基于选择性鉴别培养基、酶免疫传感器和智舌对阪崎肠杆菌快速检测的研究
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摘要
阪崎肠杆菌(Enterbacter sakazakii)是肠杆菌科的食源性致病菌,革兰氏阴性致病菌,可引起婴幼儿脑膜炎、菌血症和小肠结肠炎,婴儿和幼儿对该菌尤为更高的感染风险。在市场销售的婴幼儿配方奶粉中包括阪崎肠杆菌在内的各类肠杆菌也有检出。婴幼儿被感染后高达40-80%的死亡率非常值得关注。阪崎肠杆菌感染和传播50-80%的途径是通过婴幼儿配方奶粉(Powered Infant Formula, PIF)和其它配方奶粉。对阪崎肠杆菌快速、敏感、经济、简便的检测方法是对该菌及其所致疾病的监测、监管、预防、质控和及时诊断等多方面有效实施的保障,是多级检验检疫部门的迫切需求。
本文以选择性鉴别培养基、酶免疫传感器和智舌为手段,构建了三种快速检测阪崎肠杆菌的方法。选择性鉴别培养基是在生化反应的基础上开发的技术,将传统的细菌分离与生化反应有机的结合起来,使得检测结果直观,易于观察辨别,且具有低成本、特异性强、灵敏度高和检测快速、操作方便等优点。选择性鉴别培养基是国内外.微生物检测研究和应用的一个主要发展方向。酶免疫传感器是基于抗原-抗体特异性反应的电化学免疫传感器,在食品分析、临床诊断、环境监测等领域开展了许多研究。智舌是以多频率脉冲作为激发扫描信号,辅以特定传感器阵列,检测待测物质的整体响应信号,结合多元统计方法构建的一类新型电子舌,具有检测速度快、样品前处理简单等优点,结合每种致病菌都有其特定的酶系统及其代谢途径,其培养物具有综合种的特征,基于此,智舌在致病菌快速检测方面具有很大的潜力。分别基于生化反应结合分离培养、酶免疫传感器和智舌检测的菌种整体代谢特征研究出的三种方法共同组成了一套阪崎肠杆菌快速检测体系,上述两种或三种方法结合,可以克服各自的不足,发挥各自的优点,达到准确、敏感、快速检测阪崎肠杆菌的目的。
本论文主要围绕以下几个方面开展了一些研究工作：
1阪崎肠杆菌选择性鉴别培养基的研制
本试验以阪崎肠杆菌对某些特定抑菌成分的抵抗力为基础,设计研制了阪崎肠杆菌选择性鉴别培养基(Enterbacter sakazakii Differential Medium, EsDM)。试验中对EsDM的显色效果、平板效率、灵敏度和特异性进行评价。结果显示：EsDM对阪崎肠杆菌具有较好的选择性和特异性；用EsDM在30℃培养16 h-24 h,阪崎肠杆菌菌落为黄绿色；其它各种致病菌菌落呈其他色或未形成有效的可见菌落；EsDM对阪崎肠杆菌的出菌效率与胰大豆蛋白胨琼脂(TSA)相比为(92.42±3.43)%；检出限为101 cfu/mL;因此,EsDM对婴幼儿配方奶粉中阪崎肠杆菌分离鉴别与检测是准确、敏感的,可试验用于大批实际样品的检测,且具有操作简便、经济、结果易于观察等优点。
2基于多壁碳纳米管/海藻酸钠复合物和4-SPCE的阪崎肠杆菌酶免疫传感器的研制
为快速检测阪崎肠杆菌,将辣根过氧化物酶标记的阪崎肠杆菌抗体吸附在多壁碳纳米管/海藻酸钠复合物修饰的四通道丝网印刷碳电极表面,制得快速检测阪崎肠杆菌的酶免疫传感器。采用原子力显微镜表征不同修饰电极的表面形态,循环伏安法考察不同电极的电化学特性,采用一步免疫法检测阪崎肠杆菌和循环伏安法监测酶促反应,根据免疫反应前后还原峰峰电流的降低值来检测样品中的阪崎肠杆菌。在优化的实验条件下,酶免疫传感器与阪崎肠杆菌浓度的对数在103～108 cfu/mL范围内保持良好的线性关系,检出限为2.5×102 cfu/mL(S/N=3)。该酶免疫传感器具有较好的特异性、重现性、稳定性和准确性,可望初步用于阪崎肠杆菌的快速筛检。
3智舌对阪崎肠杆菌示踪、区分与SIMCA判别模型的建立
智舌结合主成分分析方法,对阪崎肠杆菌、鸡白痢沙门氏菌、志贺氏菌、大肠埃希氏、金黄色葡萄球菌和蜡样芽胞杆菌进行示踪和鉴别研究。结果显示,Ag电极10Hz示踪5h培养物开始出现较大离散度,这与国标的吸光度法结果类似。基于此,确定用Ag电极10Hz进行上述6种主要致病菌5h后培养物的鉴别研究,获得了6h的培养物即能达到理想的鉴别效果,6种致病菌在主成分得分图上的马氏距离均能达到2.0以上。因此,智舌Ag电极10Hz结合主成分分析法,能够对婴幼儿配方奶粉中国标规定的6种主要致病菌5h培养物进行示踪、6h培养物进行有效鉴别。用Ag电极10Hz构建阪崎肠杆菌智能识别模式单元,通过培训集样本回判和用未知样本进行检验,得到最佳的SIMCA判别模型。
Enterbacter sakazakii is an opportunistic Gram-negative, foodborne pathogen, which is a member of the family Enterbacter bacteriaceae. Enterbacter sakazakii is considered to causing meningitis, bacteremia or necrotizing enterocolitis. Specially for neonates and infants. The infant powdered milk sales in China also had a high detective rate. The 50～80% infection and transmission path of Enterbacter sakazakii was from powered infant milk and other milk. Enterbacter sakazakii on monitoring, controlling, prevention, diagnosis, and many other quality control and timely and effective implementation of protection is the urgent needs of multi-stage inspection and quarantine department.
This research used Selective Differential media, Enzymatic immunosensor and Smart Tongue to detecting Enterbacter sakazakii in three different fast methods. Selective Differential media, which is based on biochemical reaction, is a detection technology. It has successfully combined the reaction of biochemistry and the traditional separation of biochemistry of bacterium, which is easy to observe the result of detection. In addition, there are some advantages-low cost, high sensitivity, specificity and rapidness. Selective Differential media is becoming the main trend of microbial detection. Enzymatic immunosensor based on the specificity of antigen-antibody interactions with electrochemical transduction, have become an attractive subject for food analysis, clinical diagnosis, environmental monitoring and other fields. The Smart tongue applicants a new electrochemical method-----the multifrequency large amplitude pulse voltammetry (MLAPV) which is very useful for discriminating samples in the voltammetric electronic tongue by applying three frequency segments. Multivariate data analysis (MVDA) such as Principal component analysis (PCA), and Soft Independent Modeling of Class Analogy (SIMCA) were often used to analyzing the data from the electronic tongue. With the advantages of rapid detection and simple sample preparation, it seems to be suitable for detecting the liquid cultures of the pathogens.
The comments of this paper was described as follows:
1 Study on Enterbacter sakazakii Differential Medium
Based on the specific enzyme systems and the corresponding metabolisms of Enterbacter sakazakii, according to the resistibility to different antibacterial ingredients, EsDM was designed for the rapid separation, identification or detection of Enterbacter sakazakii. The tests of color effects, plating efficiency, sensitivity and specificity were conducted to evaluate the efficiency of EsDM. The results showed that: EsDM had good selectivity and specificity for Enterbacter sakazakii. After incubating for 16～24 h at 30℃, the Enterbacter sakazakii formed yellow colonies on EsDM. The other strains did not form colonies or form other color colonies. EsDM showed a mean plating efficiency of Enterbacter sakazakii cells of (91.23±2.24)% and its detection limit was 101 cfu/mL. Accordingly, EsDM is useful to detect Enterbacter sakazakii in powered infant milk for kinds of inspection agencies with the strengthens of easily-operated, lower cost, convenience for observation and so on.
2 A disposable immunosensor for Enterbacter sakazakii based on MWCNT/Sodium Alginate composite and four-channel screen-printed carbon electrode
A novel Enterbacter sakazakii immunosensor based on HRP-anti-Enterbacter sakazakii immobilized by physical adsorption on the MWCNT/sodium alginate (MWCNT/SA) composite modified four-channel screen-printed carbon electrode surface was successfully fabricated. In this strategy, MWCNT/SA biocomposite acted as the matrix to adsorb and immobilize HRP-anti-Enterbacter sakazakii. The preparation process of modified electrodes was characterized with AFM and CV. The analytical performance of proposed immunosensor toward Enterbacter sakazakii was investigated by CV. Under optimal conditions, the concentration of Enterbacter sakazakii from 103 to 108 cfu mL-1 could be detected, with a detection limit of 2.5×102 cfu/mL (S/N=3). The specificity, reproducibility, stability and accuracy of the proposed immunosensor were also evaluated. The proposed immunosensor showed simply fabricative, economical, efficient and potential application for early assessment of Enterbacter sakazakii.
3 Used Smart Tongue to tracing, discriminate and set the SIMCA model of Enterbacter sakazakii
Used Smart Tongue to tracing and discriminate Enterbacter sakazakii, Salmonella, Shigella spp, Escherichia coli, Staphylococcus aureus, Bacillus cereus. The result showed that:Ag electrode in 10Hz had the trend to discriminate the 5 hour cultures. Therefore we used Ag electrode combined with the frequency of 10 Hz to discriminate the six species of pathogens after 5 hours cultured. The 6 hour cultures can attend a excellent effect and every Mahalanobis distance exceed 2.0. Smart tongue combined with principal component analysis can tracing and discriminate the six species of pathogens, they must be detected in infant formula milk by the national standard. Complying with SIMCA, the Ag electrode in 100 Hz was studied for classifying and identifying the Enterbacter sakazakii. The best models based on the training set for classifying were detected. The discrimination accuracy of the best SIMCA model was 100% for both the training sets and the testing sets.
本文以选择性鉴别培养基、酶免疫传感器和智舌为手段,构建了三种快速检测阪崎肠杆菌的方法。选择性鉴别培养基是在生化反应的基础上开发的技术,将传统的细菌分离与生化反应有机的结合起来,使得检测结果直观,易于观察辨别,且具有低成本、特异性强、灵敏度高和检测快速、操作方便等优点。选择性鉴别培养基是国内外.微生物检测研究和应用的一个主要发展方向。酶免疫传感器是基于抗原-抗体特异性反应的电化学免疫传感器,在食品分析、临床诊断、环境监测等领域开展了许多研究。智舌是以多频率脉冲作为激发扫描信号,辅以特定传感器阵列,检测待测物质的整体响应信号,结合多元统计方法构建的一类新型电子舌,具有检测速度快、样品前处理简单等优点,结合每种致病菌都有其特定的酶系统及其代谢途径,其培养物具有综合种的特征,基于此,智舌在致病菌快速检测方面具有很大的潜力。分别基于生化反应结合分离培养、酶免疫传感器和智舌检测的菌种整体代谢特征研究出的三种方法共同组成了一套阪崎肠杆菌快速检测体系,上述两种或三种方法结合,可以克服各自的不足,发挥各自的优点,达到准确、敏感、快速检测阪崎肠杆菌的目的。
本论文主要围绕以下几个方面开展了一些研究工作：
1阪崎肠杆菌选择性鉴别培养基的研制
本试验以阪崎肠杆菌对某些特定抑菌成分的抵抗力为基础,设计研制了阪崎肠杆菌选择性鉴别培养基(Enterbacter sakazakii Differential Medium, EsDM)。试验中对EsDM的显色效果、平板效率、灵敏度和特异性进行评价。结果显示：EsDM对阪崎肠杆菌具有较好的选择性和特异性；用EsDM在30℃培养16 h-24 h,阪崎肠杆菌菌落为黄绿色；其它各种致病菌菌落呈其他色或未形成有效的可见菌落；EsDM对阪崎肠杆菌的出菌效率与胰大豆蛋白胨琼脂(TSA)相比为(92.42±3.43)%；检出限为101 cfu/mL;因此,EsDM对婴幼儿配方奶粉中阪崎肠杆菌分离鉴别与检测是准确、敏感的,可试验用于大批实际样品的检测,且具有操作简便、经济、结果易于观察等优点。
2基于多壁碳纳米管/海藻酸钠复合物和4-SPCE的阪崎肠杆菌酶免疫传感器的研制
为快速检测阪崎肠杆菌,将辣根过氧化物酶标记的阪崎肠杆菌抗体吸附在多壁碳纳米管/海藻酸钠复合物修饰的四通道丝网印刷碳电极表面,制得快速检测阪崎肠杆菌的酶免疫传感器。采用原子力显微镜表征不同修饰电极的表面形态,循环伏安法考察不同电极的电化学特性,采用一步免疫法检测阪崎肠杆菌和循环伏安法监测酶促反应,根据免疫反应前后还原峰峰电流的降低值来检测样品中的阪崎肠杆菌。在优化的实验条件下,酶免疫传感器与阪崎肠杆菌浓度的对数在103～108 cfu/mL范围内保持良好的线性关系,检出限为2.5×102 cfu/mL(S/N=3)。该酶免疫传感器具有较好的特异性、重现性、稳定性和准确性,可望初步用于阪崎肠杆菌的快速筛检。
3智舌对阪崎肠杆菌示踪、区分与SIMCA判别模型的建立
智舌结合主成分分析方法,对阪崎肠杆菌、鸡白痢沙门氏菌、志贺氏菌、大肠埃希氏、金黄色葡萄球菌和蜡样芽胞杆菌进行示踪和鉴别研究。结果显示,Ag电极10Hz示踪5h培养物开始出现较大离散度,这与国标的吸光度法结果类似。基于此,确定用Ag电极10Hz进行上述6种主要致病菌5h后培养物的鉴别研究,获得了6h的培养物即能达到理想的鉴别效果,6种致病菌在主成分得分图上的马氏距离均能达到2.0以上。因此,智舌Ag电极10Hz结合主成分分析法,能够对婴幼儿配方奶粉中国标规定的6种主要致病菌5h培养物进行示踪、6h培养物进行有效鉴别。用Ag电极10Hz构建阪崎肠杆菌智能识别模式单元,通过培训集样本回判和用未知样本进行检验,得到最佳的SIMCA判别模型。
Enterbacter sakazakii is an opportunistic Gram-negative, foodborne pathogen, which is a member of the family Enterbacter bacteriaceae. Enterbacter sakazakii is considered to causing meningitis, bacteremia or necrotizing enterocolitis. Specially for neonates and infants. The infant powdered milk sales in China also had a high detective rate. The 50～80% infection and transmission path of Enterbacter sakazakii was from powered infant milk and other milk. Enterbacter sakazakii on monitoring, controlling, prevention, diagnosis, and many other quality control and timely and effective implementation of protection is the urgent needs of multi-stage inspection and quarantine department.
This research used Selective Differential media, Enzymatic immunosensor and Smart Tongue to detecting Enterbacter sakazakii in three different fast methods. Selective Differential media, which is based on biochemical reaction, is a detection technology. It has successfully combined the reaction of biochemistry and the traditional separation of biochemistry of bacterium, which is easy to observe the result of detection. In addition, there are some advantages-low cost, high sensitivity, specificity and rapidness. Selective Differential media is becoming the main trend of microbial detection. Enzymatic immunosensor based on the specificity of antigen-antibody interactions with electrochemical transduction, have become an attractive subject for food analysis, clinical diagnosis, environmental monitoring and other fields. The Smart tongue applicants a new electrochemical method-----the multifrequency large amplitude pulse voltammetry (MLAPV) which is very useful for discriminating samples in the voltammetric electronic tongue by applying three frequency segments. Multivariate data analysis (MVDA) such as Principal component analysis (PCA), and Soft Independent Modeling of Class Analogy (SIMCA) were often used to analyzing the data from the electronic tongue. With the advantages of rapid detection and simple sample preparation, it seems to be suitable for detecting the liquid cultures of the pathogens.
The comments of this paper was described as follows:
1 Study on Enterbacter sakazakii Differential Medium
Based on the specific enzyme systems and the corresponding metabolisms of Enterbacter sakazakii, according to the resistibility to different antibacterial ingredients, EsDM was designed for the rapid separation, identification or detection of Enterbacter sakazakii. The tests of color effects, plating efficiency, sensitivity and specificity were conducted to evaluate the efficiency of EsDM. The results showed that: EsDM had good selectivity and specificity for Enterbacter sakazakii. After incubating for 16～24 h at 30℃, the Enterbacter sakazakii formed yellow colonies on EsDM. The other strains did not form colonies or form other color colonies. EsDM showed a mean plating efficiency of Enterbacter sakazakii cells of (91.23±2.24)% and its detection limit was 101 cfu/mL. Accordingly, EsDM is useful to detect Enterbacter sakazakii in powered infant milk for kinds of inspection agencies with the strengthens of easily-operated, lower cost, convenience for observation and so on.
2 A disposable immunosensor for Enterbacter sakazakii based on MWCNT/Sodium Alginate composite and four-channel screen-printed carbon electrode
A novel Enterbacter sakazakii immunosensor based on HRP-anti-Enterbacter sakazakii immobilized by physical adsorption on the MWCNT/sodium alginate (MWCNT/SA) composite modified four-channel screen-printed carbon electrode surface was successfully fabricated. In this strategy, MWCNT/SA biocomposite acted as the matrix to adsorb and immobilize HRP-anti-Enterbacter sakazakii. The preparation process of modified electrodes was characterized with AFM and CV. The analytical performance of proposed immunosensor toward Enterbacter sakazakii was investigated by CV. Under optimal conditions, the concentration of Enterbacter sakazakii from 103 to 108 cfu mL-1 could be detected, with a detection limit of 2.5×102 cfu/mL (S/N=3). The specificity, reproducibility, stability and accuracy of the proposed immunosensor were also evaluated. The proposed immunosensor showed simply fabricative, economical, efficient and potential application for early assessment of Enterbacter sakazakii.
3 Used Smart Tongue to tracing, discriminate and set the SIMCA model of Enterbacter sakazakii
Used Smart Tongue to tracing and discriminate Enterbacter sakazakii, Salmonella, Shigella spp, Escherichia coli, Staphylococcus aureus, Bacillus cereus. The result showed that:Ag electrode in 10Hz had the trend to discriminate the 5 hour cultures. Therefore we used Ag electrode combined with the frequency of 10 Hz to discriminate the six species of pathogens after 5 hours cultured. The 6 hour cultures can attend a excellent effect and every Mahalanobis distance exceed 2.0. Smart tongue combined with principal component analysis can tracing and discriminate the six species of pathogens, they must be detected in infant formula milk by the national standard. Complying with SIMCA, the Ag electrode in 100 Hz was studied for classifying and identifying the Enterbacter sakazakii. The best models based on the training set for classifying were detected. The discrimination accuracy of the best SIMCA model was 100% for both the training sets and the testing sets.
引文
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