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.     

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。     

LAMP法检测乳粉中的阪崎肠杆菌

据WHO最新报告显示,乳粉中的阪崎肠杆菌是导致婴幼儿感染和死亡的主要原因之一。FDA于2002年公布了婴幼儿乳粉中阪崎肠杆菌检测的推荐方法,但检测周期长达1周左右,而且操作复杂,难以满足控制该疾病暴发和传播的需要。DNA环介导等恒扩增(loop-mediated isothermal amplification,LAMP)技术是一种特异性、灵敏度高、检测时间短的新型基因扩增技术。本文以阪崎肠杆菌为研究对象,建立了一套应用于婴幼儿乳粉中阪崎肠杆菌快速检测方法。针对阪崎肠杆菌16s RNA基因设计4条引物特异性识别靶基因的六个特殊区域,并在63℃进行恒温扩增。实验结果表明,使用该方法能够在5h之内检测出复原乳样品中101CFU/mL的阪崎肠杆菌。灵敏度可以达到100fg阪崎肠杆菌基因组DNA。应用环介导等温扩增法,克服了传统检测周期长、操作复杂的缺点,不仅能够用于婴幼儿乳粉中阪崎肠杆菌的检测,而且满足了对于婴幼儿乳粉中阪崎肠杆菌快速检测的迫切需要。     

食品中阪崎肠杆菌分析及其检测

阪崎肠杆菌是G^-杆菌,属于肠杆菌属中的一个种,是食源性的条件致病菌。主要危害免疫力低下的婴幼儿,导致脑膜炎、败血症和坏死性小肠结肠炎等疾病。依据FDA提供的“三管”增菌法和DFI改进法,均可以检测出样品中微量的阪崎肠杆菌。该菌具有一定耐热性,广泛分布于食品和周围环境中。通过提高原料质量、改善环境卫生和加大检测力度．可有效降低阪崎肠杆菌的污染．确保婴幼儿的身心健康。     

乳粉中阪崎肠杆菌检测能力验证结果与分析

目的提升实验室检测乳粉中克罗诺杆菌属(阪崎肠杆菌)的速度和准确性。方法依据GB4789.40-2016《食品安全国家标准食品微生物学检验克罗诺杆菌属(阪崎肠杆菌)检验》和经优化的SN/T1632.3-2013《出口奶粉中阪崎肠杆菌(克罗诺杆菌属)检验方法第3部分:荧光PCR方法》以及能力验证作业指导书对乳粉中克罗诺杆菌属(阪崎肠杆菌)进行检测和结果判断。结果通过实时荧光PCR法的快速初筛,初步判断样品CODE29为阴性,样品CODE95为阳性;再经显色平板分离和VITEK2生化鉴定,结果显示:样品CODE29检出肺炎克雷伯杆菌、弗氏柠檬酸杆菌和大肠埃希氏菌;样品CODE95检出阪崎肠杆菌和大肠埃希氏菌。结论本次乳粉样品中克罗诺杆菌属(阪崎肠杆菌)检测能力验证结果合格,为提高相关微生物实验室的检测能力提供参考。     

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。     

奶粉中阪崎肠杆菌的风险评估

阪崎肠杆菌能引起脑膜炎、NEC和菌血症等，约2．5％～14％婴儿配方奶粉含有阪崎肠杆菌，0％～12％的普通奶粉含有阪崎肠杆菌，含量从0．36-66．0cfu／100g。婴幼儿奶粉中的阪崎肠杆菌问题已受到了全世界的普遍关注。本文基于大量研究和调查数据，从奶粉中阪崎肠杆菌的危害识别、奶粉中阪崎肠杆菌的暴露评估、奶粉中阪崎肠杆菌危害特性以及阪崎肠杆菌的检测方法等方面客观地对奶粉中阪崎肠杆菌进行风险评估，并对降低我国婴儿配方奶粉中阪崎肠杆菌风险提出建议。     

阪崎肠杆菌分子检测研究进展

对分子生物学法检测阪崎肠杆菌的研究进展及在分子检测中内部扩增参比（内参）的应用作一综述。     

探究乳粉中阪崎肠杆菌活菌检测方法灵敏度和检出限

摘要：目的 研究出乳粉中阪崎肠杆菌活菌检测的灵敏度和最低检出线的实时荧光RT-PCR方法。方法 将羊奶粉作为基质,根据GB4789.40-2016中第一法中规定的增菌方法,一组加入10cfu阪崎肠杆菌,另一组加入等量的阪崎肠杆菌、大肠杆菌、鼠伤寒沙门氏菌、普通变形杆菌、粪肠球菌,培养时间分别设置为15h和18h,将不同培养时间的培养物分别取1ml进行RNA提取,以cDNA为模板进行扩增。结果 增菌培养18h后,提取RNA采用实时荧光RT-PCR技术,结果显示扩增曲线良好,Ct值稳定。结论 乳粉中阪崎肠杆菌活菌检测,增菌培养18h后,采用实时荧光RT-PCR,最低可检出10cfu的阪崎肠杆菌。     

从进口浓缩营养粉中检出阪崎肠杆菌

为保证供特殊人群食用的营养食品的安全,对进口浓缩营养粉进行监测,从中检出1株阪崎肠杆菌。提示特殊食品的监测十分必要。     

SENSITIVE AND RAPID DETECTION OF ENTEROBACTER SAKAZAKII IN INFANT FORMULA BY LOOP-MEDIATED ISOTHERMAL AMPLIFICATION METHOD

Enterobacter sakazakii is an emergent pathogen associated with ingestion of infant formula milk and was defined in category "A" of the hazard identification by FAO and WHO on microorganisms, which is based on the strength of evidence of a causal association between their presence in powdered infant formula and illness in infants. In this study, a loop-mediated isothermal amplification (LAMP) method has been developed for the detection of E. sakazakii in infant formula. The assay detected the species-specific DNA sequence of the 16S-23S rRNA internal transcribed spacer. The sensitivity of the detection is 1.2 cfu per 100 g infant formula with the selective enrichment. As the amplification is made under isothermal conditions, only a water bath or heating block is needed to maintain the required temperature. Thus, the method will be easily generalized and popularized in the future.

PRACTICAL APPLICATIONS

Enterobacter sakazakii is an emerging opportunistic pathogen associated with meningitis, bacteria and necrotizing enterocolitis in neonates. Powdered infant formulae have been implicated as the source of infection in neonatal meningitis. Thus, in order to minimize the hazard caused by E. sakazakii , it is of utmost importance to develop a rapid, sensitive and simple method for the early detection of this bacterium in infant formula.
In our study, the E. sakazakii was detected sensitively and rapidly in the infant formula by the loop-mediated isothermal amplification (LAMP) assay. LAMP is easy to perform once the appropriate primers are prepared. The LAMP method will be a simple and rapid method for the scene detection of E. sakazakii as only four primers, a Bst polymerase and a regular laboratory bath are needed.     

干燥蒜粉中苯甲酸检测的假阳性分析

以GB/T5009.29-2003液相色谱条件检测干燥蒜粉样品里的苯甲酸时,出现引起误判的干扰性物质,当HPLC方法经过适当的调整(梯度洗脱条件:0min,5%B,流速0.7mL/min;5min,5%B,流速0.7mL/min;7min,7%B,流速1mL/min;12min,5%B,流速1mL/min;15min,5%B,流速1 mL/min;30min,5%B,流速1mL/min,B相为甲醇),可避免这个问题.     

IMPROVED MEDIUM FOR DETECTION OF KLEBSIELLA IN POWDERED MILK

The selectivities to pathogenic Klebsiella strains of different isolation media were compared by known standard strains. The modified MacConkey-inositol-carbenicillin (MCIC) medium (Named MCIAC, MacConkey-inositol-adonitol-carbenicillin) supplemented with adonitol gave no false-negative colonies, and exhibited higher selectivity. MCIC and Simmons citrate agar with inositol (SCAI) media gave two false-negative colonies, respectively. These three media all gave two false-positive colonies, respectively. Salmonella Shigella medium gave four false-negative colonies and five false-positive colonies. Violet red bile glucose agar medium gave the most false-positive colonies, although it gave no false-negative colonies. One hundred samples of powdered milk were examined by MCIAC, MCIC and SCAI plates. The typical positive colonies were further identified using Vitek GNI Auto Microbic system and API 20E system. The results showed that the specificity of the MCIAC medium was higher than MCIC and SCAI media.

PRACTICAL APPLICATIONS

MacConkey-inositol-carbenicillin (MCIC) is the most commonly used selective medium for the detection of Klebsiella . But some inositol-nonfermenting Klebsiella strains would be missed when selected by this medium. We improved the MCIC medium by supplementing with 1% adonitol. The new modified medium (MacConkey-inositol-adonitol-carbenicillin, MCIAC) had advantages over other selective Klebsiella media in having a higher selectivity and an incubation time of only 16–24 h. MCIAC could be routinely used for pathogenic Klebsiella selection of powdered milk and other food samples.     

THE DETECTION OF SOYBEAN FLOUR IN COCOA POWDER

Protein analysis is evaluated for determining the presence and content of soybean flour which is used as an adulterant in cocoa powder. The coefficients of the determination were significantfor estimating the level of adulteration. A protein scale is developed for quantitative measurement of soybean flour in cocoa powder.     

DETECTION AND DISCRIMINATION OF ENTEROBACTER SAKAZAKII (CRONOBACTER SPP.) BY MID-INFRARED SPECTROSCOPY AND MULTIVARIATE STATISTICAL ANALYSES

Enterobacter sakazakii can cause rare but life-threatening diseases such as meningitis in infants and neonates. Fourier transform infrared (FT-IR) spectroscopy was used to detect and discriminate between eight E. sakazakii strains, two Enterobacter cloacae strains, three Escherichia coli strains and two Klebsiella pneumoniae strains. FT-IR vibrational combination bands reflect subtle compositional differences in the cell membranes of E. sakazakii strains, especially in the region between 1,200 and 900 cm ^{− 1} which contains absorption bands from carbohydrates. Two multivariate statistical analyses including principal component analysis (PCA) and soft independent modeling of class analogy (SIMCA) were used for data analysis. E. sakazakii strains were clearly distinguishable from the other strains by PCA. Based upon SIMCA analysis, 90% of E. sakazakii, 88% of E. cloacae, 91% of E. coli and 91% of K. pneumoniae samples were correctly classified, suggesting that this technique could be used to detect E. sakazakii strains rapidly and accurately.

PRACTICAL APPLICATIONS

Fourier transform infrared (FT-IR) coupled with multivariate statistical analyses can be used to detect, discriminate and identify Enterobacter sakazakii strains that have been implicated in food safety incidents caused by contaminated infant formula. Compared with traditional microbiological plating methods, this new approach of using FT-IR could be an alternative means for rapid and accurate detection of bacterial samples that are important in agricultural, food and medical areas.     

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.     

A COMPARISON BETWEEN TWO METHODS FOR THE RECOVERY OF LISTERIA MONOCYTOGENES IN MILK POWDER REFERENCE SAMPLES

Two methods (A and B) for the recovery of Listeria monocytogenes were compared using reference samples based on spray drying of milk containing this pathogen and competitive microflora. For method A, the sample was inoculated in a buffered liquid medium (same as IDF Pre-enrichment Broth) and then passed to IDF Enrichment Broth plus phosphate buffer. For method B, the sample was inoculated in FDA Enrichment Broth (LEB) and then passed to LEB (subenrichment). The solid selective medium used in both cases was Listeria Selective Agar (Oxford formulation). The sensitivity and specificity of method B was 95.2% and 100% vs. 76.1% and 88.8% for method A, respectively.