Schnellnachweis von Listeria monocytogenes mittels real-time PCR

Molecular biological methods like the real-time PCR are well suited for a safe and rapid detection of Listeria and especially of Listeria monocytogenes. The use of commercially available kits in combination with adapted sample preparation is advantageous for the routine testing in the food industry. In addition to its high specificity and sensitivity, its superior reproducibility and short time to result, the described method is easy to use. Depending on the food matrix, the result is available after 24 h to 48 h. The procedure consists of a short enrichment, a sample preparation to extract the DNA from the cells and the PCR. The data interpretation is carried out automatically and can be used directly for documentation purposes. Eingegangen: 31. Januar 2007; Angenommen: 8. Februar 2007     

Control of Listeria monocytogenes growth in a ready-to-eat poultry product using a bacteriophage

A bacteriophage (phage) that infected strains of the species Listeria monocytogenes as well as Listeria ivanovii and Listeria welshimeri, but not Listeria grayi or Listeria innocua, was isolated from sheep faeces. The phage had a contractile tail and an icosohedral head indicating that it was a myovirus, and was morphologically similar to phage A511. At 30 °C, phages added at 5.2 × 10⁷ PFU ml⁻¹ prevented the growth in broth of L. monocytogenes present at approximately twice this concentration for 7 h, but re-growth occurred such that the concentration after 24 h incubation was similar in both control and phage-treated cultures. At the same temperature, but on the surface of vacuum-packed ready-to-eat chicken breast roll, there was an immediate 2.5 log₁₀ CFU cm⁻² reduction in pathogen concentration following addition of phages and then re-growth. However, at a temperature reflecting that at which a chilled food might be held (5°C), this re-growth was prevented over 21 days incubation. The data suggest a dose-dependent rapid reduction in pathogen concentration followed by no continued phage-mediated effect. These results, alongside other published data, indicate that a high concentration of phages per unit area is required to ensure significant inactivation of target pathogens on food surfaces.     

用PCR技术快速检测食品中的单核细胞增生性李斯特菌

建立了食品中单核细胞增生性李斯特菌快速、敏感、特异的聚合酶链反应PCR检测方法.选择的引物具有良好的单增李氏菌种的特异性.对人工污染在冷冻食品中单增李氏菌的检测低限是4-8CFU/10g,对其增菌液的检测低限是7.2-11×103/ml,每PCR反应体系的检测低限为86-132CFU.对400份自然污染样品的检测结果显示,PCR方法的检测结果同常规培养法的结果完全相符.     

应用PCR技术快速测定食品中单核细胞增生李斯特菌毒力

目的：采用PCR 技术准确、快速测定单核细胞增生李斯特菌(单增李斯特氏菌)分离株的毒力基因,鉴别强毒株和弱毒株或无毒株,以有效地限制李斯特氏菌病的传播。方法：以单增李斯特氏菌相关毒力基因(hly、plcB、inlA、inlB、inlC、inlJ、prfA)设计引物,检测重庆市2007 - 2009 年分离的40 株单增李斯特氏菌分离株中的毒力基因携带率,并进行小鼠毒力实验。结果：40 株分离株中有15 株7 种毒力基因检测结果均为阳性,6 株hly 基因阴性,4 株plcB 基因阴性,4 株prfA 基因性,5 株inlA、inlB 基因阴性,11 株inlC 基因阴性,9 株inlJ基因阴性,1 株inlA、inlB、inlC、inlJ 基因均为阴性。4 株分离株的毒力与标准菌株ATCC191161E 相当,小鼠LD50 在1.2 × 108～6.0 × 108CFU/mL 之间,为强毒株。筛选出弱毒株09-132,LD50 为1.7 × 1011CFU/mL。结论：重庆市存在发生李斯特菌食物中毒的潜在危险,内化素基因(inlA、inlB、inlC、inlJ)的缺失可能是导致菌株毒力降低的原因,而prfA 和plcB 基因与菌株毒力的相关性较小。     

Reliability of Listeria monocytogenes Identification by Specific PCR Assessed by Phenotypic and Genotypic Techniques

This study evaluates the possibility of using polymerase chain reaction (PCR) for rapid identification of food-borne Listeria monocytogenes as an alternative to API Listeria system and estimates the incidence of API Listeria misidentifications in food-borne Listeria species. A total of 198 strains, 11 L. monocytogenes, 28 other Listeria species, and 159 food isolates were phenotypically and genotypically characterized by API Listeria profiles and randomly amplified polymorphic DNA (RAPD) profiles, respectively. They were also tested for PCR amplification using genus- and species-specific primers. Clustering analysis of phenotypic and genotypic data showed discrepancies in species identification of some isolates by API Listeria profiles. Their identities were confirmed by 16S rDNA sequencing, and thus, it was revealed that 33% of Listeria innocua and 19% of Listeria welshimeri were misidentified as L. monocytogenes by API Listeria profiles. Reliable identification of L. monocytogenes was obtained by LM1–LM2 specific primers which allowed PCR amplification only in reference strains and isolates previously identified as L. monocytogenes by RAPD and 16S rDNA sequence analysis. These results corroborate the suitability of specific PCR as a rapid and accurate test for the identification of L. monocytogenes, avoiding misidentification with other Listeria species commonly found in food products.     

食品中单核细胞增生性李斯特氏菌PCR快速检测

通过扩增hly基因建立检测单核细胞增生性李斯特氏菌(Lm)的PCR方法.该方法具有较强的特异性,35株经传统方法鉴定的菌株PCR结果均为阳性,而其他三种同属异种菌,包括英诺克李斯特氏菌、绵羊李斯特氏菌和威尔斯李斯特氏菌及非李斯特氏菌均未扩增出特异性的片段.PCR方法对上Lm纯培养物的最低检测限为7.3CFU/μl,对模拟污染的生猪肉和蔬菜的检测低限为4CFU/g,牛奶为4CFU/ml.应用该方法对285份食品样品检测,17份样品Lm呈阳性,结果与常规的分离培养方法完全一致.该种方法具有敏感、特异、快速及准确的优点,可用于食品中Lm的快速检测.     

Prevalence and level of Listeria monocytogenes and other Listeria species in retail pre-packaged mixed vegetable salads in the UK

As part of the European Commission (EC) co-ordinated programme for 2005, a study of pre-packaged ready-to-eat (RTE) mixed salads containing meat or seafood ingredients from retail premises was undertaken in the UK to determine the frequency and level of Listeria monocytogenes in these products. Almost all (99.8%; 2682/2686) samples were of satisfactory/acceptable microbiological quality. Two (0.1%) samples exceeded EC legal food safety criteria due to the presence of L. monocytogenes in excess of 100 cfu g(-1) (1.7 x 10(2), 9.9 x 10(2)cfu g(-1)) while another two (0.1%) were unsatisfactory due to L. welshimeri levels over 100 cfu g(-1) (1.2 x 10(3), 6.0 x 10(3) cfu g(-1)). Overall contamination of Listeria spp. and L. monocytogenes found in samples of mixed salads in the UK was 10.8% and 4.8%, respectively. Almost twice as many salad samples with meat ingredients were contaminated with Listeria spp. and L. monocytogenes (14.7% and 6.0%, respectively) compared to samples with seafood ingredients (7.4% and 3.8%, respectively). Pre-packaged mixed salads were contaminated with Listeria spp. and L. monocytogenes more frequently when: collected from sandwich shops; not packaged on the premises; stored or displayed above 8 degrees C. This study demonstrates that the control of L. monocytogenes in food manufacturing and at retail sale is essential in order to minimize the potential for this bacterium to be present in mixed salads at the point of consumption at levels hazardous to health.     

Genetic variation of Listeria monocytogenes isolates from domestic and imported foods in Japan

Phylogenetic analyses were carried out on a total of 118 Listeria monocytogenes isolates from foods or food processing environments, and 7 isolates from listeriosis patients in Japan to evaluate the genetic variation in the pathogen in this country. Isolates of serotypes 1/2a, 1/2b and 4b were mainly examined to assess the risk of exposure of humans to L. monocytogenes from foods in Japan. The nucleotide sequences of the part of the iap gene that contains the region encoding the threonine-asparagine repeat units were determined in order to construct phylogenetic trees of the isolates investigated. A phylogram showed high genetic diversity among lineage 2 isolates, while the lineage 1 isolates showed clonal characteristics. The results of the genetic analyses suggested the presence of rare putative lineage 3 isolates and epidemic clone I (ECI) isolates in foods in Japan. The results showed that ECI was also isolated from listeriosis patients. The genetic variation in L. monocytogenes in Japan reported here suggests the necessity of monitoring the pathogen in foods and environments in addition to surveillance of listeriosis patients.     

免疫磁珠捕获PCR快速检测单核细胞增生李斯特氏菌

利用免疫磁珠富集单核细胞增生李斯特氏菌,采用聚合酶链式反应（polymerase chain reaction,PCR）扩增进行快速检测。所制备的免疫磁珠选取在37 ℃条件下均匀振荡1 h为最佳包被条件,1 mg磁珠偶联抗体的最佳量为100 μg/mg,制备的免疫磁珠捕获率为45%。所建立的免疫磁珠捕获-PCR技术在样品细菌浓度达到104 CFU/mL即可被检出,其灵敏度是直接PCR检测限（105 CFU/mL）的10 倍,可为病原菌的富集和快速检测提供新方法。     

Review--Persistence of Listeria monocytogenes in food industry equipment and premises

To understand why Listeria monocytogenes may persist in food industry equipment and premises, notably at low temperature, scientific studies have so far focused on adhesion potential, biofilm forming ability, resistance to desiccation, acid and heat, tolerance to increased sublethal concentration of disinfectants or resistance to lethal concentrations. Evidence from studies in processing plants shows that the factors associated with the presence of L. monocytogenes are those that favor growth. Interestingly, most conditions promoting bacterial growth were shown, in laboratory assays, to decrease adhesion of L. monocytogenes cells. Good growth conditions can be found in so-called harborage sites, i.e. shelters due to unhygienic design of equipment and premises or unhygienic or damaged materials. These sites are hard to eliminate. A conceptual model of persistence/no persistence based on the relative weight of growth vs. outcome of cleaning and disinfection is suggested. It shows that a minimum initial bacterial load is necessary for bacteria to persist in a harborage site and that when a low initial bacterial charge is applied, early cleaning and disinfection is the only way to avoid persistence. We conclude by proposing that there are no strains of L. monocytogenes with unique properties that lead to persistence, but harborage sites in food industry premises and equipment where L. monocytogenes can persist.     

Characterization of the novel Listeria monocytogenes PCR serogrouping profile IVb-v1

The World Health Organization Collaborating Centre for Listeria (WHOCCL) has developed in 2004 a multiplex PCR assay that separates the 4 major Listeria monocytogenes serovars (1/2a, 1/2b, 1/2c, and 4b) into distinct PCR serogroups. A new PCR profile has been recently identified, constituted of amplified DNA fragments of prs, ORF2819, ORF2110 and lmo0737. Here we characterize 22 L. monocytogenes isolates of the WHOCCL collection with this PCR IVb variant 1 (IVb-v1) profile. The 22 isolates belong to the clinically predominant serovar 4b, exhibit 6 distinct pulsed-field gel electrophoresis ApaI/AscI combined profiles, and belong to 2 unrelated multilocus sequence types, indicating that the novel profile does not correspond to a recent clonal emergence. We have updated the WHOCCL serogroup-related PCR typing scheme to include this new profile.     

Rapid real-time PCR detection of Listeria monocytogenes in enriched food samples based on the ssrA gene, a novel diagnostic target

A real-time PCR assay was designed to detect a 162-bp fragment of the ssrA gene in Listeria monocytogenes. The specificity of the assay for L. monocytogenes was confirmed against a panel of 6 Listeria species and 26 other bacterial species. A detection limit of 1-10 genome equivalents was determined for the assay. Application of the assay in natural and artificially contaminated culture enriched foods, including soft cheese, meat, milk, vegetables and fish, enabled detection of 1-5 CFU L. monocytogenes per 25g/ml of food sample in 30h. The performance of the assay was compared with the Roche Diagnostics 'LightCycler foodproof Listeria monocytogenes Detection Kit'. Both methods detected L. monocytogenes in all artificially contaminated retail samples (n=27) and L. monocytogenes was not detected by either system in 27 natural retail food samples. The method developed in this study has the potential to enable the specific detection of L. monocytogenes in a variety of food types in a time-frame considerably faster than current standard methods. The potential of the ssrA gene as a nucleic acid diagnostic (NAD) target has been demonstrated in L. monocytogenes. We are currently developing NAD tests based on the ssrA gene for a range of common foodborne and clinically relevant bacterial pathogens.     

实时荧光PCR定量检测食品中单增李斯特菌

目的建立快速、敏感、特异的食品中单增李斯特菌检测方法。方法针对单增李斯特菌溶素A基因（hlyA）设计一对引物和一条探针，并用该引物和探针运用实时荧光PCR技术对单增李斯特菌的DNA、细胞、质粒和样品进行实时荧光PCR定量检测。结果利用实时荧光PCR技术，建立了DNA校正曲线、细胞校正曲线和质粒校正曲线。DNA校正曲线在1～32CFU/ml、细胞校正曲线在32—320CFU／ml、质粒校正曲线在1—37Copies／ml，线形关系良好，且三种校正曲线检测样品得出的结果基本吻合。结论本试验建立起来的实时荧光PCR定量检测单增李斯特菌的方法灵敏度高、特异性好、准确，可应用于食品中单增李斯特菌的检测。     

荧光假单胞菌、沙门氏菌和单增李斯特菌多重PCR检测方法的建立

针对肉制品中易污染的荧光假单胞菌、沙门氏菌和单增李斯特菌等有害微生物,通过3 种标准菌株及肉制品建立多重聚合酶链式反应方法,实现肉制品中这3 种菌的同时、快速检测。利用荧光假单胞菌的gyrB基因、沙门氏菌的invA基因和单增李斯特菌的hlyA基因设计3 对特异性引物,在确定引物特异性的基础上,对3 种标准菌株及在冷却肉上过夜富集后进行灵敏度检测。结果表明,该多重聚合酶链式反应方法对于同时检测这3 种有害微生物具有高度的特异性;同时检测这3 种菌时,纯菌DNA检测限可达1 pg/μL;将3 种菌一起接种到冷却肉中35 ℃过夜培养后,荧光假单胞菌、沙门氏菌和单增李斯特菌的检测限分别可达到9、5、70 CFU/mL。     

Development of a rapid, simple paddle-style dipstick dye immunoassay specific for Listeria monocytogenes

We have developed two types of new paddle-style dipstick dye immunoassays. The first is genus Listeria specific and the second is specific to Listeria monocytogenes. They are based respectively, on antisera raised against heat-killed L. monocytogenes cells and against internalin B crude extract, a virulence protein found only in the pathogenic L. monocytogenes. The minimum detectable level for L. monocytogenes is 2×10⁷ CFU ml⁻¹ for strain number 88/049 in pure culture. Detection is unaffected by the presence of high numbers (approximately log 8.0 CFU/ml) of the other microorganisms tested. When the dipsticks were applied to milk samples inoculated with L. monocytogenes reference material (ALM92), there was a strong response to the enrichment cultures. The new assay may prove useful in detection of L. monocytogenes in enrichment cultures of milk and ice cream food samples.     

PCR Detection of Listeria monocytogenes in different Food Products compared with the mini-VIDAS LMO System and the Standard Procedure ISO 11290-1

The presence of Listeria monocytogenes in 225 natural samples, including different food types, was investigated by three methods: (i) culture-based standard procedure ISO 11290-1, (ii) mini-VIDAS (Vitek Immuno Diagnostic Assay System) LMO, an enzyme linked fluorescent assay (ELFA) commercially available, and (iii) PCR using a previously established procedure. Identification of isolates recovered from the standard method and mini- VIDAS, on Oxford and PALCAM selective plates, was carried out using the API-Lis system and also by PCR, using L. monocytogenes specific primers. In all, 65 samples (64 meat products and one smoked salmon) were positive with any of the three procedures assayed. Taking into account the results based on PCR identification, the standard culture-based method found 22 and the mini- VIDAS 23, while PCR detected 60 positive samples in a total of 225 food samples. For comparative purposes, a set of “known positive samples” was established as reference that included 33 natural samples from which L. monocytogenes isolates (identified by specific PCR) had been recovered. The mini-VIDAS and ISO 11290- 1 methods were equally sensitive. Compared to them PCR was clearly the more accurate and efficient procedure for detection of L. monocytogenes in food. Moreover, it showed no false negative results. The PCR approach can be completed in 48 working hours, and because of its specificity might eventually be cheaper than the other two procedures. Consequently we recommend PCR for routine detection of L. monocytogenes in food.

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Zusammenfassung (Redaktion). 225 Lebensmittelproben wurden auf das Vorhandensein von Listeria monocytogenes mit Hilfe von drei verschiedenen Methoden untersucht: (i) durch die auf Kultivierung basierende Standard-Methode nach ISO 11290-1, (ii) durch mini-VIDAS (Vitek Immuno Diagnostic Assay System) LMO, einem Enzym-basierten Verfahren mittels Fluoreszenz-Nachweis (ELFA; im Handel erh?ltlich) und (iii) durch ein PCR-Verfahren, das jüngst von den Autoren etabliert worden ist. Die Identifikation von Isolaten durch Einsatz der Standard- oder der mini-VIDAS- Methode wurde nachvollzogen durch Verwendung des API-Lis-Systems und auch durch die PCR unter Verwendung von L. monocytogenes-spezifischen Primern. Insgesamt wurden 65 Lebensmittelproben durch jede der drei Methoden als positiv identifiziert. Durch die Standard-Methode wurden 22, durch die mini-VIDAS 23 und durch die PCR 60 positive Proben unter den 225 Lebensmittelproben identifiziert. Zu Vergleichszwecken wurden Referenzproben (belastet mit L. monocytogenes) durch PCR positiv identifiziert. Das mini-VIDAS- und das Verfahren nach ISO 11290-1 waren von gleicher Sensitivit?t; aber im Vergleich zu ihnen war die PCR deutlich exakter und effizienter, um L. monocytogenes in Lebensmitteln nachzuweisen. Das Ergebnis des Nachweises mittels PCR kann nach 48 Arbeitsstunden vorliegen. Daher empfehlen die Autoren ihre PCR als Routine-Verfahren für den Nachweis von L. monocytogenes in Lebensmitteln.

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数字PCR定量检测食品中单核细胞增生李斯特氏菌方法的研究

目的建立微滴式数字PCR技术(droplet digital PCR,ddPCR)定量检测食品中单核细胞增生李斯特氏菌的分析方法。方法选择基因hly A为靶序列,设计PCR扩增引物和Taq Man探针,优化反应条件和反应体系,通过细菌分离和dd PCR方法对靶标基因的检测特异性、灵敏度和重复性进行实验,并对定量结果进行分析。结果 ddPCR反应中的最佳探针浓度为5 pmol/μL,特异性好,检出限为(3.6±0.1)copies/20μL,重复性实验良好,标准偏差为0.067%。ddPCR的拷贝数(copy number)与细菌密度(colony forming units,CFU/mL)形成了较好的线性关系。结论本研究建立dd PCR的拷贝数和菌液密度或菌落数的线性关系,可以为单核细胞增生李斯特氏菌的快速定量检测提供参考。     

EMA结合实时荧光PCR方法检测单核细胞增生李斯特氏菌

建立叠氮溴化乙锭（EMA）结合实时荧光PCR（qPCR）方法检测单核细胞增生李斯特氏菌（Listeria monocytogenes）活菌。以李斯特溶血素O（LLO）基因hly设计引物、TaqMan探针,用李斯特属典型菌、沙门氏菌等56株致病菌株验证特异性,不同质量浓度EMA处理进行qPCR检测。尝试脱氧胆酸钠溶液（SD）强化抑制效果,73份不同的人工污染食品、环境样本（卤鸡肉、牛奶、肉馅、垃圾渗滤液）测试实用性。结果表明,引物探针准确检测L. monocytogenes,对其他菌株无特异性扩增。EMA最适质量浓度2.5 μg/mL,经过15 min光激活与死菌DNA共价结合明显抑制了扩增,方法检出限为150 CFU/mL。SD处理L. monocytogenes活菌Ct值增加。与传统培养法比较,EMA-qPCR方法检测100 %准确,操作简单、省时高效,在食品、环境方面应用前景广阔。     

Improved Sample Preparation for Real-Time PCR Detection of Listeria monocytogenes in Hot-Smoked Salmon using Filtering and Immunomagnetic Separation Techniques

This work evaluated the application of filtration and immunomagnetic separation (IMS) as sample pretreatments for use in combination with real-time polymerase chain reaction (PCR) to detect and quantify Listeria monocytogenes in hot-smoked salmon. Salmon was artificially inoculated with L. monocytogenes at levels ranging from 8 × 10⁰ to 8 × 10⁵ cfu/g of sample, and homogenates obtained from these samples were filtered to recover bacterial cells without a pre-enrichment step. High recovery of bacterial cells was achieved using standard coffee filters. IMS significantly reduced the co-extraction of PCR inhibitors present in the samples to increase the assay sensitivity with regression line parameters applicable for quantification. The limit of detection and quantification were equal to 2 × 10¹–4 × 10¹ and 2 × 10² cfu/g of sample, respectively. The entire detection procedure could be completed within 3.5 h. This study demonstrated that coupling filtration and IMS with real-time PCR has contributed to improve the sensitivity of L. monocytogenes detection from hot-smoked salmon.