DETECTION OF LISTERIA MONOCYTOGENES BY ADSORPTION AND ELUTION FROM HYDROPHOBIC MATRICES

The binding of L. monocytogenes Scott A strain to three hydrophobic matrices, octyl, phenyl and butyl Sepharose, was investigated. Optimal adsorption of L. monocytogenes to octyl Sepharose was obtained at pH 3.5 and 4 M NaCl. However, it was difficult to elute the bacteria from octyl Sepharose, even after changing the pH and lowering the salt concentration. Good adsorption of L. monocytogenes to phenyl Sepharose at pH 3.5 and 4 M NaCl was also observed. L. monocytogenes was found to adsorb weakly to butyl Sepharose, which is less hydrophobic than phenyl Sepharose. Bacteria were eluted under various conditions. The best elution was obtained with 10 mM sodium phosphate, followed by an increasing gradient of ethylene glycol. To test the potential application of hydrophobic chromatography for separating L. monocytogenes from food matrices, milk was inoculated with L. monocytogenes and then passed through a column of phenyl Sepharose at pH 3.5 and 4 M NaCl. Nearly all L. monocytogenes were bound to the hydrophobic gel and were eluted in a pure and viable form by changing the pH and lowering the salt concentration, and by using a polar reducing agent, ethylene glycol. This study shows that hydrophobic interaction chromatography can be used to separate L. monocytogenes from milk and may be applicable to other food suspensions. It is a gentle method that makes use of the hydrophobic surface properties of Listeria for attachment to hydrophobic gels, as well as using mild elution conditions to avoid inactivation of the organism.     

A COLLABORATIVE STUDY FOR DIRECT ENUMERATION OF LOW LEVELS OF LISTERIA MONOCYTOGENES FROM VARIOUS FOODS

A direct plating method for the enumeration of low levels of foodborne Listeria monocytogenes was evaluated in a collaborative study involving 18 laboratories across Canada. Shrimp, coleslaw, ice cream and wieners were inoculated with low levels (5 × 10² and 10³/g) of L. monocytogenes and shipped to participants. Foods were diluted and then plated onto either lithium chloride phenylethyl and moxa-lactam agar (LPM), Oxford agar (OXA), modified Oxford agar (MOX) or Palcam agar (PAL). Recovery was good for all foods, except coleslaw. Of the four plating media tested, all were more or less equivalent in their ability to recover colonies for enumeration, except that more colonies were enumerated on LPM than on PAL agar. Recovery of L. monocytogenes ranged from <50 to 1250 cfu/g for wieners, <50 to 800 cfu/g for shrimp, <100 to 1440 cfu/g for ice cream and <50 to 700 cfu/g for coleslaw. Results indicate that the direct plating method can be used for the recovery of low levels of Listeria monocytogenes in Category 3 foods, as presently suggested for use in the Canadian Listeria compliance guide.     

RAPID DETECTION OF LISTERIA MONOCYTOGENES IN GROUND TURKEY BY IMMUNOMAGNETIC SEPARATION AND POLYMERASE CHAIN REACTION

The aim of this study was to determine the prevalence of Listeria monocytogenes in packaged fresh ground turkey in Turkey using immunomagnetic separation (IMS) as a selective enrichment step in method and polymerase chain reaction (PCR). A total of 180 ground turkey samples were collected during a 1-year period. Thirty-two (17.7%) of the samples contained L. monocytogenes, 24 (13.3%) contained Listeria innocua, 7 (3.8%) had Listeria ivanovii and 5 (2.7%) had Listeria seeligeri by means of IMS-based cultivation method. A PCR assay was performed, based on hlyA gene-specific primers. In all L. monocytogenes isolates, hlyA gene was confirmed, indicating that the correlation between IMS-based cultivation and PCR methods was 100%. The results suggest that the prevalence of L. monocytogenes in ground turkey is relatively high in Turkey and that ground turkey should be produced under appropriate hygienic and technological conditions for the prevention of public health hazards.

PRACTICAL APPLICATIONS

Using fast and reliable methods to detect and identify foodborne pathogenic bacteria, including Listeria monocytogenes , is important to detect the risk of contaminated product and protect public health. In some ways it is time-consuming to isolate and identify the pathogenic microorganisms from food products using conventional techniques. Different methods or techniques can be used both for redounding the isolation chance and to gain time for this purpose. Immunomagnetic separation (IMS) and polymerase chain reaction (PCR) techniques are effective and rapid methods for separation, detection and confirmation of Listeria spp. from foods. In this study rapid, specific and sensitive IMS method was used to determine the prevalence of L. monocytogenes in fresh ground turkey and PCR technique was used for the verification of the L. monocytogenes isolates.     

ENUMERATING LISTERIA MONOCYTOGENES IN FOOD: A PROBLEM OF LOW NUMBERS

Listeria monocytogenes is recognized as a serious foodborne pathogen in humans. However, an adequate enumeration method is still lacking for the examination of food products which are usually contaminated at low levels, < 100 CFU g⁻¹. The improvement of Listeria enumeration has given place to a considerable sum of research tasks, leading to the proposal of several alternative methods. Several attempts to enumerate L. monocytogenes with the most probable number technique or with methods based on molecular biology or bacteria concentration techniques have been reported. The objective of this paper is to synthesize the current knowledge concerning L. monocytogenes enumeration, by focusing particularly on the problem of the enumeration of low numbers.     

直接杂交法检测基因组的单拷贝基因

本文叙述一种用于基因组限制性图谱研究的Southern印迹术的改良方法。正常和地中海贫血DNA经限制性内切核酸酶完全消化及凝胶电泳分离后,进行干印,变性与中和处理,再和相应的珠蛋白基因探针杂交。修改过的凝胶杂交法具有简便、经济和省时等优点,可对单拷贝基因进行灵敏、可靠的检测,因此能用于遗传病的产前诊断和DNA多态性的分析。作者还就此法与现存的探针制备系统合用的可行性作了扼要讨论。     

COMPARISON OF THREE POLYMERASE CHAIN REACTION-BASED METHODS FOR THE DETECTION OF LISTERIA MONOCYTOGENES IN FOOD

Three PCR-based methods for the detection of Listeria monocytogenes in food (BAX for Screening, Probelia and a method according to Kaclíková et al. (2003) were compared on the basis of the determination of detection limits for 15 artificially contaminated food products. Detection limits of all methods for all samples were 10⁰ cfu per 10 g, with the exception of three cheese samples which did not produce valid results because of the inhibition of Probelia PCR. Detection limits for nonviable L. monocytogenes cells were sufficiently high ( 10⁹ cfu per 10 g) for BAX and the method according to Kaclíková et al. (2003), but considerably low ( 10⁶ cfu per 10 g) for Probelia. The results demonstrate that BAX for Screening as well as the Kaclíková et al. (2001) method fulfill the sensitivity requirements for a rapid alternative method for the detection of L. monocytogenes in food, which would be equivalent to the standard method EN ISO 11290–1.     

INFLUENCE OF TEMPERATURE ON THE FATTY ACID PROFILE OF LISTERIA MONOCYTOGENES

Variation in the fatty acid profile of two Listeria monocytogenes strains grown at varying temperatures was determined. The fatty acid profiles varied greatly at different temperatures. General decreases in relative percentages of branched and medium chain (up to C16:0) fatty acids and variable changes in long chain fatty acids were found with increasing growth temperature. Individual fatty acid percentages between strains were variable. The relative percentages of unknown long chain fatty acids, detected in both strains at various temperatures, were greatest in Scott A (7.07%) and ATCC 19114 (13.15%) at 35C. Results demonstrated that L. monocytogenes had altered fatty acid profile in response to changes in growth temperature.     

快速检测猪肉中单核细胞增多症李氏菌PCR试剂盒的研制

快速检测猪肉中单核细胞增多症李氏菌的ＰＣＲ试剂盒与分离培养法平行检测１５６份猪肉样品,结果ＰＣＲ的阳性率为５．８％,分离培养法的阳性率为３．８％,两者符合率为９７％。实验证明,该试剂盒具有良好的特异性和可重复性,可测出样品中至少３２ＯＣＦＵ细菌,并在２ｄ内取得结果,具有推广应用价值。     

THE CELL CYCLE IN TUMOURS: AN EXAMINATION OF DATA GAINED BY THE TECHNIQUE OF LABELLED MITOSES

Published data from a large series of investigations by the technique of labelled mitoses have been analysed using a process of computer simulation. The results of the analyses of tumour data are discussed, first from the point of view of the quality of simulation that can be achieved and secondly as a review of the extent of our knowledge of the intermitotic time of cells in tumours. Particularly in primary tumors the data are often consistent with a broad distribution of intermitotic times within a tumour and the use of the term 'cell cycle time'may be misleading.