应用表面增强拉曼技术快速检测6种食源性致病菌的研究
发布时间：2018-08-02 16:08
【摘要】：临床诊断和生物安全领域迫切的需要不需经过耗时培养过程的直接的细菌检测方法。开发快速、灵敏和准确的细菌诊断方法是保障公众以及动物健康的主要目标。传统的细菌检测包括染色,光学显微镜检查,微生物培养等,而传统检测方法只能判别出Gram+(G+)和Gram-(G-),并且微生物培养过程需要24-72小时;以及新的扩增技术,包括免疫测定(酶联免疫吸附法(ELISA),核酸鉴定(聚合酶链式反应,PCR)等,虽然扩增技术可以达到高特异性和灵敏度,但都需要繁琐的操作过程,不适于现场检测;高通量测序和微阵列可以同时检测多种病原菌,但这些技术需要事先分离出细菌的DNA,以及需要昂贵的核酸扩增仪器,这些都阻碍了这些技术在实际检测中的应用。于此相比,分子光谱,如红外光谱和拉曼光谱,都可以快速扫描得到微生物的生化指纹。但因为红外光谱对水极度敏感,从而限定了它在微生物范畴的应用,而拉曼光谱可以提供与红外光谱互补的微生物的指纹信息,而且它不受水的影响,可以检测有水干扰的生物样本,这使得拉曼光谱在微生物检测中的应用前景良好。本研究将表面增强拉曼技术应用于食源性致病菌的检测。本研究主要为以下两方面:(1)利用表面增强拉曼光谱技术快速检测布鲁氏菌S2株、鼠伤寒沙门氏菌、金黄色葡萄球菌、大肠杆菌O157:H7和福氏志贺氏菌。本研究中应用表面增强拉曼(SERS)技术,以原位包覆纳米银为基底,获得5种细菌的SERS谱,达到增强细菌拉曼信号,检测食源性致病菌的目的。在400-2000 cm-1范围内,布鲁氏菌S2株有10处明显的拉曼谱峰,鼠伤寒沙门氏菌有9处明显的拉曼谱峰,金黄色葡萄球菌有5处明显的拉曼谱峰,大肠杆菌O157:H7有9处明显的拉曼谱峰,福氏志贺氏菌有7处明显的拉曼谱峰。5种食源性致病菌的拉曼谱峰位置以及强度区别明显。本研究利用SERS技术可以实现10 min内快速检测并识别了五种食源性致病菌。本研究是国内首次报道对福氏志贺氏菌和布鲁氏菌S2株进行SERS光谱研究,为临床感染诊断供了基础依据。(2)适配体原位纳米银表面增强拉曼光谱的细菌快速检测方法建立。在本研究中,报道了一种新的细菌检测方法,利用表面增强拉曼技术对适配体特异性识别细菌后进行原位还原进行检测,可以快速、直观的检测出目标细菌。并且,发现Bacteria-aptamer@AgNP的SERS信号与适配体浓度(R~2=0.9773)和细菌浓度(R~2=0.9671)均具有良好的线性关系,并且检测限可以低到10 cfu/mL。同时,该方法是通过适配体特异性识别细菌后,有目的的对目标菌株进行SERS增强处理,所以,通过图谱便可以直观的鉴别细菌,无需复杂的化学计量学分析。并且,利用该SERS技术成功的在多种混合细菌里面检测到了金黄色葡萄球菌。
[Abstract]:In the field of clinical diagnosis and biosafety, there is an urgent need for direct bacterial detection without time-consuming culturing. The development of rapid, sensitive and accurate bacterial diagnostics is the primary goal of protecting public and animal health. Traditional bacterial tests include staining, optical microscopy, microbial culture, and so on, while traditional methods can only identify Gram (G) and Gram- (G-), and the process of microbial culture takes 24-72 hours; and new amplification techniques. Including immunoassay (enzyme linked immunosorbent assay) (ELISA), nucleic acid identification (PCR), although the amplification technology can achieve high specificity and sensitivity, but all need a cumbersome operation process, not suitable for field detection; High-throughput sequencing and microarray can simultaneously detect a variety of pathogens, but these techniques require the isolation of bacterial DNA in advance, as well as the need for expensive nucleic acid amplification instruments, which hinder the application of these techniques in practical detection. In contrast, molecular spectra, such as IR and Raman spectra, can quickly scan microbiological fingerprints. But because the infrared spectrum is extremely sensitive to water, which limits its application in the microbial category, the Raman spectrum can provide fingerprint information of microorganisms complementary to the infrared spectrum, and it is not affected by water. Biological samples with water interference can be detected, which makes the application of Raman spectroscopy in microbial detection promising. In this study, surface-enhanced Raman technique was applied to the detection of foodborne pathogens. The main contents of this study were as follows: (1) S _ 2 strain of Brucella, Salmonella typhimurium, Staphylococcus aureus, Escherichia coli O157:H7 and Shigella flexneri were detected by surface-enhanced Raman spectroscopy. In this study, the surface-enhanced Raman (SERS) technique was used to obtain the SERS spectra of five kinds of bacteria on the basis of in situ coated silver nanoparticles, which enhanced the Raman signal of bacteria and detected foodborne pathogenic bacteria. In the range of 400-2000 cm-1, there were 10 obvious Raman peaks in S2 strain of Brucella, 9 obvious Raman peaks in Salmonella typhimurium, 5 obvious Raman peaks in Staphylococcus aureus and 9 obvious Raman peaks in Escherichia coli O157:H7. There are 7 obvious Raman peaks of Shigella flexneri. 5. The location and intensity of Raman peaks of 5 species of foodborne pathogenic bacteria are obviously different. In this study, SERS was used to detect and identify five foodborne pathogens within 10 min. This study is the first time in China to report the SERS spectra of Shigella flexneri and Brucella S2 strains, which provides a basis for clinical diagnosis. (2) A rapid method for the detection of aptamer in situ nano-silver surface-enhanced Raman spectroscopy was established. In this study, a new method of bacterial detection was reported. Surface enhanced Raman technique was used to detect the specific identification of aptamer and in situ reduction, which could be used to detect the target bacteria quickly and intuitively. It was also found that there was a good linear relationship between the SERS signal of Bacteria-aptamer@AgNP and the aptamer concentration (R _ (2) O _ (0.9773) and the bacterial concentration (R _ (2) O _ (0.9671), and the detection limit could be as low as 10 cfur ~ (-1) 路mL ~ (-1). At the same time, the method is to identify bacteria by aptamer specificity, and then the target strain is enhanced by SERS. Therefore, the identification of bacteria can be intuitively identified by map without complex chemometrics analysis. Staphylococcus aureus was successfully detected in a variety of mixed bacteria using the SERS technique.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.61
本文编号：2159972
[Abstract]:In the field of clinical diagnosis and biosafety, there is an urgent need for direct bacterial detection without time-consuming culturing. The development of rapid, sensitive and accurate bacterial diagnostics is the primary goal of protecting public and animal health. Traditional bacterial tests include staining, optical microscopy, microbial culture, and so on, while traditional methods can only identify Gram (G) and Gram- (G-), and the process of microbial culture takes 24-72 hours; and new amplification techniques. Including immunoassay (enzyme linked immunosorbent assay) (ELISA), nucleic acid identification (PCR), although the amplification technology can achieve high specificity and sensitivity, but all need a cumbersome operation process, not suitable for field detection; High-throughput sequencing and microarray can simultaneously detect a variety of pathogens, but these techniques require the isolation of bacterial DNA in advance, as well as the need for expensive nucleic acid amplification instruments, which hinder the application of these techniques in practical detection. In contrast, molecular spectra, such as IR and Raman spectra, can quickly scan microbiological fingerprints. But because the infrared spectrum is extremely sensitive to water, which limits its application in the microbial category, the Raman spectrum can provide fingerprint information of microorganisms complementary to the infrared spectrum, and it is not affected by water. Biological samples with water interference can be detected, which makes the application of Raman spectroscopy in microbial detection promising. In this study, surface-enhanced Raman technique was applied to the detection of foodborne pathogens. The main contents of this study were as follows: (1) S _ 2 strain of Brucella, Salmonella typhimurium, Staphylococcus aureus, Escherichia coli O157:H7 and Shigella flexneri were detected by surface-enhanced Raman spectroscopy. In this study, the surface-enhanced Raman (SERS) technique was used to obtain the SERS spectra of five kinds of bacteria on the basis of in situ coated silver nanoparticles, which enhanced the Raman signal of bacteria and detected foodborne pathogenic bacteria. In the range of 400-2000 cm-1, there were 10 obvious Raman peaks in S2 strain of Brucella, 9 obvious Raman peaks in Salmonella typhimurium, 5 obvious Raman peaks in Staphylococcus aureus and 9 obvious Raman peaks in Escherichia coli O157:H7. There are 7 obvious Raman peaks of Shigella flexneri. 5. The location and intensity of Raman peaks of 5 species of foodborne pathogenic bacteria are obviously different. In this study, SERS was used to detect and identify five foodborne pathogens within 10 min. This study is the first time in China to report the SERS spectra of Shigella flexneri and Brucella S2 strains, which provides a basis for clinical diagnosis. (2) A rapid method for the detection of aptamer in situ nano-silver surface-enhanced Raman spectroscopy was established. In this study, a new method of bacterial detection was reported. Surface enhanced Raman technique was used to detect the specific identification of aptamer and in situ reduction, which could be used to detect the target bacteria quickly and intuitively. It was also found that there was a good linear relationship between the SERS signal of Bacteria-aptamer@AgNP and the aptamer concentration (R _ (2) O _ (0.9773) and the bacterial concentration (R _ (2) O _ (0.9671), and the detection limit could be as low as 10 cfur ~ (-1) 路mL ~ (-1). At the same time, the method is to identify bacteria by aptamer specificity, and then the target strain is enhanced by SERS. Therefore, the identification of bacteria can be intuitively identified by map without complex chemometrics analysis. Staphylococcus aureus was successfully detected in a variety of mixed bacteria using the SERS technique.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.61
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