【作者】 覃昱；

【导师】 罗炳德；

【作者基本信息】 南方医科大学 ， 营养与食品卫生学， 2014， 博士

【摘要】 一、研究背景和目的克罗诺杆菌(Cronobacter spp.)是一种重要的新型食源性致病菌,是囊括了原来所有阪崎肠杆菌(Enterobacter sakazakii)的新属,该属的细菌是人和动物肠道内寄生的一种有周生鞭毛,能运动,兼性厌氧的革兰阴性无芽孢杆菌。该菌呈世界性分布,主要通过婴幼儿配方奶粉经消化道感染儿童,引起新生儿脑膜炎、败血症和坏死性结肠炎,甚或遗留神经系统后遗症或导致死亡。自1961年首次报道由克罗诺杆菌引起的两例脑膜炎病例,随后在全球范围内相继出现了由该菌引起的疾病报道。2001年4月,在美国某医院新生儿重症监护室一早产儿发烧、心动过速,脑脊液培养发现克罗诺杆菌,诊断为脑膜炎,用抗生素治疗无效,9d后死亡。扩大检查该院49名婴儿的粪便和尿液,发现10人为阪崎肠杆菌阳性,其原因是使用了某批Portagen的婴儿配方奶粉。而且在该批奶粉中也检查出了克罗诺杆菌,结果导致Portagen婴儿配方奶粉于2002年4月被召回。1998年,在比利时有12名婴儿因哺喂同一牌号的婴儿配方奶粉而发生小肠结肠炎坏死。在这些婴儿的粪便和该批奶粉中同时分离出克罗诺杆菌,当该批奶粉停用后,未有新的病例发生。2004年安徽阜阳劣质婴儿配方奶粉事件引起我国政府的高度重视,并且首次从87份婴儿配方奶粉中分离到11株该菌,检出率达12.16%。尽管专家目前认为是由于奶粉的营养比例问题导致小孩畸形和死亡,但是从生病小孩的临床症状看,小孩头部肿大和反应迟钝与克罗诺杆菌感染的症状相似,可能该菌在致病中起到重要作用。近期研究表明,克罗诺杆菌是一种广泛存在的微生物,不仅能够存在于污染的食品和食品工厂当中,而且在几乎所有的环境中均能够分离得到。到目前为止已从奶酪、猪肉、蔬菜、谷类、草药、香辛料,和经过UHT灭菌的牛奶等食品中分离出克罗诺杆菌。克罗诺杆菌致死率为10～80%,但是作为一种条件性致病菌,对新生儿具有更为严重的危害性,其严重威胁到公共卫生安全和人类健康。目前,针对克罗诺杆菌的检测方法主要有FDA推荐的分离培养、生化及血清学鉴定、免疫学方法和PCR。传统的检测和定量方法主要是采用选择性培养基,通过增菌和选择培养来实现,但其中主要存在三大问题,一是通常情况下,致病菌在食品检测样品中的量很少,在样品处理和检测中容易出现人为的差错；二是非常繁琐和耗时,检测速度慢,通常需要6d以上；三是许多致病菌在常规检测中属难培养或不可培养微生物,灵敏度较低；免疫学方法的特异性和灵敏度均较低；PCR法敏感、准确、快速,可替代传统检测方法,但由于需要昂贵的仪器设备、繁琐的电泳过程,以及对检测人员较高的技术要求,而使其难以在基层单位推广和普及。因此,建立一种特异富集、快速、简便、准确的检测方法,对克罗诺杆菌感染的流行病学调查和防治工作十分重要。1、样品富集技术——免疫磁珠分离法(immunomagnetic separation,IMS)生物样品往往是复杂的混合物,除少数特殊样品外,不能直接用于检测,因此需要进行样品前处理这一重要步骤。传统的样品处理方法是从样品中分离目的微生物,通常使用选择性培养基来获得单个纯化的目的微生物。如果对于受到损伤的和热应激微生物还需经过增菌培养,增菌过程应包括前增菌、选择性增菌和后增菌,因此从样品中检测病原微生物需很长的时间,而实际检测时间主要是等待增菌的时间,这样就远远不能适应快速检测的需要。由于免疫磁珠分离法能特异性吸附目的细菌,受到了不少学者的关注。免疫磁珠是由直径3-5微米,有一定磁性,且分散度良好的微珠经不同单克隆抗体包被而成的颗粒,而免疫磁珠分离方法是于70年代中期发展起来的一项免疫学技术,其分离原理是通过磁珠表面包被的细胞特异性抗体与细胞表面标记分子间特异结合,使抗原阳性细胞与抗原阴性细胞相分离,具备了固相化试剂特有的优点以及免疫学反应的高度专一性,被广泛应用于多种血液细胞的分离,蛋白质的纯化,以及对食品、水、生物样品、环境等标本中病原微生物的分离和检测工作中,显示出良好的开发应用前景。IMS用于微生物检测领域的原理是：用特异性抗体包被在磁珠表面来捕获检样(或增菌液)中的目标微生物,然后在磁场的作用下被吸附沉淀,使菌株与影响和干扰检测灵敏度的检样残渣和其他杂菌分离开,起到浓缩作用,从而提高检测的灵敏度和检出率。IMS方法操作简便,只需在检测样品或者增菌液中加入免疫磁珠,室温振荡混匀一定的时间后,在磁场作用下反复用缓冲液洗涤即可,捕获目标菌的免疫磁珠不用与目标菌分离,直接进行后续实验。具有分离速度快、效率高、可重复性好；操作简单,不需要昂贵的仪器设备；不影响被分离细胞或其它生物材料的生物学性状和功能等优点,从而在微生物检测方面具有很大的优势。2、快速检测技术——环介导等温扩增(loop-mediated isothermal amplification, LAMP)环介导等温扩增(LAMP)是日本学者Notomi等建立的一种新的核酸等温扩增技术,其原理是,针对靶基因的6或8个区域设计4或6种特异性引物,利用一种链置换DNA聚合酶(Bst DNA polymerase)在等温条件(约65℃)保温几十分钟,即可完成核酸扩增反应。LAMP能够高效、快速、灵敏、特异地扩增靶DNA,操作简便,成本低,适于大规模样品的检测和基层单位推广应用,现已广泛应用于疾病诊断、病原微生物检测以及动物胚胎的性别鉴定等。因此,本实验将结合上述两种技术,采用免疫磁珠分离富集样品中克罗诺杆菌,建立其快速的LAMP检测技术。首先建立克罗诺杆菌免疫磁珠特异性捕获技术,并进行磁珠模拟研究。然后针对克罗诺杆菌的特异性外膜蛋白OmpA基因,设计内外引物,运用LAMP技术检测被富集的克罗诺杆菌,建立用于克罗诺杆菌快速灵敏的IMS-LAMP检测方法。并利用已经建立的免疫磁珠分离乳制品中克罗诺杆菌的方法,对人工污染克罗诺杆菌的乳制品进行LAMP检测,同时采集209份市售配方奶粉进行检测,结果与传统国标法进行比较。二、研究方法1、建立克罗诺杆菌免疫磁珠特异性捕获技术,并进行磁珠模拟研究。制备克罗诺杆菌(ATCC29544、ATCC51329、奶粉分离株)、非目标菌(大肠杆菌、副溶血性弧菌、痢疾志贺菌、普通变形杆菌、沙门菌、金黄色葡萄球菌等)的菌悬液,利用克罗诺杆菌特异性免疫磁珠进行捕获,接种平板后进行菌落计数,并计算免疫磁珠的特异性吸附效率。同时设计干扰试验,在含有104cfu/ml的大肠杆菌菌悬液中,加入不同浓度的目标菌,用磁珠进行捕获,评估免疫磁珠的特异性富集效率。在经3h和6h预增菌的含有已知量目标菌的奶粉模拟标本及未增菌的粪便模拟标本中,分别加入磁珠进行捕获,评估基于免疫磁珠捕获法的克罗诺杆菌检测流程的可行性。2、根据克罗诺杆菌外膜蛋白ompA基因序列(Accession number:DQ000206)的保守区,设计出特异的LAMP引物,对各扩增条件进行优化,建立25μL的LAMP反应体系。3、以20株实验菌株基因组DNA为模板,建立LAMP与PCR法检测方法,并对比两者的特异性；将过夜培养的克罗诺杆菌ATCC29544标准菌株进行稀释,分别进行LAMP和PCR扩增,比较两者的灵敏度。4、模拟样品的检测：购买奶粉样品(克罗诺杆菌阴性)3份,分别用ATCC29544进行接种,随后做LAMP和PCR检测,并与国标法进行比较。5、收集国产婴幼儿配方奶粉209份,通过免疫磁珠富集,IMS-LAMP技术检测,并与国标法进行比较。三、结果1、菌落计数表明,制备的克罗诺杆菌免疫磁珠对克罗诺杆菌ATCC29544、 ATCC51329和实验室分离株的捕获效率均超过30%,对6种非目标菌的非特异性吸附效率<3%。干扰试验结果显示,与常规直接平板接种法相比,免疫磁珠捕获法的敏感性提高了约100倍；奶粉和粪便模拟试验的结果显示,免疫磁珠捕获法的最低检测限分别可达101cfu/25g和101cfu/mL,并提高了标本中目标菌的检出率。2、成功建立优化的克罗诺杆菌LAMP反应体系,具体为(25μL):6mmol/L MgCl2,0.6mmol/L dNTPs,0.8mol/L甜菜碱,1.6μmo/L内引物,0.4μmo/L外引物,1μL的Bst DNA聚合酶(8U),10×Thermopol buffer以及2μL模板；最佳反应条件：58℃,反应60min。经过免疫磁珠富集后,LAMP法检出克罗诺杆菌ATCC29544的特异性较PCR法高,LAMP的检测限为101cfu/ml。3、对于模拟样品,LAMP和PCR与国标检测结果一致,均可检出3份模拟克罗诺杆菌阳性样品。4、对209份实际婴幼儿配方奶粉进行检测,其中IMS-LAMP检测为16份阳性,国标法检测15份阳性,无统计学差异,因此其检测结果一致、可信,但大大缩短了检测时间。四、结论本研究建立克罗诺杆菌免疫磁珠特异性捕获技术,同时以克罗诺杆菌的外膜蛋白OmpA基因序列作为靶序列,设计一套LAMP引物进行扩增,并对各反应条件进行优化,保证了方法的可靠性和特异性。对大肠杆菌、沙门氏菌、志贺氏菌等肠道致病菌以及金黄色葡萄球菌等非克罗诺杆菌共10株进行LAMP扩增,均未出现阳性结果,特异性强。对细菌培养物检测限为101cfu/mL,在检测灵敏度上具有更大的优势。建立的IMS-LAMP法能直接对奶粉等食品中的克罗诺杆菌进行检测,与传统国标检测方法相比,大大缩短了检测时间,节省了人力、物力。而且IMS-LAMP方法操作简单,反应快速,在恒温条件下进行,标准培养物2-3h完成,奶粉样品26h完成(国标法需要6d),耗时短；同时,使用恒温水浴装置即可完成反应,不需要使用昂贵、精密的仪器设备,实现反应及产物检测一步完成,操作简便,检测成本低。综上所述,本研究建立的克罗诺杆菌IMS-LAMP检测方法具有特异性强、灵敏度高、方便快捷、成本低等特点,为克罗诺杆菌的检测提供了新的发展方向,有望成为简易的常规检测手段,尤其适用于基层检验检疫机构,对于提高食品卫生水平、保证食品安全和推动食品国际贸易发展具有重要的意义。更多还原

【Abstract】 1. Background and objectiveCronobacter spp. is an important new type of foodborne pathogen, which includes all Enterobacter sakazakii of the previous taxonomy system. This genus of bacteria resides in the intestinal tract of human and animals, is peritrichous, motile, facultative anaerobic, gram-negative and sporeless. This bacteria is distributed worldwide, and infects the digestive tract of children primarily through formula milk powder, resulting in neonatal meningitis, sepsis, and necrotizing enterocolitis, or even neurological sequelae and death.Since the first two cases of Cronobacter spp. infection-caused meningitis in1961, Cronobacter spp. infection-related diseases have been reported worldwide. In April2001, at a hospital in the US, a premature infant was sent to neonatal intensive care unit because of fever and tachycardia. Cronobacter spp. was found in the cerebrospinal fluid, and it was diagnosed as meningitis. Antibiotics failed to control the infection, and the baby died9days later. Expanded inspection was carried out for the49infants using feces and urine as samples, and10of them were found to be Cronobacter spp.-positive, which was believed to be from a batch of Portagen infant formula milk powder. Cronobacter spp. was actually detected in that batch of milk powder, which lead to the recall of Portagen infant formula milk powder in April2002. In1998,12infants that were fed with the brand of infant formula milk powder developed necrotizing enterocolitis, and Cronobacter spp. was isolated in feces of all these infants and that batch of milk powder. The2004Anhui Fuyang inferior infant formula milk powder incident aroused great attention of our government, and11strains of Cronobacter spp. were isolated from87samples of infant formula milk powder, the detection rate was up to12.16%. Although experts have declared that it was disproportional nutrition of the milk powder that caused deformities and death of the infants, the manifestations of these infants, including head enlargement and unresponsiveness indicated Cronobacter spp. infection, which might have played an important part in the disease. Cronobacter spp. is widely distributed, not only in contaminated food and food factories, it can actually be isolated from almost all places of the environment. Cronobacter spp. have been separated so far from chees, pork, vegetables, grains, herbs, spices, UHT-sterilized milk and other foods. Cronobacter spp. infection has a fatality rate of10-80%, seriously threating public health and safety, and as a conditional pathogen, it is most dangerous for infants.Currently, methods to detect Cronobacter spp. include isolation and culture (recommended by FDA), biochemical and serological identification, immunological methods and PCR. Traditional methods to detect and quantify Cronobacter spp. use selective medium, and involve enrichment of bacteria and selectively culture of target bacteria. This method has three major shortcomings:first, the pathogenic bacteria generally exists in trace amount, so it can be easily missed during sample handling and detection; second, it’s remarkably tedious and time-consuming, the detection process generally requires over6days; and third, many pathological bacterial are difficult to culture or nonculturable, so this method has low sensitivity. As for the other methods, immunological methods has low specificity and sensitivity; PCR is sensitive, accurate and fast, and can replace traditional detection method, but it requires expensive instrument, tedious electrophoresis process, and professional technique of the personnel, which make it difficult to promote and popularize this method in grassroots units. Therefore, it is of great importance for epidemiology study and prevention of Cronobacter spp. infection to establish a highly specific, rapid, simple and accurate detection method.1) Sample enrichment by immunomagnetic separation (IMS)Biological samples are often complex mixtures, which can’t be directly used for detection and requires preparation except for a few special samples. Traditional sample processing aims to isolate the target microorganism by selective culture to obtain purified microorganism. If damaged or thermal-stressed, the microorganism would need extra enrichment culture, including pre-enrichment, selective enrichment, and post-enrichment, so it would cost a lot of time to detect specific microorganism from the samples. In practice, most of the time is spent on enrichment of the microorganism, which makes rapid detection virtually impossible. Immunomagnetic separation can specifically absorb and enrich target bacteria, and has attracted much attention of the scholars.Immunomagnetic beads are3-5μm, paramagnetic beads with good dispersion and coated with specific monoclonal antibody. Immunomagnetic separation was developed since the mid-1970s, and the separation principle is that the coating antibody binds specific marker on the surface of target cells, so that antigen-positive and antigen-negative cells can be separated. This method combines the intrinsic advantages of solidified agent and the high specificity of immuno reaction, is widely applied in separation and purification of multiple proteins, and is well suited for isolation and detection of pathogenic microorganisms in food, water, biological samples, and environmental samples, showing good perspective of development and application.The principle of application of IMS in detection of microorganism:magnetic beads are coated with specific antibody that captures target microorganism in the sample or enriched culture, then the beads are attracted and precipitated with magnetic field. So target bacteria can be isolated from interfering bacteria and sample residue, getting concentrated, so that detection sensitivity and detection rate can be improved. IMS can be easily operated, the immunomagnetic beads are added to sample or enriched culture, incubated at room temperature for a certain period of time, then washed with butter several times in magnetic field, and the target bacteria is captured and concentrated, which can be directly used for subsequent experiments without extra separation from the beads. This method has the advantages of rapid separation, high efficiency, good repeatability, simple operation without expensive instruments, and little effects on the biological characteristics and function of the target cell or biological substances, and is thus extremely suited for detection of microorganism.2) Rapid detection by loop-mediated isothermal amplification (LAMP)Loop-mediated isothermal amplification is a novel isothermal nucleic acid amplification technology developed by Japanese scholar Notomi and his colleagues. Briefly, the template is incubated with4or6primers specific for6or8region of target gene and a special strand displacement DNA polymerase (Bst DNA polymerase) under isothermal condition (about65℃), and the amplification reaction is completed. LAMP can amplify target DNA rapidly with high efficiency, high sensitivity and high specificity, in addition, this method can be easily operated and cost-efficient, thus is well suited for large-scale analysis and grassroots units, and it is now widely used in diagnosis of disease, detection of pathogenic microorganism, and sex identification of animal embryo.So, in this study, we combined the two techniques, that is, Cronobacter spp. is isolated and concentrated from sample, then used for rapid LAMP detection. First, the technique using IMS to capture Cronobacter spp. was established and tested with simulated sample. Then internal and external primers specific for the OmpA gene encoding a specific outer membrane protein of Cronobacter spp., and LAMP technique was used to detect Cronobacter spp. from the enriched sample, thus establishing the rapid and highly sensitive IMS-LAMP method for detection of Cronobacter spp. Then we tested this method with simulated dairy product sample and209samples of formula milk powder in market, and the results were compared with that of traditional national standard method.2. Method:1) The technique using IMS to capture Cronobacter spp. was established and tested using simulated sample. Bacterial suspension of Cronobacter spp. and other bacteria(escherichia coli, Vibrio parahaemolyticus, Shigella flexneri, Proteus vulgaris, salmonella typhimurium, staphylococcus aureus, etl) were prepared, and were captured using Cronobacter spp.-specific immunomagnetic beads, then the captured bacteria were seeded on plates for colony count, to calculate the recovery rate of IMS capturing. Meanwhile, interference assay was performed, in which different concentrations of Cronobacter spp. were added to E. coli suspension with a concentration of104cfu/ml, and then captured with IMS to assess its efficiency of specific enrichment. Simulated milk powder sample containing known amount of target bacteria were tested for IMS capturing after3h or6h of pre-enrichment and simulated feces sample was also tested without pre-enrichment, to evaluate the practical applicability of IMS-based method in detection of Cronobacter spp.2) Primers specific for a conserved region of the OmpA gene (Accession number:DQ000206) encoding an outer membrane protein of Cronobacter spp. were designed for LAMP. The parameters of amplification were optimized and a25μL LAMP reaction system was established.3) Genomic DNA of20strains of experimental bacteria were used as template for LAMP and PCR analyses, the specificities of the two were compared; Cronobacter spp. cultured overnight was diluted to density gradient and used for amplification by LAMP and PCR, to compare sensitivities of the two methods.4) Test using simulated sample:three kinds of Cronobacter spp.-negative milk powder were inoculated with ATCC29544Cronobacter spp. strain, and were used for detection by LAMP and PCR, and the results were compared with that of national standard method.5)209samples of domestic infant formula milk powder were collected, and tested using the IMS-LAMP technique, and the results were compared with that of national standard.3. Results1) Colony count showed that recovery rates of IMS for the ATCC29544, ATCC51329and lab isolated strains of Cronobacter spp. were all over30%, and non-specific absorption of non-target bacteria was lower than3%. Interference assay showed that sensitivity of IMS is100times more sensitive than conventional direct plating method. Test using simulated samples of milk powder and feces showed that detection limit of IMS was101cfu/25g and101cfu/mL respectively, and the detection rate was improved.2) LAMP reaction system for detection of Cronobacter spp. was established. Each reaction mixture (total volume of25μL) contained2.5μL of6mM MgCl2,0.6mM dNTPs,0.8M Betaine,16pmol each of FIP and BIP primers,4pmol each of the F3 and B3primers, and2μL of DNA sample. And reaction for60min at58℃. After enrichment with IMS, LAMP showed higher specificity than PCR in detection of the ATCC29544strain of Cronobacter spp., and the detection limit of LAMP was101cfu/ml.3) Both LAMP and PCR successfully detected the3Cronobacter spp.-positive samples, which was consistent with results of national standard method.4) Of the209samples of infant formula milk powder in market,16were Cronobacter spp.-positive in IMS-LAMP test, while15were positive as shown by national standard method. The IMS-LAMP detection method showed the same result as that of national standard method, thus is similarly reliable, but it greatly reduced time required for detection.4. ConclusionThis study established the IMS technique to capture Cronobacter spp., and primers specific for the OmpA gene encoding the outer membrane protein of Cronobacter spp. were designed for LAMP analysis, and the parameters of reaction were optimized to ensure reliability and specificity of this method. LAMP assay were also performed for10other bacteria including E. coli, Salmonella, Shigella and some other pathogenic bacteria of the intestinal tract, as well as staphylococcus aureus, the results were all negative, indicating high specificity of this method. Detection limit of this method for bacterial culture was101cfu/mL, showing large advantage in detection sensitivity. The IMS-LAMP method we established for detection of Cronobacter spp. can be directly used for analysis of milk powder and other foods, and compared to conventional national standard method, it largely reduces time requirement, saving manpower and money. The IMS-LAMP method is simple, fast, performed under constant temperature, and requires only2-3h for detection of standard culture or26h for milk powder (national standard method requires6days). In addition, application of this method requires no expensive delicate instruments-a constant temperature water bath would be sufficient, and reaction and product testing can be performed in a single step, which is extremely simple and cost-efficient.In summary, the IMS-LAMP method we established for detection of Cronobacter spp. is highly sensitive, highly specific, simple and fast, and cost-efficient, shedding light on a new direction of development in Cronobacter spp. detection. This method has the potential to become a simple routine test, and is especially suited for grassroots inspection and quarantine institution. This method is of great significance in improving hygiene standard of food, ensuring food safety, and promoting international trade.更多还原