利用环介导等温扩增技术对奶粉中阪崎肠杆菌进行检测

利用阪崎肠杆菌16S-23S rDNA间区(ITS)序列,在比较阪崎肠杆菌与其近源株ITS序列的基础上,设计了4条阪崎肠杆菌LAMP检测特异性引物,建立了奶粉中阪崎肠杆菌LAMP检测方法.用15株阪崎肠杆菌,61株近源菌验证试验表明,所建立的LAMP方法准确且灵敏度高.加菌试验表明,奶粉样品中阪崎肠杆菌检测低限为1.2 CFU/100 g.新建的LAMP方法与FDA BAM方法比较试验表明,两种方法的检测结果完全符合.     

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。     

恒温实时荧光法快速检测奶粉中阪崎肠杆菌方法的建立

为实现奶粉中快速检测阪崎肠杆菌,本文建立了检测阪崎肠杆菌的恒温实时荧光法。针对阪崎肠杆菌16S r RNA设计三组LAMP引物,采用Deaou-308C恒温实时荧光检测平台,选取常见病原菌标准株进行引物特异性检测;选取阪崎肠杆菌标准菌株进行基因组DNA灵敏度和最低检测限测定,同时利用人工污染方式检测此方法在脱脂和全脂奶粉中的灵敏度和最低检测限,利用Real Amp法和国标法对20份市售奶粉进行对比实验。结果显示,引物组16S-11扩增效率最优,与常见病原菌无交叉反应,对阪崎肠杆菌基因组DNA、阪崎肠杆菌污染的脱脂和全脂奶粉的灵敏度分别达到102 CFU/m L、102 CFU/m L和103 CFU/m L;对阪崎肠杆菌基因组DNA和阪崎肠杆菌污染的脱脂和全脂奶粉的最低检测限分别达到103 CFU/m L、103 CFU/m L和104 CFU/m L;在20份市售奶粉样品中Real Amp检测结果与传统国标培养结果一致,表明本文建立的阪崎肠杆菌Real Amp检测方法适用于阪崎肠杆菌的快速检测。     

多重PCR快速检测婴幼儿奶粉中的病原菌

为了建立同时检测婴幼儿奶粉中阪崎肠杆菌、金黄色葡萄球菌、蜡样芽孢杆菌的多重PCR方法,根据阪崎肠杆菌ompA基因、金黄色葡萄球菌nuc基因、蜡样芽孢杆菌hblA基因分别设计3对引物进行多重PCR扩增,并对反应条件进行优化。结果多重PCR扩增出长度为514、156、235bp的特异性目的条带。不增菌的情况下,多重PCR同时检测3种病原菌的灵敏度是103cfu/mL,3种病原菌在奶粉中的检出限是104cfu/g。建立的多重PCR反应准确、快速、高效,为同时检测婴幼儿奶粉中的阪崎肠杆菌、金黄色葡萄球菌、蜡样芽孢杆菌提供了新方法。     

婴儿配方粉中阪崎肠杆菌荧光定量PCR检测方法的建立

建立奶粉中阪崎肠杆菌荧光定量PCR检测方法,为准确、快速地定量检测阪崎肠杆菌奠定基础。根据阪崎肠杆菌16S rDNA基因序列,设计特异引物和荧光标记探针并合成,用PCR扩增产物克隆的重组质粒作为阳性对照,经紫外分光光度计测定重组质粒浓度．梯度稀释后作为标准品绘制标准曲线,其Ct值与模板浓度有良好的线形关系,相关系数R^2值为：0．997,可以用于阪崎肠杆菌浓度鉴定。用该方法检测阪崎肠杆菌样品21份,阳性4份,阳性检出率为19％,与常规检测方法结果完全符合。本文建立的检测阪崎肠杆菌的荧光定量PCR方法,较常规技术更为简便、快速,有很好的应用前景和研究价值。     

应用环介导等温扩增检测阪崎肠杆菌

建立环介导等温扩增检测阪崎肠杆菌的方法,通过肉眼可见的白色沉淀,判断检测结果。将阪崎肠杆菌(ATCC29544)的16S-23SrRNA间区序列作为靶基因,设计2对特异引物,优化反应条件,进行LAMP扩增。对产物进行酶切分析,与理论上的预期结果相一致。通过测序比对,与GenBank上报道的同源性达到99%。用25株食源性致病菌证实该引物特异性强。LAMP扩增20min,其灵敏度为4.3×102 fg/tube,结果表明,LAMP方法检测阪崎肠杆菌,灵敏度高、特异性强、耗时短、方法简便。     

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。     

环介导等温扩增法快速检测乳中阪崎肠杆菌

利用环介导等温扩增方法的快速、简便等优点,建立一种检测乳中阪崎肠杆菌的方法.以阪崎肠杆菌的OmpA序列为靶基因,设计特异性引物.优化并建立LAMP检测乳中阪崎肠杆菌的方法.结果表明,LAMP检测阪崎肠杆菌纯培养物的灵敏度为3.7×101cfu/mL,其灵敏度是PCR方法的10倍.人工污染阪崎肠杆菌灭菌乳的检测限为4.3×101 cfu/mL.对23株致病菌进行特异性实验,特异性良好.该方法具有特异性强、灵敏度高、设备简便、耗时短等优点,在食品检测中具有良好的应用前景.     

奶粉中阪崎肠杆菌的污染及快速检测方法的研究

目的：了解福建省市售婴幼儿配方奶粉中阪崎肠杆菌的污染状况、污染途径并寻找快速准确的检测方法。方法：应用分离鉴定、PCR 和荧光PCR 等方法检测,分离鉴定采用阪崎肠杆菌显色培养基和全自动微生物生化仪。结果：阪崎肠杆菌在192 份市售婴幼儿配方奶粉中的检出率为1.56%,在60 份原料奶粉中的检出率为13.33%,30 份生产车间环境样本均未检出。结论：福建省市售婴幼儿配方奶粉中存在阪崎肠杆菌的安全隐患,某工厂奶粉中阪崎肠杆菌的污染主要来自原料奶粉。荧光PCR 和显色培养基可用于阪崎肠杆菌的快速筛选和分离。     

LAMP法在阪崎肠杆菌快速检测中的应用

针对阪崎肠杆菌的ompA基因设计4条特异性引物(2条内引物、2条外引物),建立一种快速检测阪崎肠杆菌的DNA环介导恒温扩增法(LAMP)。通过对2株阪崎肠杆菌标准菌株、24株阪崎肠杆菌分离株和18株非阪崎肠杆菌测试,结果表明此方法具有很高的特异性;对其灵敏度进行检验,发现该方法的最低检测限可以达到8CFU/mL,具有较好的敏感性。在1份实验室能力验证盲样和26份实际奶粉样品中应用发现,该方法与传统生化方法结果吻合,具有较好的可靠性。     

Characterization of the gene encoding the 16S rRNA of Enterobacter sakazakii and development of a species-specific PCR method

The gene encoding the 16S rRNA of Enterobacter (E.) sakazakii (ATCC 29544, plus four strains isolated from powdered infant formula) was studied, and the sequence compared with those of other Enterobacteriaceae in aspects of genetic variability. Sequence differences between E. sakazakii and other Enterobacteriaceae within the hypervariable regions V1, V2, and V3, respectively, were used to develop two PCR methods for E. sakazakii. PCR1 employed a primer pair located in V1/V2, while PCR2 utilized a primer pair located in V1/V3, respectively. The two PCR methods were tested against a set of 57 E. sakazakii and 148 non-E. sakazakii isolates. PCR1 gave an amplicon with a size of 406 bp and resulted in 100% positive results for E. sakazakii, but also detected Citrobacter koseri/amalonaticus and all nine tested Salmonella enterica serovars. In contrast, PCR2 (amplicon size of 952 bp) gave positive results only for E. sakazakii, thus allowing specific identification of this species.     

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 infant milk formula by gamma irradiation: determination of D10-value

ABSTRACT:  Enterobacter sakazakii is an emerging foodborne pathogen that has caused several cases of meningitis and necrotizing entercolitis in infants and has been associated with infant formulas. Five strains of E. sakazakii were inoculated individually into brain heart infusion broth and rehydrated or dehydrated infant milk formula and exposed to ionizing radiation. E. sakazakii strains in brain heart infusion broth and rehydrated infant milk formula (RIMF) were exposed to irradiation dose of up to 1 kGy while strains in dehydrated infant milk formula (DIMF) were exposed to irradiation dose of up to 9 kGy. The D₁₀-values were determined by using a linear regression model. Average calculated D₁₀-values ranged from 0.21 to 0.29 kGy, 0.24 to 0.37 kGy, and 1.06 to 1.71 kGy in brain heart infusion broth, RIMF, and DIMF, respectively. The results obtained from this study will be useful for powdered infant milk formula industries to reduce the risk associated with E. sakazakii .     

奶粉中阪崎肠杆菌的分离与鉴定

目的了解某地区婴儿配方奶粉中阪崎肠杆菌的污染情况,为我国制定婴儿配方奶粉中阪崎肠杆菌的限量标准及为政府加强对婴儿配方奶粉的管理提供科学依据。方法常规生理生化鉴定法和实时荧光PCR法。结果检出2株阪崎肠杆菌。结论我国市售的婴儿配方奶粉中存在少量的阪崎肠杆菌污染。     

Development of an immobilization and detection method of Enterobacter sakazakii from powdered infant formula

Enterobacter sakazakii is an emerging opportunistic pathogen that is associated with rare but life-threatening cases of meningitis, necrotizing enterocolitis, and sepsis in premature and full-term infants. In the present study, a procedure was developed for immobilization of E. sakazakii with zirconium hydroxide coupled with detection by a species-specific duplex PCR, based on 16s-23s rDNA internal transcribed spacer (ITS) and ompA gene. Specificity of duplex PCR was tested against two-type strains, six isolates of E. sakazakii and other eight non-E. sakazakii species. When pure culture of E. sakazakii was used for immobilization, total recovery rate ranged from 79.4% to 99.6% of input bacteria, and the detection limit of duplex PCR was 3x10(5)CFU/ml. Different levels of E. sakazakii were inoculated into 90ml reconstituted powdered infant formula (PIF), and detection limit of duplex PCR was 3x10(0)CFU/ml with 24-30h enrichment after immobilization. When the experiment was performed in the presence of 10(2)CFU/ml Salmonella typhimurium, the detection limit of duplex PCR was not affected after enrichment. Seven out of 13 commercial PIF were detected positive by duplex PCR after immobilization, while only three were positive by biological methods. This study demonstrates that the combination of immobilization method with duplex PCR is easy, rapid, and efficient, and may have applications for the detection of E. sakazakii in more PIF samples.     

乳粉中阪琦肠杆菌的PCR检测

针对我国食品进出口和生产卫生的需要,使用以16 S rRNA基因为靶序列设计合成了一对可扩增282 bp的目的片段的引物,建立了检测阪琦肠杆菌的PCR方法.结果表明,阪琦肠杆菌PCR产物有282 bp的特异性片段,而金黄色葡萄球菌无此目的条带,证实该引物具有特异性.该方法快速、可靠、灵敏,特异性强,可用于食品中阪琦肠杆菌的检测.     

Characterization of a collection of Enterobacter sakazakii isolates from environmental and food sources

Enterobacter sakazakii has emerged as a rare cause of neonatal meningitis, septicemia and enterocolitis. Contaminated infant milk formula (IMF) has been identified as one infection route. A small number of clinical outbreaks have been epidemiologically linked to IMF contaminated post-pasteurization during manufacture and/or mishandled when reconstituted. Currently no agreed standardized typing protocol has been developed to trace E. sakazakii. The objectives of this study were to apply biochemical and genetic methods to characterize 51 environmental and food E. sakazakii isolates and 6 E. sakazakii type strains. Isolates were presumptively identified using biochemical profiles based on API 20E and ID32E methods and by culture on differential selective Druggan Forsythe Iversen (DFI) agar. Identification was subsequently confirmed by real time polymerase chain reaction (PCR). All but one of the isolates was identified as E. sakazakii by biochemical profiling. One isolate was identified as Escherichia vulneris by ID 32E and as Pantoea agglomerans by API 20E. All isolates produced green/blue colonies on DFI medium characteristic of this organism. Real time PCR could differentiate between E. sakazakii, Enterobacter spp. and other Enterobacteriacae. Analysis of RAPD banding patterns revealed 3 major clusters of E. sakazakii. There was a large degree of diversity noted amongst the remaining isolates. Our findings indicate that RAPD may be applied as a useful and reliable tool for direct comparison of E. sakazakii isolates providing traceability through the infant formula food chain.     

THERMAL RESISTANCE, SURVIVAL AND INACTIVATION OF ENTEROBACTER SAKAZAKII (CRONOBACTER SPP.) IN POWDERED AND RECONSTITUTED INFANT FORMULA

Enterobacter sakazakii has recently been recognized as an opportunistic foodborne pathogen, and dry infant formula serves as the mode of transmission. The objectives of this study were to investigate the heat resistance, survival and inactivation under room and refrigeration temperatures storage of dry and reconstituted infant formula milk (IFM). E. sakazakii strains (eight strains) showed a wide variability in heat resistance at different temperatures (55, 60 and 63C). The D-values at 55C ranged from 1.51 to 14.83 min, at 60C from 0.17 to 2.71 min and at 63C from 0.05 to 0.88 min. The calculated z values for the studied E. sakazakii strains ranged from 3.76–10.11C. Microwave oven heating of 60-mL portions of reconstituted IFM for 40–50 s was effective in eradicating inoculated E. sakazakii. Storing powdered IFM for 15 days at 4C resulted in at least a 1-log reduction in E. sakazakii strains, whereas storing reconstituted IFM at 4C for 2 weeks resulted in more than a 2-log reduction in E. sakazakii.

PRACTICAL APPLICATIONS

This study shows that E. sakazakii strains differ widely in their heat resistance. No differences were observed between biofilm formers and nonformers in terms of heat-resistance in thermal inactivation kinetics experiments. Conventional high temperature short-time pasteurization processes are considered sufficient to inactivate all E. sakazakii strains, and a household microwave oven (40–50 s for 60-mL portions) can be used to inactivate E. sakazakii if present in reconstituted infant formula milk (IFM). Growth of E. sakazakii can be inhibited in powdered and reconstituted IFM by refrigeration. Also, it is recommended that reconstituted IFM be discarded or refrigerated if not immediately consumed. The probiotic L. acidophilus ATCC 4356 was not effective in inhibiting E. sakazakii in powdered or reconstituted IFM.     

Growth and survival of Enterobacter sakazakii in human breast milk with and without fortifiers as compared to powdered infant formula

Enterobacter sakazakii infections often involve debilitated neonates consuming contaminated reconstituted powdered infant formula. There is the possibility that expressed human breast milk can become contaminated with E. sakazakii in the hospital or home setting and through the use of contaminated breast milk fortifiers. In addition, although breast milk has been shown to have some antimicrobial effects, this has not been extensively researched in regards to E. sakazakii. Thus, we examined the survival and growth of 9 strains of E. sakazakii in breast milk, human breast milk with fortifiers and powdered infant formula at 10, 23 and 37 degrees C. The average generation times for clinical, food and environmental isolates in breast milk were 0.94+/-0.04, 0.75+/-0.04 and 0.84+/-0.04 h at 23 degrees C; and 0.51+/-0.03, 0.33+/-0.03 and 0.42+/-0.03 h at 37 degrees C, respectively. E. sakazakii was able to survive up to 12 days in breast milk with fortifiers at 10 degrees C. However, its average generation times among replicates and isolate sources ranged from 11.97+/-3.82 to 27.08+/-4.54 h in breast milk at 10 degrees C. Interestingly, average generation times in breast milk with fortifiers at 23 degrees C (0.83+/-0.05, 0.93+/-0.06 and 0.96+/-0.06 h) and 37 degrees C (0.41+/-0.04, 0.51+/-0.05 and 0.54+/-0.05 h) were longer than in powdered infant formula and breast milk at the same temperatures, indicating that human breast milk fortifiers may have an inhibitory effect on the growth of E. sakazakii. However, the intrinsically ascribed antimicrobial properties of breast milk do not appear to inhibit the growth of this foodborne pathogen in-vitro.