PDF(Pubmed)
Abstract：
Achieving effective control over microbial contamination necessitates the precise and concurrent identification of numerous pathogens. As a common bacterium in the environment, Pseudomonas is rich in variety. It not only has pathogenic strains, but also spoilage bacteria that cause food spoilage. In this research, we devised a remarkably sensitive duplex droplet digital PCR (dddPCR) reaction system to simultaneously detect pathogenic Pseudomonas aeruginosa (P. aeruginosa) and spoilage Pseudomonas fragi (P. fragi). By employing comparative genomics, we identified four genes of P. fragi. Through a specific analysis, the RS22680 gene was selected as the detection target for P. fragi, and the lasR gene was chosen for P. aeruginosa, which were applied to construct a dddPCR reaction. In terms of specificity, sensitivity and anti-interference ability, the constructed dddPCR detection system was verified and analyzed. The assay showed excellent sensitivity and applicability, as evidenced by a limit of detection of 100 cfu/mL. When the concentration of natural background bacteria in milk or fresh meat was 100 times that of the target detection bacteria, the method was still capable of completing the absolute quantification. In the simulation of actual sample contamination, P. aeruginosa could be detected after 3 h of enrichment culture, and P. fragi could be detected after 6 h. The established dddPCR detection system exhibits exceptional performance, serving as a foundation for the simultaneous detection of various pathogenic bacteria in food products.
摘要：
实现对微生物污染的有效控制需要精确和同时识别多种病原体。作为环境中常见的细菌，假单胞菌品种丰富。它不仅有致病菌株，还有导致食物腐败的腐败细菌。在这项研究中,我们设计了一个非常灵敏的双链液滴数字PCR(dddPCR)反应系统来同时检测致病性铜绿假单胞菌(P.铜绿假单胞菌）和腐败脆弱假单胞菌（P.frafi）。通过使用比较基因组学，我们确定了4个基因。通过具体分析,选择RS22680基因作为Fragi的检测目标，选择了铜绿假单胞菌的lasR基因,用于构建dddPCR反应。就特异性而言，灵敏度和抗干扰能力，对构建的dddPCR检测系统进行了验证和分析。该方法具有良好的灵敏度和适用性,如100cfu/mL的检测限所示。当牛奶或鲜肉中天然背景菌的浓度是目标检测菌的100倍时，该方法仍能完成绝对定量.在模拟实际样品污染时,富集培养3小时后可检测到铜绿假单胞菌，和P.fragi可以在6小时后检测到。建立的dddPCR检测系统表现出卓越的性能，作为同时检测食品中各种致病菌的基础。
