环介导等温扩增技术快速检测肉中单增李斯特菌

通过建立的环介导恒温扩增（Loop-Mediated Isothermal Amplification,LAMP）方法以达到肉中单增李斯特菌快速、灵敏的检出。以特异性的hlyA毒力基因作为靶基因,与6株非单增李斯特菌进行特异性试验,同时对不同培养浓度的单增李斯特菌进行了LAMP和PCR方法的灵敏度比较,进而应用LAMP法检测人工污染肉中的单增李斯特菌。结果表明：纯培养物中单增李斯特菌LAMP检出限为8.8×100CFU/mL,其灵敏度比普通PCR高100倍;在人工污染肉中单增李斯特菌的检出限为8.8×101CFU/mL,在1h内即可完成扩增反应。LAMP方法具备快速、特异、简单、灵敏度高等优势,在食品基质中单增李斯特菌的检测方面具有较好的应用前景。     

免疫磁珠富集技术联合选择性培养基快速检测单增李斯特菌

单增李斯特菌是一种危害极大的食源性致病菌,建立快速及特异的检测方法对于食品安全监控尤为重要。文中联合免疫磁珠与选择性培养基对不同浓度(101~105CFU/mL)单增李斯特菌进行检测,并对3种李斯特菌、金黄色葡萄球菌及副溶血弧菌进行交叉试验;同时模拟食物污染,探索免疫磁珠-平板法检测样品的检测限以及该方法的最快检测时间。结果显示特异性免疫磁珠联合选择性平板法可检出浓度为103CFU/mL及以上的单增李斯特菌;牛奶样品仅需6 h增菌能被检出,检测限为0.7 CFU/mL。联合使用免疫磁珠富集技术与选择性培养基,能在30 h内完成对牛奶样品的检测,较国标法减少38 h以上,且具有同等的灵敏度。     

食品中单核增生李斯特氏菌检测研究进展

单核增生李斯特氏菌和李斯特菌病的危害近年来引起世界各国食品和卫生部门的广泛关注.关于如何找到一种快速、敏感、准确、合理的检验方法,是当今各国食品卫生部门亟待解决的重要研究课题.对该菌的传统分离方法、免疫学检测方法、核酸检测等方法的最新进展进行了综述,为进行该菌的准确、快速检测奠定了基础.     

PCR技术检测单核细胞增生李斯特氏菌研究进展

单核细胞增生李斯特氏菌(Listeria monocytogenes)是危害公共卫生和食品行业的一种重要人畜共患病致病菌.PCR技术是近年来被广泛地运用到食源性致病菌快速检测的分子生物学方法之一.就PCR技术检测单核细胞增生李斯特氏菌中的特异性鉴别基因、模板DNA制备等关键因素及多重PCR、巢式PCR、反转录PCR与定量PCR等主要技术进行了综述.     

原核表达MurA蛋白并制备其多克隆抗体用于免疫磁珠快速检测单增李斯特菌

【目的】制备MurA多抗,结合免疫磁珠与选择平板进行单增李斯特菌的快速检测,建立单增李斯特菌的免疫磁珠快速检测方法。【方法】构建MurA的原核表达载体,转化大肠杆菌进行优化表达。镍柱纯化表达产物,质谱鉴定重组蛋白,再免疫小鼠,制备其多克隆抗体。用所获多抗制备免疫磁珠,建立单增李斯特菌免疫磁珠-选择性培养基检测方法,并对人工污染牛奶样品进行检测。【结果】在大肠杆菌中高效表达了分子量约为72 kD的可溶性融合蛋白,质谱鉴定其为MurA蛋白;免疫小鼠获得的抗血清效价达1:10 000,与伤寒沙门氏菌、副溶血弧菌、大肠杆菌及属内其它病原菌均无交叉;所建立的免疫磁珠-选择性培养基检测法可检出浓度为103 CFU/mL及以上的单增李斯特菌,仅与英诺克李斯特菌存在一定交叉反应;牛奶样品单次仅需9 h增菌就能被检出,较常规增菌时间缩短39 h;检测限为0.4 CFU/mL。【结论】表达并纯化得到高纯度的单增李斯特菌MurA蛋白,制备的鼠源多克隆抗体亲和力高,特异性好;建立了快速检测单增李斯特菌的免疫磁珠联合选择性培养基法,在灵敏度不变的情况下,实现24 h内成功对牛奶样品的检测,较国标法减少42 h以上。     

荧光重组酶介导等温扩增检测食品中单增李斯特菌

【背景】单增李斯特菌为肉类及乳制品中常见的食源性致病菌,传统的培养法检测无法满足口岸大批量食品的快速检测要求,建立简便、灵敏、快速及现场可操作的技术至关重要。【目的】建立快速简便的荧光重组酶介导等温扩增(Recombinase-Aided Amplification,RAA)法检测单增李斯特菌,以适应口岸快速通关及监管的实际需求。【方法】根据单增李斯特菌hlyA基因保守区设计特异性引物、探针,通过引物两两组合结合探针筛选出扩增效率及灵敏度最佳的引物组合,优化反应温度及引物探针浓度,确定最佳反应条件。将建立的荧光RAA法应用于食品基质及实际样品检测中,同时与国标GB 4789.30-2016进行比对验证。【结果】单增李斯特菌荧光RAA最佳反应温度为42℃,最佳引物、探针终浓度均为400 nmol/L。建立的荧光RAA法特异性强,纯菌灵敏度达到3×102 CFU/mL。加标食品基质牛肉、大西洋鲑鱼及再制干酪LB2增菌只需4 h,即可检测原始浓度分别达到0.3、3、30 CFU/mL的单增李斯特菌。荧光RAA法只需5 min即可观察结果,20-30 min完成扩增,速度及灵敏度明显高于国标法...     

选择性增菌液对单核增生性李斯特氏菌检出效果的比较

为了了解食品中单核增生李斯特氏茵(Listeria monocytogenes)的污染状况,比较不同选择性增茵方法对单核增生李斯特氏茵的检出效果,并进一步比较不同增茵方法在不同类食品中检出单核增生李斯特氏茵的差异性,进而确定特定食品最合适的增茵方法,随机采集本市生鲜肉、水产品、果蔬及冷冻食品4类135份食品.采用国标LB二次增茵法、EB法、最新改良FDA法及Fraser肉汤增菌法进行增菌,采用PALCAM选择性平板进行分离,先用行标多重PCR法进行初步验证后再进行国标生化鉴定.4种方法共检出单核增生李斯特氏茵23株,其中LB二次增菌法检出5株、Fraser肉汤增菌法检出6株、EB法检出5株、最新改良FDA法检出7株.结论是4种方法总的检出率没有较大的差异性,但对于不同类食品的检出率有所不同.     

PCR检测鼠疫耶尔森氏菌研究进展

快速确诊鼠疫对鼠疫的防治工作至关重要。传统的细菌学“四步检查法”和血清学方法检测虽可确诊鼠疫,但存在烦琐、费时、费用高、不能进行快速诊断等弊病。PCR方法具有快速、特异、敏感的特点,尤其对培养条件苛刻、生长缓慢或已死亡的病原体检测优势更为明显,国内外学者现已建立了多种PCR方法用于鼠疫的检测,鼠疫PCR方法简便安全,是流行病学调查和紧急情况下检测鼠疫的有力手段。     

应用乳酸菌生物保护剂控制肉制品中单增李斯特菌的研究进展

肉制品营养丰富,极易被微生物污染,单增李斯特菌是污染肉制品主要病原菌之一。乳酸菌做为生物保护剂已经被广泛应用于食品中控制单增李斯特菌。本文首先分析了我国肉制品中单增李斯特菌的污染状况,总结了乳酸菌应用于肉制品安全控制的概况;然后进一步详细介绍了乳酸菌对单增李斯特菌的抑菌机理,着重探讨了乳酸菌对单增李斯特菌致病能力(生长、抗性和毒性)的影响;文章最后指出了乳酸菌在食品应用中存在的问题,并对未来的研究方向提供了建议,以期为乳酸菌在食品安全控制中的应用提供参考。     

Survival of Listeria monocytogenes and Enterococcus faecium in sludge evaluated by real-time PCR and culture methods

AIMS: This study evaluates the behaviour in spiked sludge of a pathogenic bacteria, Listeria monocytogenes, by cultural and molecular techniques, and compares its survival with the one of a faecal indicator, Enterococcus faecium. METHODS AND RESULTS: Listeria monocytogenes strain Scott A and E. faecium(T) were followed for 17 days after inoculation in sludge. Kinetics of survival depended on the bacteria and on the technique used [most probable number method, direct plate count or real-time quantitative PCR (qPCR)]. The concentration of L. monocytogenes decreased rapidly regardless of the technique, but the decrease was much more dramatic with culture techniques than with qPCR. On the contrary, the concentrations of culturable E. faecium(T) were stable. CONCLUSIONS: The results suggest that the cells of L. monocytogenes strain Scott A might have entered a viable, but nonculturable (VBNC) status, whereas cells of the indicator bacteria, E. faecium(T), maintained themselves better and stayed culturable. SIGNIFICANCE AND IMPACT OF THE STUDY: The difference of survival kinetics in the sludge of a faecal indicator (E. faecium) and a pathogenic bacterium (L. monocytogenes) may be linked to the fact that they either enter or do not enter into a VBNC status.     

Screening and characterization of monoclonal antibodies to the surface antigens of Listeria monocytogenes serotype 4b

Aims:  This study aims to develop and characterize monoclonal antibodies (Mabs) with high specificity and affinity for surface antigens of an epidemiologically important serotype 4b of Listeria monocytogenes .
Methods and Results:  Hybridoma clones were derived from B lymphocytes of mice immunized with L. monocytogenes serotype 4b and screened against this strain by an enzyme-linked immunosorbent assay. Twenty-nine clones secreting Mabs reactive with formalin-killed bacteria were obtained; 15, 8, 5 and 1 Mabs were immunoglobulin subclasses IgG2a, IgG2b, IgM and IgG1, respectively. Immuofluoresence or immunogold labelling demonstrated all except five IgM and one IgG2a Mabs bound to the surface of a live L. monocytogenes serotype 4b. The majority of the 23 surface-binding Mabs recognized linear epitopes on a 77-kDa protein. These surface-binding Mabs exhibited little or no cross-reactivity with non-4b serotypes (1/2a, 1/2b, 3a, etc.) of L. monocytogenes , five other Listeria species, Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium.
Conclusions:  The Mabs recognizing a 77-kDa surface protein are novel antibodies with specificity and affinity for L. monocytogenes serotype 4b.
Significance and Impact of the Study:  These anti-77 kDa surface protein Mabs may be explored as reagents for the development of Mabs-based diagnostic immunoassays for L. monocytogenes serotype 4b strains.     

Detection of viable and dead Listeria monocytogenes on gouda-like cheeses by real-time PCR

AIMS: Surface contamination by Listeria monocytogenes of gouda-like cheeses during processing represents a potential public health problem. The aim of this work was to develop novel real-time PCR diagnostics to detect the presence of viable, dead or viable but not culturable (VBNC) cells on gouda-like cheeses. METHODS AND RESULTS: We used ethidium monoazide bromide (EMA)-PCR for direct quantification of viable and dead cells, while semiquantitative detection of culturable cells below the PCR detection limit (c. 100 CFU g(-1)) was obtained by combining growth and real-time PCR. We were able to quantify the fraction of >0.5% viable cells in a background of dead cells by EMA-PCR, given that the viable cell concentration was above the PCR detection limit. The combined growth and real-time PCR complemented the EMA-PCR, and enabled semiquantitative detection of low levels of culturable cells (10 and 100 CFU g(-1)). SIGNIFICANCE AND IMPACT OF THE STUDY: The significance of this work is that we have developed a novel concept for detection of viable and potentially infectious L. monocytogenes.     

PCR detection of Listeria monocytogenes: a study of multiple factors affecting sensitivity

AIMS: To test, under comparable conditions, several parameters affecting sensitivity of PCR detection in order to establish a PCR procedure suitable for the routine detection of Listeria monocytogenes in food. METHODS AND RESULTS: Beef samples artificially inoculated were used to determine sensitivity of PCR detection under different parameters. As few as 1 CFU g(-1) were detected by DNA extraction using a DNeasy Tissue Kit (Qiagen GmhH, Hilden, Germany) of 1 ml aliquot and PCR amplification with primers directed to the hlyA gene. This PCR protocol was applied in 60 naturally contaminated foods, comparing two enrichment procedures with the traditional culture method. The highest number of positives was recorded by PCR following a 24-h pre-enrichment step at 30 degrees C and a 24-h enrichment step at 37 degrees C. Afterwards, it was applied in 217 naturally contaminated foods and 56 of them tested positive for L. monocytogenes in which only 17 tested positive using the culture method. CONCLUSIONS: The PCR procedure described has proved to be a rapid and sensitive method suitable for the routine analysis of different types of food. SIGNIFICANCE AND IMPACT OF THE STUDY: The method proposed for the detection of L. monocytogenes, has been validated in naturally contaminated food and is suitable to implement in the food industry.     

Listeria monocytogenes in spontaneous abortions in humans and its detection by multiplex PCR

AIM: To assess the extent of Listeria monocytogenes in causation of human spontaneous abortions by isolation methods and PCR analysis for the presence of virulence-associated genes. METHODS AND RESULTS: A total of 305 samples comprising blood, urine, placental bits, faecal and vaginal swabs were collected from 61 patients with spontaneous abortions. Listeria spp. were isolated from 10 samples collected from nine (14.8%) patients. Confirmation of these isolates was based on biochemical tests, haemolysis on blood agar, CAMP test, phosphatidylinositol-specific phospholipase C (PI-PLC) assay followed by in vivo pathogenicity tests and multiplex PCR to detect virulence-associated genes (prfA, plcA, hlyA, actA and iap). Three isolates were confirmed as L. monocytogenes. Of these, two isolates turned out to be pathogenic and found to posses all five genes. However, the remaining two haemolytic L. monocytogenes isolates lacking the plcA gene and activity in the PI-PLC assay were found to be nonpathogenic by in vivo tests. CONCLUSIONS: The occurrence of pathogenic L. monocytogenes in cases of spontaneous abortions was 3.3%. It seems that the plcA gene and its expression have an important role as essential virulence determinants in pathogenic Listeria spp. SIGNIFICANCE AND IMPACT OF THE STUDY: The recovery of pathogenic L. monocytogenes isolates from cases of spontaneous abortion indicates the significance of listeric infection in pregnant women.     

Novel cost-efficient real-time PCR assays for detection and quantitation of Listeria monocytogenes

Listeriosis is a serious food-borne infection with mortality rates approaching 30%. Therefore, the rapid, cost-effective, and automated detection of Listeria monocytogenes throughout the food chain continues to be a major concern. Here we describe three novel quantitative real-time PCR assays for L. monocytogenes based on amplification of a target hlyA gene with SYBR Green I chemistry and hydrolysis probe (TaqMan MGB probe). In order to offer sensitive, rapid and robust tool of additional economical value the real-time PCR assays were designed and optimized to only 5 μl-reactions. All assays were evaluated by using different non-reference Listeria strains isolated from various food matrices. Results demonstrated specificity to L. monocytogenes with accurate quantification over a dynamic range of 5-6 log units with R² higher than 0.98 and amplification efficiencies reaching above 92%. The detection and quantification limits were as low as 165 genome equivalents. Comparison of novel assays to commercially available TaqMan® Listeria monocytogenes Detection Kit and previously published studies revealed similar specificity, sensitivity and efficiency, but greater robustness and especially cost-efficiency in the view of smaller reaction volumes and continuous increase in sample throughput.