构建冻干无细胞生物传感器快速检测临床铜绿假单胞菌感染  

Developing a lyophilized cell-free biosensor for rapid diagnostics of clinical Pseudomonas aeruginosa infections

作　　者：黄慧文 俞鼎辰 李一荣[2] 刘天罡 HUANG Huiwen;YU Dingchen;LI Yirong;LIU Tiangang(Key Laboratory of Combinatorial Biosynthesis and Drug Discovery,Ministry of Education,School of Pharmaceutical Sciences,Wuhan University,Wuhan 430071,Hubei,China;Department of Clinical Laboratory,Zhongnan Hospital,Wuhan University,Wuhan 430071,Hubei,China;TaiKang Center for Life and Medical Sciences,Wuhan University,Wuhan 430071,Hubei,China)

机构地区：[1]武汉大学药学院组合生物合成与新药发现教育部重点实验室,湖北武汉430071 [2]武汉大学中南医院检验科,湖北武汉430071 [3]武汉大学泰康生命医学中心,湖北武汉430071

出　　处：《微生物学报》2023年第5期1959-1969,共11页Acta Microbiologica Sinica

基　　金：武汉大学泰康生命医学中心;国家自然科学基金(31971341)。

摘　　要：【目的】铜绿假单胞菌(Pseudomonas aeruginosa)是常见于医院感染的条件致病革兰氏阴性细菌,其群体感应信号3-氧代十二烷酰基高丝氨酸内酯(3-oxo-dodecanoyl-homoserine lactone,3OC12-HSL)可作为铜绿假单胞菌感染的生物标志物。本研究期望开发针对3OC12-HSL的冻干无细胞生物传感器,以实现临床铜绿假单胞菌感染的快速诊断。【方法】首先构建报告质粒以重建3OC12-HSL的应答过程,而后将该质粒加入冻干无细胞表达系统中以实现生物传感器的制备;接着利用梯度浓度的3OC12-HSL表征该传感器的灵敏度与动力学特征,并测试其底物特异性;最后通过临床样本测试验证其效果,并优化临床样本的预处理方法。【结果】本研究构建的冻干无细胞生物传感器能够在60 min内实现对临床呼吸道样本中铜绿假单胞菌感染的诊断,具有高灵敏度和高特异性。【结论】本研究构建了针对3OC12-HSL的冻干无细胞生物传感器,并借助RNase抑制蛋白预表达的策略提升了其对体液样本的耐受性,最终证明其具备开发成临床铜绿假单胞菌感染的快速检测方法的潜力。[Objective]Pseudomonas aeruginosa is a Gram-negative bacterium prevalent that is conditionally pathogenic and commonly associated with nosocomial infections.Its quorum sensing signal,3-oxo-dodecanoyl-homoserine lactone(3OC12-HSL),has been reported as a biomarker for its infection.Here,we aimed to develop a 3OC12-HSL biosensor for facilitating rapid diagnostics of clinical P.aeruginosa infections.[Methods]We constructed a reporter plasmid responsive to 3OC12-HSL and prepared a lyophilized cell-free biosensor based on that plasmid.We characterized the biosensor with 3OC12-HSL concentration gradient,validated its utility by testing clinical samples,and attempted to optimize the preprocessing procedure for clinical samples.[Results]Our cell-free biosensor is sensitive,specific,and capable of diagnosing P.aeruginosa infections in clinical respiratory samples within 60 min.[Conclusion]In this study,we developed a lyophilized cell-free 3OC12-HSL biosensor.By pre-expressing RNase inhibitor,we increased our biosensor’s tolerance to body fluid,and further demonstrated its potential to be developed as a rapid diagnostic method for clinical P.aeruginosa infections.

关 键 词：无细胞生物传感器 铜绿假单胞菌 N-酰基高丝氨酸内酯 

分 类 号：R446.5[医药卫生—诊断学]