的目的是开发一种双链重组酶辅助扩增（RAA）结合侧向流动试纸条（LFD）的方法，用于快速、同时检测[病原体名称1]和[病原体名称2]。本研究开发了用于[病原体名称1]和[病原体名称2]共培养的SKII培养基。通过在引物浓度、扩增温度和反应时间方面优化双链RAA-LFD反应条件，当反应在39℃进行20分钟时，双链RAA-LFD检测法能够成功检测到[病原体名称1]和[病原体名称2]。双链RAA-LFD方法显示出良好的特异性，与其他病原体无交叉反应。此外，双链RAA-LFD对[病原体名称1]和[病原体名称2]的检测限分别为60 fg基因组DNA以及纯培养物中1.78×10[细菌数量单位1]CFU/mL和2.46×10[细菌数量单位2]CFU/mL的细菌。此外，双链RAA-LFD技术能够在富集6小时后，即使在初始浓度分别非常低至1.78×10[细菌数量单位1]CFU/mL和2.46×10[细菌数量单位2]CFU/mL的人工加标牛奶样品中鉴定出[病原体名称1]和[病原体名称2]。实际样品的结果表明，双链RAA-LFD方法与生化鉴定方法和PCR方法的总符合率可达92.98%至98.25%，一致性较高。本研究结果表明，双链RAA-LFD检测法是一种精确、灵敏且简单的现场检测技术，可用于鉴定[病原体名称1]和[病原体名称2]，有望用于疾病诊断。
