荧光标记噬菌体快速检测K1荚膜大肠杆菌方法的建立及应用

Establishment and application of fluorescence labeled phage for rapid detection of Escherichia coli K1

摘要:

[背景] 大肠杆菌（Escherichia coli，E.coli）是引发新生儿脑膜炎和禽类脑膜炎最常见的革兰氏阴性菌，其中含K1荚膜大肠杆菌是重要的病原菌。目前，K1荚膜大肠杆菌的检测方法存在一些弊端。[目的] 利用PNJ1809-36噬菌体的宿主特异性建立快速检测K1荚膜大肠杆菌的方法。[方法] 用荧光染料SYBR Gold标记PNJ1809-36噬菌体，侵染33株受试菌，在荧光显微镜下观察，测定该方法的特异性；倍比稀释宿主菌DE058，用荧光标记噬菌体侵染，测定该方法的灵敏度；用荧光标记噬菌体检测8份模拟粪样，测定该方法的临床应用效果；测定4℃避光保存4个月的荧光标记噬菌体的效价和检测效果。[结果] 33株受试菌中的9株K1荚膜大肠杆菌有8株可见环状荧光，1株未能检出；20株非K1荚膜大肠杆菌以及4株非大肠杆菌属细菌均不能观察到荧光，检测灵敏度达100 CFU/mL。8份模拟粪样的检测结果显示，3份含有K1荚膜大肠杆菌的粪样均可见环状荧光，5份不含K1荚膜大肠杆菌的粪样均无荧光。荧光标记噬菌体4℃避光保存4个月后效价无明显下降，检测效果无明显变化，表明该荧光标记噬菌体在4℃避光条件下较稳定。[结论] 用荧光标记的PNJ1809-36噬菌体能够在15 min内特异、快速、直观地检测K1荚膜大肠杆菌。

关键词:噬菌体;荧光标记;大肠杆菌;检测;K1荚膜

Abstract:

[Background] Escherichia coli (E. coli) is one of the most common Gram-negative bacteria causing neonatal meningitis and poultry meningitis. The strain E. coli capsular K1 is an important pathogen. Current methods for detecting E. coli capsular K1 have some disadvantages.[Objective] A rapid detection method for E. coli capsular K1 was established by using the host specific phage PNJ1809-36. [Methods] Phage PNJ1809-36 was labeled with fluorescent dye SYBR gold, and 33 tested strains were infected by this fluorescent phage. The specificity was determined by observing the 33 test strains using the fluorescence microscopy. The sensitivity was determined by observing the host bacteria DE058 with gradient tenfold dilution after being infected with fluorescence labeled phage. Eight simulated feces were detected by fluorescent labeled phages. Moreover, the titer change and detection efficiency of fluorescent labeled phages stored at 4℃ in darkness were investigated. [Results] Among the 33 strains tested, 8 strains of 9 E. coli can be observed with fluorescence around the bacteria, but one could not be detected. No fluorescence was observed in 20 non-E. coli capsular K1 strains and 4 non-E. coli strains. The detection sensitivity was 100 CFU/mL. The detection results of 8 simulated fecal samples showed that fluorescence could be observed in 3 samples containing E. coli capsular K1, and no fluorescence could be detected in 5 samples without E. coli capsular K1. After stored at 4℃ for 4 months, the titer of fluorescence labeled phages showed no obvious decrease, and the detection efficiency did not change significantly. [Conclusion] The fluorescence labeled phage PNJ1809-36 could be used to detect E. coli capsular K1 rapidly and intuitively within 15 min.

Key words:phage;fluorescence labeling;Escherichia coli;detection;K1 capsule