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.     

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.     

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.     

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.     

猪肉中沙门氏菌的PCR检测

选用合适的引物,利用PCR快速扩增反应检测猪肉中的沙门氏菌.根据沙门氏菌的Fimy基因设计一对引物,对不同血清型的沙门氏菌和非沙门氏菌进行PCR检测,沙门氏菌均能检测出特异条带,而非沙门氏菌无一能检测出特异条带.将沙门氏菌和非沙门氏菌混合培养,对混合培养物提取DNA模板进行检测,结果呈阳性,而不含沙门氏菌的混合培养物则没有特异条带;对模板进行梯度稀释后PCR扩增检测,当DNA含量只有0.045 ng/μL时仍能扩增出条带,将菌液进行梯度稀释后提取DNA模板,沙门氏菌含量在102cfu/mL时能被检测出来.     

SENSITIVE AND RAPID DETECTION OF ESCHERICHIA COLI O157:H7 IN GROUND BEEF BY NESTED PCR INCORPORATING IMMUNOMAGNETIC SEPARATION

A protocol for detection of low numbers of E. coli O157:H7 in ground beef by nested PCR incorporating immunomagnetic separation (IMS) was developed. The protocol enabled detection of 24 colony-forming-units (CFU) in 10 g of seeded ground beef without enrichment cultivation. Differential centrifugation was used for maximally recovering the target CFU. Partial digestion of the resulting cell pellet with proteinase K at 37°C was used for the removal of beef tissue, which was required for the proper function of IMS. Within the range of 24 to 2400 CFU/10 g, a log linear relationship between the numbers of inoculated CFU and the integrated intensity of the nested PCR products was obtained with both shiga-like toxin (SLT) 1 and 2 primer pairs.     

TLC薄层色谱法同步检测功能红曲中酸型和内酯型莫那呵啉K(MonacolinK)

功能红曲产品中的Monacolin—K包括酸型和内酯型两种成分。采用TLC法，以60％乙腈溶液作为提取剂，V（氯仿）：V（甲醇）：V（氨水）=25：3：1作为展开剂，以10％硫酸-乙醇溶液显色，紫外检测波长为365nm，可同步检测酸型和内酯型莫奈呵啉K。该法适用于任何含莫奈呵啉K的样品的检测，特别适用于大批量经常性的定性或半定量检测莫奈呵啉K。     

RECENT ADVANCES IN THE RAPID DETECTION OF BREWERY MICRO-ORGANISMS AND DEVELOPMENT OF A MICROCOLONY METHOD

Recent developments in the rapid detection of low concentrations of micro-organisms are reviewed. A modification of the microcolony method, involving uptake of optical brighteners by developing yeast colonies, is described. The method includes the use of incident light microscopy and appears to offer advantages over other detection methods.     

THE DETECTION OF ENTEROBACTER AGGLOMERANS (ERWINIA HERBICOLA) IN BREWERY WORTS BY GAS CHROMATOGRAPHY

A gas chromatographic procedure is described to detect Enterobacter agglomerans, a bacterial contaminant in beer breweries. Ethanol, acetic acid, acetoin and 2,3-butanediol produced by bacteria in a growth medium was used to detect the contaminant at levels as low as three organisms/ml. A correlation was found between the initial numbers of contaminants present and the amounts of volatiles produced.     

RAPID DETECTION OF A SMALL NUMBER OF VIABLE CELLS BY THE FLUORESCENT GLUCOSE METHOD

A fluorescent glucose, 2NBDG, was rapidly consumed by 4 different species of microorganisms: Escherichia coli, Lactobacillus acidophilus, Saccharomyces cerevisiae, and Candida tropicalis. E. coli and L. acidophilus became fluorescent enough for microscopic observation within 1 min, while S. cerevisiae and C. tropicalis became fluorescent in a longer time span. All of the 14 coliforms that were isolated from various foods consumed 2NBDG and became fluorescent within 1 min. Therefore, 2NBDG is expected to be a useful indicator of viable cells irrespective of species as long as they could assimilate glucose. This 2NBDG method is valid in counting small amount of cells, such as 10–100 cells, accurately and carefully. Also, the 2NBDG method was successfully applied to the rapid detection of a small number of E. coli in milk. The feasibility of the 2NBDG method is discussed from the viewpoint of food safety control.