應(yīng)用表面增強(qiáng)拉曼技術(shù)快速檢測6種食源性致病菌的研究
發(fā)布時(shí)間：2018-08-02 16:08
【摘要】：臨床診斷和生物安全領(lǐng)域迫切的需要不需經(jīng)過耗時(shí)培養(yǎng)過程的直接的細(xì)菌檢測方法。開發(fā)快速、靈敏和準(zhǔn)確的細(xì)菌診斷方法是保障公眾以及動(dòng)物健康的主要目標(biāo)。傳統(tǒng)的細(xì)菌檢測包括染色,光學(xué)顯微鏡檢查,微生物培養(yǎng)等,而傳統(tǒng)檢測方法只能判別出Gram+(G+)和Gram-(G-),并且微生物培養(yǎng)過程需要24-72小時(shí);以及新的擴(kuò)增技術(shù),包括免疫測定(酶聯(lián)免疫吸附法(ELISA),核酸鑒定(聚合酶鏈?zhǔn)椒磻?yīng),PCR)等,雖然擴(kuò)增技術(shù)可以達(dá)到高特異性和靈敏度,但都需要繁瑣的操作過程,不適于現(xiàn)場檢測;高通量測序和微陣列可以同時(shí)檢測多種病原菌,但這些技術(shù)需要事先分離出細(xì)菌的DNA,以及需要昂貴的核酸擴(kuò)增儀器,這些都阻礙了這些技術(shù)在實(shí)際檢測中的應(yīng)用。于此相比,分子光譜,如紅外光譜和拉曼光譜,都可以快速掃描得到微生物的生化指紋。但因?yàn)榧t外光譜對(duì)水極度敏感,從而限定了它在微生物范疇的應(yīng)用,而拉曼光譜可以提供與紅外光譜互補(bǔ)的微生物的指紋信息,而且它不受水的影響,可以檢測有水干擾的生物樣本,這使得拉曼光譜在微生物檢測中的應(yīng)用前景良好。本研究將表面增強(qiáng)拉曼技術(shù)應(yīng)用于食源性致病菌的檢測。本研究主要為以下兩方面:(1)利用表面增強(qiáng)拉曼光譜技術(shù)快速檢測布魯氏菌S2株、鼠傷寒沙門氏菌、金黃色葡萄球菌、大腸桿菌O157:H7和福氏志賀氏菌。本研究中應(yīng)用表面增強(qiáng)拉曼(SERS)技術(shù),以原位包覆納米銀為基底,獲得5種細(xì)菌的SERS譜,達(dá)到增強(qiáng)細(xì)菌拉曼信號(hào),檢測食源性致病菌的目的。在400-2000 cm-1范圍內(nèi),布魯氏菌S2株有10處明顯的拉曼譜峰,鼠傷寒沙門氏菌有9處明顯的拉曼譜峰,金黃色葡萄球菌有5處明顯的拉曼譜峰,大腸桿菌O157:H7有9處明顯的拉曼譜峰,福氏志賀氏菌有7處明顯的拉曼譜峰。5種食源性致病菌的拉曼譜峰位置以及強(qiáng)度區(qū)別明顯。本研究利用SERS技術(shù)可以實(shí)現(xiàn)10 min內(nèi)快速檢測并識(shí)別了五種食源性致病菌。本研究是國內(nèi)首次報(bào)道對(duì)福氏志賀氏菌和布魯氏菌S2株進(jìn)行SERS光譜研究,為臨床感染診斷供了基礎(chǔ)依據(jù)。(2)適配體原位納米銀表面增強(qiáng)拉曼光譜的細(xì)菌快速檢測方法建立。在本研究中,報(bào)道了一種新的細(xì)菌檢測方法,利用表面增強(qiáng)拉曼技術(shù)對(duì)適配體特異性識(shí)別細(xì)菌后進(jìn)行原位還原進(jìn)行檢測,可以快速、直觀的檢測出目標(biāo)細(xì)菌。并且,發(fā)現(xiàn)Bacteria-aptamer@AgNP的SERS信號(hào)與適配體濃度(R~2=0.9773)和細(xì)菌濃度(R~2=0.9671)均具有良好的線性關(guān)系,并且檢測限可以低到10 cfu/mL。同時(shí),該方法是通過適配體特異性識(shí)別細(xì)菌后,有目的的對(duì)目標(biāo)菌株進(jìn)行SERS增強(qiáng)處理,所以,通過圖譜便可以直觀的鑒別細(xì)菌,無需復(fù)雜的化學(xué)計(jì)量學(xué)分析。并且,利用該SERS技術(shù)成功的在多種混合細(xì)菌里面檢測到了金黃色葡萄球菌。
[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.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S852.61
本文編號(hào)：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.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S852.61
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