基于聚合酶螺旋反应技术快速检测铜绿假单胞菌

Rapid detection of Pseudomonas aeruginosa by polymerase spiral reaction (PSR)

摘要:

【背景】铜绿假单胞菌是一种重要的水源和食源性致病菌，可引起急性肠道炎、脑膜炎、败血症和皮肤炎症等疾病。加强铜绿假单胞菌的快速检测，对保障食品安全具有重要的意义。【目的】建立聚合酶螺旋反应(Polymerase spiral reaction，PSR)方法快速检测铜绿假单胞菌。【方法】针对铜绿假单胞菌外毒素A调控基因——ETA基因(toxA)设计引物，通过引入加速引物、优化反应条件和筛选颜色指示剂，建立快速检测铜绿假单胞菌的PSR方法，并研究方法的特异性、敏感性和可靠性。【结果】建立的方法在等温65 °C条件下，40 min内可完成PSR反应，且可通过钙黄绿素和羟基萘酚蓝直接判读结果。方法特异性强、灵敏度高，最低检出限分别为20 CFU/mL细菌和1.011 5 pg/μL基因组DNA。可视化PSR方法检测包装饮用水来源的分离菌株与传统生化方法检测结果一致。【结论】研究建立的可视化PSR方法为铜绿假单胞菌DNA快速检测提供了一种可行的有效手段。

Abstract:

[Background] Pseudomonas aeruginosa is an important water- and food-borne pathogenic bacterium causing acute enteritis, meningitis, sepsis and skin inflammation. It is important to detect rapidly P. aeruginosa for food safety. [Objective] A rapid and simple method for detection of P. aeruginosa using polymerase spiral reaction (PSR) technique was established. [Methods] The primers were designed according to the P. aeruginosa exotoxin A gene—ETA gene (toxA), the visual PSR method for rapid detection of P. aeruginosa comprised primer screening, accelerated primer introduction, reaction conditions optimization and color indicator screening. The specificity, sensitivity and reliability of the method were evaluated. [Results] The PSR assay to detect toxA of P. aeruginosa could fulfill within 40?min at 65 °C, and visualize the results by Calcein and HNB. This method specifically detected P. aeruginosa without showing cross-reaction with closely related Pseudomonas species or other bacteria. The sensitivity of the method was high, with detection limit 20 CFU/mL bacteria and 1.011 5 pg/μL genomic DNA. The detection of isolated strain of packaged drinking water showed that the PSR method was consistent with the results of traditional biochemical methods for the P. aeruginosa detection. [Conclusion] The established visual PSR method provides a potential method to be used for rapid detection in the field.