种特异性PCR快速检测奶粉中阪崎肠杆菌研究

阪崎肠杆菌是一种目前认为以奶粉为传播媒介的食源性条件致病菌,通过α-1,4-葡萄糖苷酶基因和ompA基因分别设计引物ESF-ESR和ESSF-ESSR,进行单重和双重PCR方法研究,结果显示所有阪崎肠杆菌菌株PCR扩增均为阳性,阴性对照均未扩增出目的片段;纯菌单重PCR灵敏度分别为102cfu/mL和101cfu/mL,双重PCR灵敏度为103cfu/mL;在有或无其他细菌存在时,人工污染阪崎肠杆菌模拟样品单重PCR检测灵敏度分别为103cfu/mL和102cfu/mL,双重PCR检测灵敏度为104cfu/mL;实际样品检测显示PCR方法与传统方法具有很好的一致性。结果表明:该PCR方法具有很好的种特异性和灵敏度,能够克服奶粉中杂菌对快速检测阪崎肠杆菌造成的干扰,减少以保守序列来设计引物导致假阳性结果的出现,可以较好地应用于奶粉中阪崎肠杆菌的检测与鉴定。     

应用阳离子磁珠捕集法快速检测奶粉中阪崎肠杆菌

目的:研究应用阳离子磁珠捕集法检测奶粉中阪崎肠杆菌,缩短阪崎肠杆菌检测周期,提高效率.方法:对阳离子磁珠捕集法检测阪崎肠杆菌的检测限和抗干扰性进行了实验,并通过检测乳粉样品与传统培养法相进行比较.结果:阳离子磁珠捕集法检测阪崎肠杆菌的检测限达到20～30 CFU/250ml,抗干扰性能强,检测周期比传统培养法缩短28 h～36 h,检测结果与传统培养法保持一致.结论:应用阳离子磁珠捕集法检测奶粉中的阪崎肠杆菌的方法是完全可行的.     

乳及乳制品中阪崎肠杆菌PCR-DHPLC检测新技术的建立

为了应用PCR结合变性高效液相色谱(DHPLC)技术建立食品中阪崎肠杆菌的快速检测方法,根据阪崎肠杆菌16S-23S rRNA特异基因序列的特点设计特异性引物,PCR扩增的产物经DHPLC技术进行快速检测.以阪崎肠杆菌等59株参考菌株做特异性试验;阪崎肠杆菌菌株稀释成不同梯度,做灵敏度试验,结果表明该方法具有很好的特异性,方法灵敏度较高,检测低限可达到为25 CFU/mL;该方法可以快速、准确检测阪崎肠杆菌,是食品中致病菌快速检测的新技术.     

益生菌拮抗阪崎肠杆菌的初步研究

目的研究鼠李糖乳杆菌和植物乳杆菌等8种常见益生菌对阪崎肠杆菌的拮抗作用。方法采用牛津杯法测定益生菌耗尽上清对阪崎肠杆菌的抑菌圈,获得对阪崎肠杆菌具有较强抑菌能力的鼠李糖乳杆菌和植物乳杆菌;采用混合培养法对2株益生菌与阪崎肠杆菌的拮抗竞争能力进行测试。结果 8种益生菌耗尽上清均能抑制阪崎肠杆菌,其抑菌能力具有热稳定性且依赖于酸性pH环境。阪崎肠杆菌(107CFU/mL)与鼠李糖乳杆菌(108CFU/mL或109CFU/mL)共孵育至24 h,其活菌量开始逐渐下降,至120 h孵育结束下降到105CFU/mL;菌量比为1:10的阪崎肠杆菌与植物乳杆菌共孵育至24 h,其活菌量开始逐渐下降,菌量比为1:100时则提前至8 h,至120 h孵育结束活菌量均下降到102CFU/mL。结论鼠李糖乳杆菌和植物乳杆菌均能有效地竞争拮抗阪崎肠杆菌。     

改良环介导等温扩增技术快速检测肉类中的大肠杆菌O157: H7

针对大肠杆菌O157:H7(Escherichia coli O157:H7,E.coli O157:H7)传统检测方法检测周期长的问题,建立了肉类中的E.coli O157:H7的改良环介导等温扩增(LAMP)快速检测方法。以E.coli O157:H7的O157特异性抗原rfbE基因、鞭毛H7特异性抗原fliC基因序列作为靶序列,分别设计2套增加了环引物的改良LAMP引物序列,单管同时检测,通过肉眼观察白色沉淀,判断检测结果。采用36株细菌验证了该改良LAMP引物的特异性。热裂解法提取的DNA经改良LAMP体系扩增20 min,检测E.coli O157:H7的灵敏度为1.4 CFU/mL,人工污染肉中的E.coli O157:H7检出限为1.8 CFU/g。137份实样中,检测出1份E.coli O157:H7假阳性,与行业标准SNT0973-2000符合率达到99.3%。     

阪崎肠杆菌的抵抗力及其机制研究

阪崎肠杆菌属于肠杆菌科肠杆菌属,作为一种条件致病菌,能引起严重的新生儿脑膜炎、小肠结肠炎和菌血症,死亡率高。婴儿配方奶粉被认为是主要的污染来源和传播工具。本文以阪崎肠杆菌对高热、高渗透压、干燥等应激条件的抵抗力及其机制研究进展进行了综述。阪崎肠杆菌的抗热性并没有明显高于其它菌株,但似乎具有更高的抵抗干燥和渗透压的能力。该菌出现这样的抗性表型的机制尚不完全清楚,其机制研究更待深入。     

阪崎肠杆菌噬菌体的分离及其生物学特性

[目的]以阪崎肠杆菌模式菌株及分离菌株为指示菌,从污水中分离出该菌噬菌体,并对其基本生物学特性进行研究.[方法]以双层琼脂法从污水中分离噬菌体,通过同属和同科参考菌株测定噬菌体的特异性和宿主谱;电镜观察噬菌体颗粒形态;随机扩增多态性DNA(RAPD)实验分析噬菌体的分子生物学特性.[结果]从污水中分离得到5株噬菌体,表现出较窄的宿主范围,仅裂解阪崎肠杆菌,以ATCC 51329分离的噬菌体SK2可裂解27株阪崎肠杆菌中的24株(89%),负染经电镜观察,5株噬菌体都是由多面体头部和尾部组成;随机引物(5′-GAAACGGGTG-3′)扩增DNA分析,5株噬菌体DNA明显不同.[结论]分离出的5株噬菌体仅对阪崎肠杆菌敏感,在阪崎肠杆菌的分型、预防、治疗、以及生态环境的净化等方面具有潜在用途.     

环介导等温扩增技术快速检测肉中单增李斯特菌

通过建立的环介导恒温扩增（Loop-Mediated Isothermal Amplification,LAMP）方法以达到肉中单增李斯特菌快速、灵敏的检出。以特异性的hlyA毒力基因作为靶基因,与6株非单增李斯特菌进行特异性试验,同时对不同培养浓度的单增李斯特菌进行了LAMP和PCR方法的灵敏度比较,进而应用LAMP法检测人工污染肉中的单增李斯特菌。结果表明：纯培养物中单增李斯特菌LAMP检出限为8.8×100CFU/mL,其灵敏度比普通PCR高100倍;在人工污染肉中单增李斯特菌的检出限为8.8×101CFU/mL,在1h内即可完成扩增反应。LAMP方法具备快速、特异、简单、灵敏度高等优势,在食品基质中单增李斯特菌的检测方面具有较好的应用前景。     

阪崎肠杆菌能力验证样品均匀性和稳定性的研究

目的制备出以脱脂奶粉为基质的均匀性好、稳定性强的阪崎肠杆菌(Enterobacter sakazakii)能力验证样品。方法研究奶粉基质的粒度、奶粉和菌粉的混合比例,得到阪崎肠杆菌能力验证样品的最佳均匀性条件;研究包装形式及贮存温度对样品稳定性的影响,得到阪崎肠杆菌能力验证样品的最佳稳定性条件。结果要保证样品足够均匀,奶粉最佳粒径范围为120μmD180μm,1 g菌粉最多与300 g奶粉进行混合;贮存温度对样品稳定性有较大影响,高温明显降低样品稳定性;真空包装可以显著提高样品稳定性。结论以奶粉为基质的阪崎肠杆菌能力验证样品的制备能够更准确地考查乳品微生物检验人员的检测水平,为国内微生物能力验证水平的提高奠定了基础。     

奶粉伴侣中微生物污染特点分析

目的：了解婴幼儿经常食用的奶粉伴侣中微生物的污染状况。方法：2016年在我国8个省（直辖市）采集奶粉伴侣864份,按照食品安全国家标准方法检测菌落总数、大肠菌群、阪崎肠杆菌、单核细胞增生李斯特菌、金黄色葡萄球菌和沙门氏菌。结果：104%（9/864）的奶粉伴侣菌落总数＞104 cfu/g,012%（1/864）大肠菌群＞102 cfu/g,未检出单核细胞增生李斯特氏菌、金黄色葡萄球菌和沙门氏菌,但1份样品检出阪崎肠杆菌。结论：奶粉伴侣微生物污染水平较低。     

Thermal resistance and inactivation of Enterobacter sakazakii isolates during rehydration of powdered infant formula

Enterobacter sakazakii may be related to outbreaks of meningitis, septicemia, and necrotizing enterocolitis, mainly in neonates. To reduce the risk of E. sakazakii in baby foods, thermal characteristics for Korean E. sakazakii isolates were determined at 52, 56, and 60 degrees C in saline solution, rehydrated powdered infant formula, and dried baby food. In saline solution, their D-values were 12-16, 3-5, and 0.9-1 min for each temperature. D-values increased to 16-20, 4-5, and 2-4 min in rehydrated infant formula and 14-17, 5-6, and 2-3 min in dried baby food. The overall calculated z-value was 6-8 for saline, 8-10 for powdered infant formula, and 9-11 for dried baby food. Thermal inactivation of E. sakazakii during rehydration of powdered infant formula was investigated by viable counts. Inactivation of cultured E. sakazakii in infant formula milk did not occur for 20 min at room temperature after rehydration with the water at 50 degrees C and their counts were reduced by about 1-2 log CFU/g at 60 degrees C and 4-6 log CFU/ml with the water at 65 and 70 degrees C. However, the thermostability of adapted E. sakazakii to the powdered infant formula increased more than two times. Considering that the levels of E. sakazakii observed in powdered infant formula have generally been 1 CFU/100 g of dry formula or less, contamination with E. sakazakii can be reduced or eliminated by rehydrating water with at least 10 degrees C higher temperature than the manufacturer-recommended 50 degrees C.     

Development of a loop-mediated isothermal amplification assay for detection of <Emphasis Type="Italic">Cronobacter</Emphasis> spp. (<Emphasis Type="Italic">Enterobacter sakazakii</Emphasis>)

Contamination of Cronobacter spp. (Enterobacter sakazakii) in infant formulas and other food products is a severe problem. Here a loop-mediated isothermal amplification (LAMP) assay was developed for rapidly detecting Cronobacter spp. in powdered infant formula. Sequences of 16S/23S rDNA internal intergenic spacer of Cronobacter spp. were used as the target template to design LAMP primers. The detection outcome can be evaluated by the white precipitate or the fluorescence intensity under ultraviolet irradiation, both visible to naked eyes. The sensitivity and specificity of the LAMP assay was further analyzed in comparison with that of regular PCR and real time quantitative PCR. The results showed that all of Cronobacter spp. strains display positive reaction to the detections while all of the non-Cronobacter spp. strains were negative, and that the LAMP assay exhibits a high sensitivity of 9.1 fg/μL (The sensitivity of regular PCR and real time quantitative PCR is 91 and 9.1 pg/μL, respectively.). The amplified reaction could be accomplished in about 1 h, with the results visible to naked eyes. Hence, the LAMP assay developed by this study can provide a rapid and simple approach for the detection of Cronobacter spp. in infant formula.     

Real time PCR using TaqMan and SYBR Green for detection of Enterobacter sakazakii in infant formula

Enterobacter sakazakii is an emerging pathogen that causes meningitis, bacteremia, sepsis, and necrotizing enterocolitis in neonates and children. Powdered milk-based infant formulas have been associated with the E. sakazakii-related outbreaks in premature or other immunocompromised infants. In this study, we developed two real time PCR assays using TaqMan and SYBR Green to identify the pathogen after selective enrichment in mLST and BHI. The accuracy of two detections was tested by 35 strains of E. sakazakii and 88 non-E. sakazakii bacterial strains. The results showed that all of these E. sakazakii strains were positive reaction to the detections and all of the non-E. sakazakii strains were negative. The newly developed assays enable us to detect 1.1 CFU/100 g infant formula. And both of the assays can be accomplished within 2 business days. Compared to the traditional detection, the real time PCR procedures are quicker and simpler. In this study, we also developed a new method to design the primers, which can support multiple real time PCR with one pair of primers in SYBR Green detection. The detection methods are more sensitive and effective based on Two-Tm-Value of PCR.     

Detection of Enterobacter sakazakii in dried infant milk formula by cationic-magnetic-bead capture

Enterobacter sakazakii has been associated with life-threatening infections in premature low-birth-weight infants. Contaminated infant milk formula (IMF) has been implicated in cases of E. sakazakii meningitis. Quick and sensitive methods to detect low-level contamination sporadically present in IMF preparations would positively contribute towards risk reduction across the infant formula food chain. Here we report on the development of a simple method, combining charged separation and growth on selective agar, to detect E. sakazakii in IMF. This protocol can reliably detect 1 to 5 CFU of E. sakazakii in 500 g of IMF in less than 24 h.     

阪崎肠杆菌显色培养基的应用研究

阪崎肠杆菌(Enterobacter sakazakii)是新近引起广泛关注的一种危险的条件致病菌, 主要存在于婴幼儿奶粉、婴幼儿补充食品中。由于目前日常使用的传统检验方法存在检测周期长等方面的不足之处, 本实验室研究设计出一种新的显色培养基(HKMCES), 通过与OXOID公司的同类产品(OXCES)比较, 分别应用于质控菌株、污染样品和实际样品的测试, 对这2种显色培养基的灵敏度、特异性、检测效果以及前增菌方法进行了初步评价。结果表明, 合适的增菌方法更有利于样品中阪崎肠杆菌的检出, 本实验室研制的显色培养基和OXOID公司的显色培养基均具有较好的选择性和特异性, 检测效果相当。这种新的显色培养基能使检测周期缩短, 具有较好的应用价值。     

Cloning and sequencing of the ompA gene of Enterobacter sakazakii and development of an ompA-targeted PCR for rapid detection of Enterobacter sakazakii in infant formula

Enterobacter sakazakii is an emerging, infant formula-borne pathogen that causes severe meningitis, meningoencephalitis, sepsis, and necrotizing enterocolitis in neonates and infants, with a high fatality rate. Traditional detection methods take up to 7 days to identify E. sakazakii. The outer membrane protein A gene (ompA), along with its flanking sequences from E. sakazakii (ATCC 51329), was cloned in the pGEM-T Easy vector and sequenced. Comparison of the nucleotide and deduced amino acid sequences of the ompA gene with other sequences available in the GenBank database revealed a high degree of homology with ompA genes of other gram-negative bacteria belonging to the Enterobacteriaceae. Based on regions of the ompA gene unique to E. sakazakii, two primers were synthesized to develop and optimize an E. sakazakii-specific PCR. The PCR amplified a 469-bp DNA product from all E. sakazakii strains tested but not from other bacteria. Experiments to determine the sensitivity of the PCR indicated that it could detect as few as 10(3) CFU/ml of E. sakazakii bacteria in infant formula directly and 10(-1) CFU/ml after an 8-h enrichment step. We conclude that this PCR, combined with enrichment culturing, has the potential to be used as a rapid tool for detecting the presence of E. sakazakii in infant formula.     

Rapid and sensitive detection of heat-labile I and heat-stable I enterotoxin genes of enterotoxigenic Escherichia coli by Loop-Mediated Isothermal Amplification

We developed a technique for detecting the heat-labile I (LTI) and heat-stable I (STI) genes of enterotoxigenic Escherichia coli (ETEC) using a novel DNA amplification procedure designated Loop-Mediated Isothermal Amplification (LAMP). The detection limit of accelerated LAMP utilizing loop primers was 4 CFU/test for LTI and was 40 CFU/test for STI, which are 10-fold higher than those of conventional PCR assay (detection limit, 40 CFU/test and 400 CFU/test, respectively). No DNA amplification was observed in LT and ST non-producing E. coli or other bacterial strains; thus, high specificity was verified. The specificity of LAMP assay was also confirmed by digestion of LAMP products using restriction enzymes and DNA sequence analysis. In the accelerated LAMP assay, DNA amplification was detected within 35 min, and thus LAMP is superior to conventional PCR in terms of rapidity. It was confirmed that increased concentrations of primers and Bst DNA polymerase could further facilitate the reaction. Furthermore, with the high amplification efficiency of the LAMP assay, amplification can be visually observed by the turbidity caused by magnesium pyrophosphate, a byproduct of the reaction. Detection of LTI and STI in ETEC by LAMP is thus an extremely rapid procedure with high sensitivity and specificity that requires no specialized equipment. This assay is expected to become a valuable tool for rapid diagnosis in ETEC infection.     

水霉菌环介导等温扩增检测方法的建立

【目的】建立一种快速简单检测水霉病病原菌的方法。【方法】针对水霉菌ITS区基因序列设计4条特异性引物,包括两条外引物和两条内引物,优化反应条件,观察检测结果。对该方法的特异性和敏感性进行研究。【结果】建立了环介导等温扩增技术检测水霉菌的方法,确定了其最适反应条件。该方法能够检测到浓度低至103个/mL的水霉菌孢子,其灵敏度是普通PCR方法的100倍。【结论】建立的检测水霉菌的LAMP技术,具有操作简便快速等特点,可用做特异性水霉及其孢子的快速鉴定。     

Development of sandwich enzyme-linked immunosorbent assay for the detection of Cronobacter muytjensii (formerly called Enterobacter sakazakii)

This study aimed to produce a polyclonal antibody against Cronobacter muytjensii (C. muytjensii, formerly called Enterobacter sakazakii) and to develop an immunoassay for its detection. The optimum production of rabbit anti-C. muytjensii immunoglobulin G (IgG) and chicken anti-C. muytjensii IgY was reached in weeks 8 and 9, respectively. Purification of rabbit anti-C. muytjensii IgG from immunized rabbit sera was accomplished using the caprylic acid and ammonium sulfate precipitation method. As a result, sodium dodecyl sulfate-polyacrylamide gel electrophoresis produced two bands around 25 and 50 kDa, corresponding to a light and a heavy chain, respectively. The optimized conditions for sandwich enzyme-linked immunosorbent assay were using rabbit anti-C. muytjensii IgG (1 μg/mL) as a detection antibody and chicken anti-C. muytjensii IgY (10 μg/mL) as a capture antibody. In this assay, no cross-reactivity was observed with the other genera of pathogenic bacteria tested, which included Escherichia coli O157:H7, Salmonella typhimurium, Staphylococcus aureus, Bacillus cereus and Listeria monocytogenes. The developed assay did not show cross-reactivity with other tested species of Cronobacter and Enterobacter genera such as C. turicensis, C. sakazakii, E. aerogenes, E. pulveris and E. helveticus. The detection limit of sandwich ELISA for C. muytjensii was found to be 2.0 × 10(4) colony forming units (CFU)/mL. In addition, detection of C. muytjensii in infant formula powder showed a low matrix effect on the detection curve of sandwich ELISA for C. muytjensii, the detection limit being found to be 6.3 × 10(4) CFU/mL. These findings demonstrate that the developed method is able to detect all strains of C. muytjensii. Hence, this ELISA technique has potent application for the rapid and accurate detection of C. muytjensii in dietary foods.