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.     

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.     

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.     

DETERMINATION AND SIGNIFICANCE OF GENETIC DIVERSITY IN LISTERIA ISOLATED FROM FOOD AND ENVIRONMENT

Listeriosis in humans and animals is caused by the opportunistic bacterium Listeria monocytogenes. The repeated outbreaks in 1980s have increased the interest in the epidemiology of the disease. The organism is widely distributed in the environment, and healthy carriers are found among humans and animals. It has been isolated from a variety of foods including chicken, cheese, milk, sausages, and smoked, fermented and marinated fish products.
The ubiquity of L. monocytogenes and risk to the target populations make study of the epidemiology of the etiological agent important. Phage and serotyping have been employed in conjunction with other methods in modern molecular biology to isolate, identify and type the various strains of L. monocytogenes. Information regarding the genetic diversity among different species of the genus Listeria and among the same species is lacking. This report attempts to address this problem.     

套式聚合酶链反应在HIV-1检测中的应用

根据HIV-1 gag.pol和env基因序列设计了套式聚合酶链反应(nesled polymerasechain reaction,nPCR)引物。将外周血单个核细胞裂解液先用外侧引物扩增,其产物再经内侧引物扩增,直接走凝胶电泳判定结果。nPCR的敏感性。较常规PCR高100～1000倍,可检测到0.5fg质粒DNA和50μ1HIV-1感染者外周血样中的HIV基因。用本法检测63名HIV-1感染者全为阳性,而61名健康人和可疑者均为阴性,后者经追踪检测抗体排除了HIV-1感染。实验证明,nPCR是一种敏感性极高、特异性很强、操作简便易行的HIV-1基因检测技术。它已经在早期诊断和外周血病毒含量检测中发挥了重要作用,而且有着更广阔的应用前景。     

聚合酶链反应快速检测胃组织和唾液中的幽门螺杆菌

针对幽门螺杆菌（ＨＰ）尿素酶Ａ基因设计一对引物进行聚合酶链反应,检测１株ＨＰ标准株和７株临床分离株均阳性,而４株其它肠道菌均阴性,特异性１００％。１０倍系列稀释试验表明敏感性达到１００ｐｇＤＮＡ水平。从３５例胃镜检查者取幽门旁组织块进行快速和常规尿素酶试验,细菌培养及ＰＣＲ检测,１５例ＨＰ阳性者ＰＣＲ检测也为阳性,其中７例阳性者有３例唾液ＰＣＲ检测为阳性,表明ＨＰ确存在于口腔中。本研究采用直接热裂解法处理临床标本,取其粗提物行ＰＣＲ,免除复杂的酚一氟仿抽提步骤,该法简便快速,且损失小,成功率高,在临床实验诊断中有推广价值。     

GENETIC AND ENVIRONMENTAL DETERMINANTS OF FOOD PREFERENCE IN DROSOPHILA TRIPUNCTATA

Mark-release-recapture field experiments involving two isofemale strains of Drosophila tripunctata revealed that strain identity strongly and consistently affected the preferences of both males and females for mushrooms versus tomatoes. Females, but not males, showed an augmented preference for the type of food on which they had been kept prior to release. The behavior of F2 flies from reciprocal crosses between the two strains demonstrated that genetic variation for food preference is autosomal and largely additive. Because mating often occurs in the vicinity of food in the wild, positive assortative mating with respect to genes for food preference may lead to greater phenotypic variance in preference, which could increase the variety of food resources used by a population.     

用捕捉ELISA法检测类脊髓灰质炎患者血清中肠道病毒71型IgM抗体以进行早期诊断

以肠道病毒71型(EV71)感染Vero细胞制备的EV71抗原,用于IgM捕捉ELISA法检测类脊髓灰质炎(类脊灰)患者血清中EV71 IgM抗体,特异性高,敏感性及稳定性良好。154例脊髓灰质炎(脊灰)病毒IgM阴性的可疑脊灰患者血清中EV71 IgM抗体阳性检出率为14.9％(23/154)。病后第2天的标本即可测出EV71 IgM,5至12天者抗体滴度较高,病后第40天的标本尚可测出EV71 IgM。本法是EV71感染的早期快速诊断方法,可用于与脊灰的鉴别诊断。     

SIMULTANEOUS PCR DETECTION OF BACTERIA AND MOLD DNA SEQUENCES IN PHARMACEUTICAL SAMPLES BY USING A GRADIENT THERMOCYCLER

A gradient thermocycler, the Stratagene RoboCycler 96-Gradient, was evaluated for the simultaneous PCR amplification of microbial genes which indicated the presence of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Aspergillus niger in pharmaceutical samples. Suspensions of pharmaceutical products were inoculated with pure cultures of bacteria and mold. After a 24 h incubation, bacterial DNA was extracted from each enrichment broth using a mild lysis in Tris-EDTA-Tween 20 buffer containing proteinase K while mold DNA was extracted by boiling the samples in Tris-EDTA-SDS buffer for 1 h. A 10 μL aliquots of extracted DNA was added to Ready-To-Go PCR beads and specific primers for E. coli, S. aureus, and P. aeruginosa. However, 50 μL aliquots of extracted mold DNA were used for amplification of specific A. niger DNA sequences. The individual samples were loaded into the RoboCycler 96-Gradient thermocycler. Simultaneous detection of all microbial genes was performed by using a gradient profile that allowed the use of DNA primers with different annealing temperatures. Standard methods for quality control evaluation of pharmaceutical products required 6–8 days while simultaneous PCR detection of bacteria and mold DNA sequences was completed within 27 h.