实时荧光定量PCR法检测阪崎肠杆菌和肠杆菌科

使用全新实时荧光定量PCR的方法与传统的国家标准方法共同对180个样品中的阪崎肠杆菌以及肠杆菌科进行检测。包括婴幼儿配方奶粉(包括含益生菌或谷物的婴幼儿配方食品以及环境样品等)。分别从重复性,排除死亡细菌的干扰(假阳性)以及相对准确性、相对灵敏性和相对特异性等方面进行对比研究。结果表明,使用两种方法所得结果完全一致,并且实时荧光定量PCR方法有着明显的时间短,易操作等优点。     

基于内参系统的阪崎肠杆菌实时荧光定量PCR检测方法的建立

根据阪崎肠杆菌ompA靶基因设计特异性引物和探针,并加入内参(IAC),建立能够实时监控反应过程的荧光定量PCR检测方法。结果表明,该方法对阪崎肠杆菌基因组DNA的最低检测限为1pg;对细菌的最低检测限为1×10~4 CFU;对含有靶基因质粒的最低检测限为10~3拷贝;Ct值与模板拷贝数均呈良好的线性关系(R~2=0.999)。人工污染试验结果表明,在初始菌量为10CFU/25g奶粉样品时,采用水洗加试剂盒法和水煮法提取DNA,阪崎肠杆菌均在增菌10h时检出。研究结果为进一步优化和完善阪崎肠杆菌分子生物学方法的标准化提供了参考。     

婴幼儿奶粉中阪崎肠杆菌双重荧光PCR快速检测方法的建立

旨在建立针对阪崎肠杆菌的双重荧光PCR快速检测方法。以阪崎肠杆菌局部大分子合成(MMS)操纵子和外膜蛋白A(ompA)为靶基因,建立双重荧光PCR反应体系,探讨该体系的特异性、灵敏度和抗干扰能力。结果表明,双重荧光PCR体系对阪崎肠杆菌的灵敏度为4.3×10³ CFU/mL,人工污染初始菌量为2 CFU/100 g奶粉样品增菌24 h即可检出;39株实验菌中的15株阪崎肠杆菌出现特异性扩增,24株非阪崎肠杆菌未出现特异性扩增。本研究所建立的双重荧光PCR体系特异好、灵敏度较高及抗干扰能力强,可用于婴幼儿奶粉中阪崎肠杆菌的快速检测。     

奶粉中阪崎肠杆菌PCR和荧光PCR检测方法的研究

建立了奶粉中可致婴幼儿高死亡率的阪崎肠杆菌的PCR和荧光PCR检测方法。利用细菌16S和23SrDNA的保守区设计通用引物,对6株阪崎肠杆菌16S-23SrDNA间区序列(ITS)进行扩增和测序,在比对阪崎肠杆菌ITS序列的基础上,设计了11条PCR和荧光PCR检测引物,组合成30对PCR引物,并筛选出一对种特异性引物,建立了奶粉中阪崎肠杆菌PCR和荧光PCR检测方法。用10株阪崎肠杆菌,18株近源菌株验证实验表明,本文所建立的PCR和荧光PCR方法特异性强;加菌实验表明,奶粉样品中阪崎肠杆菌检测低限为(2.2～5.4)CFU/100g,灵敏度高;新建的PCR和荧光PCR方法与FDABAM(美国食品及药品管理局微生物分析手册)方法比对实验表明,三种方法的检测结果完全一致。由于PCR和荧光PCR检测方法快速、可靠,因此可替代传统检验方法。     

进口乳粉中阪崎肠杆菌检测分析

应用检验检疫行业标准<奶粉中阪崎肠杆菌的检验方法>中分离计数及普通PCR两种方法时50份进口乳粉进行检测,两种方法检测结果均准确可靠,但增茵方法不统一,标准有待完善.近三年来,天津口岸共检出31份阪崎肠杆菌阳性的进口乳粉.对这些阳性样品及阪崎肠杆茵进行了地域、污染情况及生化性状的总结分析,为完善检验方法及阪崎肠杆菌的进一步深入研究奠定基础.     

乳粉中阪崎肠杆菌的检测方法

为乳品企业开展阪崎肠杆茵出厂检验寻找更加便捷、准确,且检测费用低廉的检测方法.参照SN/T1632.1-2005标准,在定性检测基础上有所改进.选择3种基础肠道分离培养基伊红美蓝、麦康凯、山梨醇麦康凯分离培养及生化鉴定阪崎肠杆菌,对两种修复培养基灵敏度进行比较及阪崎肠杆菌在5种肠道分离培养基上生长情况比较.结果表明.A和B两组修复培养对婴幼儿乳粉中阪崎肠杆菌均能起到修复作用,B组使用BP修复培养比A组蒸馏水修复培养灵敏度高出1个稀释度(10倍).阪崎肠杆菌在5种肠道分离培养基上均能生长.选择上述3种基础肠道分离培养基分离鉴定阪崎肠杆茵,费用低廉,用于乳品企业对每批次产品进行自检,有更大的经济效益和社会效益.     

检测阪崎肠杆菌间接ELISA方法的建立

建立阪崎肠杆菌(Enterobacter Sakazakii,ES)特异性抗体间接ELISA检测方法,以期作为融合后杂交瘤细胞株的筛选体系。应用灭活的阪崎肠杆菌菌体为抗原,筛选最佳反应条件,建立检测该病原菌的酶联免疫吸附检测方法。通过对ELISA反应条件优化,确定菌体抗原包被的最适工作浓度为2×105mL-1,4℃包被过夜;5%脱脂奶粉进行封闭;血清最佳稀释比为1∶2 000;酶标抗体最适稀释度为1∶1 000倍;最后确定37℃显色10 min,检测灵敏度达到103mL-1。该方法的建立为阪崎肠杆菌单克隆抗体制备过程中阳性杂交瘤细胞株的筛选提供有效手段和奠定实验基础。     

3种方法检测婴幼儿配方奶粉中的阪崎肠杆菌

目的对市售婴幼儿配方奶粉中阪崎肠杆菌检测方法进行研究。方法分别用常规培养鉴定方法、常规PCR方法和实时荧光PCR方法对32份奶粉样品进行阪崎肠杆菌分离鉴定。结果32份样品中检出2株阳性株，阳性率为6．25％，3种鉴定方法结果相符。结论联合使用多种鉴定方法可提高阪崎肠杆菌检测的可靠性。     

冰激凌中阪崎肠杆菌的检测

目的 对市售冰激凌中的阪崎肠杆菌进行抽样检测。方法 按照《食品安全国家标准 食品微生物学检验 (GB 4789.40- 2010)》进行检验。结果 从冰激凌中检出阪崎肠杆菌,经过染色和生化试验, 在29份样品中有3份样品分离出阪崎肠杆菌,检出率为10.34%。结论 市售冰激凌样品中存在一定比例的阪崎肠杆菌,有较大的安全隐患。质检系统应加强对市售的冰激凌的卫生预防与控制。     

中国安徽阜阳劣质婴儿配方粉中阪崎肠杆菌的污染

2004年中国安徽阜阳劣质婴儿配方粉事件引起了我国政府的高度重视.为了调查婴儿配方粉中阪崎肠杆菌的污染状况,根据美国FDA和加拿大实验室的方法,建立了婴儿配方粉中阪崎肠杆菌的分离鉴定技术.从87份阜阳劣质奶粉样品中检测到11份阪崎肠杆菌阳性样品,污染阳性率为12.6%.用API 20E和Qualicon BAX(R)系统鉴定了11株阪崎肠杆菌.这是国内首次从婴儿配方粉中分离到阪崎肠杆菌菌株.     

柳江县市售国产婴幼儿配方粉中阪崎肠杆菌污染监测分析

目的 了解市售国产婴幼儿配方粉中阪崎肠杆菌污染状况,为消费预警提供科学依据。方法 分别按2011、2012年版国家食源性致病菌监测工作手册对市场上11家国内生产的32份婴幼儿配方粉进行检测。结果 32份样品检出2株阪崎肠杆菌,检出率为6.25%。其中婴幼儿配方奶粉中阪崎肠杆菌检出率4.35%(1/23);婴幼儿谷物食品中阪崎肠杆菌检出率11.11%(1/9)。结论 柳江县市售部分婴幼儿配方粉中存在阪崎肠杆菌污染,食用安全隐患不容忽视,应加强监测力度,预防和控制阪崎肠杆菌引起的食物中毒事件发生。     

乳粉中坂崎肠杆菌的快速检验

采用一种选择性显色培养基开展了乳粉中坂崎肠杆菌的检测，该培养基针对坂崎肠杆菌产生α-葡萄糖苷酶活性的特点．在显色培养基中添加了一种底物5-溴-4-氯-吲哚-α-D-吡喃葡萄糖苷（XαGlc），α-葡萄糖苷酶水解XαGlc,使菌落呈现蓝绿色．与传统检测方法相比，该检测方法选择性更强，操作更为简便，检测时间明显缩短，是一种较为理想的检测乳粉中坂崎肠杆菌的方法．     

Rapid, specific detection of Enterobacter sakazakii in infant formula using a real-time PCR assay

Enterobacter sakazakii is a rare cause of invasive infection with high mortality rates in neonates. Powdered milk-based infant formulas have been associated with the E. sakazakii-related outbreaks in premature or other immunocompromised infants. In this study, an assay was developed for the specific detection of E. sakazakii in infant formula using an application of the fluorogenic 5' nuclease assay (TaqMan). A set of primers and probe was designed using the E. sakazakii partial macromolecular synthesis operon: the rpsU gene 3' end and the primase (dnaG) gene 5' end. The specificity of the assay was evaluated using 68 Enterobacter and 55 non-Enterobacter strains. The newly developed assay enables us to detect 100 CFU/ ml in pure culture and in reconstituted infant formula in 50 cycles of PCR without enrichment. The assay was specific enough to discriminate E. sakazakii from all other Enterobacter and non-Enterobacter strains tested. The developed real-time PCR assay could save up to 5 days and eliminate the need for plating samples on selective or diagnostic agars and for biochemical confirmation steps. The real-time PCR assay could be used to rapidly screen infant formula samples for E. sakazakii and would be a boon to food industries and regulatory agencies.     

Enterobacter sakazakii in food and beverages (other than infant formula and milk powder)

The ubiqitous microorganism Enterobacter sakazakii is a rare contaminant of infant formula and may cause severe systemic infection in neonates. So far, other food is not known to cause E. sakazakii-infections. The scarce information about the ecology of E. sakazakii and the uncertainty concerning the source of infection in children and adults warrant a summary of the current knowledge about the presence of this opportunistic microorganism in food other than infant formula. This review systematizes publications on the presence of E. sakazakii in food and beverages until June 2006. Food other than infant formula has been rarely investigated for the presence of E. sakazakii. Nevertheless, this microorganism could be isolated from a wide spectrum of food and food ingredients. E. sakazakii was isolated from plant food and food ingredients like cereal, fruit and vegetables, legume products, herbs and spices as well as from animal food sources like milk, meat and fish and products made from these foods. The spectrum of E. sakazakii-contaminated food covers both raw and processed food. The kind of processing of E. sakazakii-contaminated food was not restricted to dry products. Fresh, frozen, ready-to-eat, fermented and cooked food products as well as beverages and water suitable for the preparation of food, were found to be contaminated by E. sakazakii. Although E. sakazakii-contaminated food do not have general public health significance, measures for prevention should consider the presence of E. sakazakii in food, food ingredients, their processing and preparation as possible source of contamination, colonization or infection.     

Thermal inactivation of Enterobacter sakazakii in rehydrated infant formula

The presence of low levels of Enterobacter sakazakii in dried infant formula have been linked to outbreaks of meningitis, septicemia, and necrotizing enterocolitis in neonates, particularly those who are premature or immunocompromised. In the current study, the ability of 12 strains of E. sakazakii to survive heating in rehydrated infant formula was determined at 58 degrees C with a submerged coil apparatus. The observed D58-values ranged from 30.5 to 591.9 s, with the strains appearing to fall into two distinct heat resistance phenotypes. The z-value of the most heat-resistant strain was 5.6 degrees C. When dried infant formula containing this strain was rehydrated with water preequilibrated to various temperatures, a more than 4-log reduction in E. sakazakii levels was achieved by preparing the formula with water at 70 degrees C or greater.     

Real-time PCR von Enterobacter sakazakii in Säuglingsanfangsnahrung

Enterobacter sakazakii represents a rare opportunistic pathogen that causes a high mortality rate. Especially newborns are infected by contaminated infant formula. To reduce this risk, the EU directive 2073/2005 demands for the absence of the pathogen in 30 ×10 g infant formula. We describe a PCR-/real-time PCR that specifically detected 32 E. sakazakii strains among 21 species of Enterobacteriaceae as well as contaminated specimens among 30 different brands of infant formula. This real-time PCR, performed after a selective enrichment of E. sakazakii, reduces the working time by 1 to 3 days in comparison to the detection methods recommended by the US Food and Drug Administration (FDA) and ISO/DTS 22964 that is still under revision. In contrast to the latter procedure, we detected contaminated specimens of infant formula by the use of the procedure described in this report and the FDA method.

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Zusammenfassung: Enterobacter sakazakii repr?sentiert einen seltenen opportunistischen Erreger, der Infektionen mit hoher Mortalit?tsrate hervorruft. Insbesondere Neugeborene erkranken durch kontaminierte Anfangsnahrung. Zur Risikominimierung fordert die EU-Verordnung 2073/2005 die Abwesenheit des Erregers in 30 ×10 g S?uglingsanfangsnahrung. Ein PCR-/Real-Time PCR-Verfahren wird beschrieben, das zun?chst mit 32 E. sakazakii -St?mmen und weiteren 20 Arten der Enterobacteriaceen geprüft und anschlie?end zur Untersuchung 30 unterschiedlicher Marken von S?uglingsanfangsnahrung verwendet wurde. Nach der Selektivanreicherung führt das Real-Time PCR-Verfahren zu einer Ersparnis von 1 bis 3 Arbeitstagen beim Nachweis von E. sakazakii im Vergleich zum Verfahren der amerikanischen Food and Drug Administration (FDA) und dem noch in Bearbeitung befindlichen Verfahren ISO/DTS 22964. Im Gegensatz zur letztgenannten Untersuchungsmethode erfolgten mehrere Positivnachweise mit dem hier beschriebenen Verfahren und dem der FDA.

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Eingegangen: 18. Januar 2007     

Detection of Enterobacter sakazakii strains by real-time PCR

A precise 5' nuclease (TaqMan) real-time PCR was developed and validated in house for the specific detection of Enterobacter sakazakii isolates. Specifically designed nonpatented primers and a hydrolysis (TaqMan) probe were used to target the 16S rRNA gene. All 27 E. sakazakii and 141 non-E. sakazakii strains tested with the real-time PCR were identified correctly. To monitor false-negative results, an internal amplification control was coamplified with the same primers used for the E. sakazakii DNA. The detection probability of the assay was 56% when an E. sakazakii cell suspension containing 10(2) CFU/ml was used as template in the PCR (0.5 CFU per reaction) and 100% with a 10(3) CFU/ml suspension. This PCR assay should be very useful for the diagnostic detection of E. sakazakii in foods, especially powdered infant formula, after cultural enrichment.     

Inactivation of Enterobacter sakazakii in reconstituted infant formula by monocaprylin

Enterobacter sakazakii is an emerging pathogen that causes meningitis, bacteremia, sepsis, and necrotizing enterocolitis in neonates and children, with a mortality rate of 14%. Epidemiological studies have implicated dried infant formula as the principal source of the pathogen. Caprylic acid is a natural eight-carbon fatty acid present in breast milk and bovine milk and is approved as generally recognizable as safe by the U.S. Food and Drug Administration. The objective of this study was to determine the antibacterial effect of monocaprylin (monoglyceride ester of caprylic acid) on E. sakazakii in reconstituted infant formula. A five-strain mixture of E. sakazakii was inoculated into 10-ml samples of reconstituted infant formula (at 6.0 log CFU/ml) followed by 0, 25, or 50 mM (1%) monocaprylin. The samples were incubated at 37 or 23 degrees C for 0, 1, 6, and 24 h and at 8 or 4 degrees C for 0, 6, 24, and 48 h, and the surviving populations of E. sakazakii at each sampling time were counted. The treatments containing monocaprylin significantly reduced the population of E. sakazakii (P < 0.05) compared with the controls. Monocaprylin (50 mM) reduced the pathogen by >5 log CFU/ml by 1 h of incubation at 37 or 23 degrees C and by 24 h of incubation at 8 or 4 degrees C. Results indicate that monocaprylin could potentially be used to inactivate E. sakazakii in reconstituted infant formula; however, sensory studies are warranted before its use can be recommended.