食品中单核细胞增生李斯特氏菌调查及毒力研究

为了解和掌握各类食品中单核细胞增生李斯特氏菌（Lm)污染状况及菌株的毒力情况,采集5类265件食品,菌株鉴定应用API细胞鉴定系统;毒力测定采用溶血实验、多聚酶链反应（PCR）以及小鼠致病力实验的方法进行,结果显示Lm检出率为4.90%;其中从生肉、熟肉、灌汤和速冻食品中的检出率分别为15.00%、2.48%、9.52%;从熟肉和速冻食品中还检出其它李斯特氏菌,检出率分别为4.96%、5.92%;乳制品和水产品中未检出Lm。13株Lm毒力测定显示,溶血实验与小鼠的致病力和PCR测定的溶血素基因无必然联系,而溶血素基因与内化素基因阳性结果相同,Lm对多种抗生素敏感,其中对氨氨苄青霉素、头孢唑啉和环丙沙星敏感率为100%。     

单核细胞增生李斯特氏菌食品分离株协同 溶血现象研究

目的对从食品中分离的88株单核细胞增生李斯特氏菌(Listeria monocytogenes)进行协同溶血(christie,atkins and munch-petersen,c AMP)测试。方法根据食品安全国家标准食品微生物学检验单核细胞增生李斯特氏菌检验GB 4789.30-2010的方法进行c AMP测试。结果所测试的88株单核细胞增生李斯特氏菌分离株与GB 4789.30中描述的结果一致,即在靠近金黄色葡萄球菌(Staphylococcus aureus)的接种端溶血增强,78株靠近马红球菌一端呈阴性反应。同时,发现10株分离株c AMP测试结果与传统的测试结果不同,在靠近马红球菌(Rhodococcus equi)的接种端溶血增强。结论对88株单核细胞增生李斯特氏菌分离株c AMP测试结果表明,有大约11%的菌株在靠近马红球菌的接种端溶血增强。     

单核细胞增生李斯特菌的毒力因子

目的单核细胞增生李斯特菌(Listeria monocytogenes,Lm)属于革兰阳性无芽孢杆菌李斯特菌属,主要通过食物传播。Lm的致病性与毒力因子密切相关,研究其毒力因子对认识致病机理有着重要意义。方法对Lm重要的毒力因子(溶血素、磷脂酶、内化素、肌动蛋白、P60蛋白等)进行综述。结果溶血素是一个多功能的毒力因子,对于Lm逃离吞噬细胞囊是必需的。Lm能产生两种磷脂酶C:磷脂酰肌醇磷脂酶C和磷脂酰胆碱磷脂酶C,协助细菌的细胞内复制。肌动蛋白使得Lm在宿主细胞间能够扩散。内化素与Lm的侵袭力有关。P60蛋白是Lm的主要免疫原性抗原。结论Lm毒力因子研究的深入对李斯特菌病的防治将带来深远的影响。     

The occurrence,growth, and biocontrol of Listeria monocytogenes in fresh and surface-ripened soft and semisoft cheeses

Listeria monocytogenes continues to pose a food safety risk in ready-to-eat foods, including fresh and soft/semisoft cheeses. Despite L. monocytogenes being detected regularly along the cheese production continuum, variations in cheese style and intrinsic/extrinsic factors throughout the production process (e.g., pH, water activity, and temperature) affect the potential for L. monocytogenes survival and growth. As novel preservation strategies against the growth of L. monocytogenes in susceptible cheeses, researchers have investigated the use of various biocontrol strategies, including bacteriocins and bacteriocin-producing cultures, bacteriophages, and competition with native microbiota. Bacteriocins produced by lactic acid bacteria (LAB) are of particular interest to the dairy industry since they are often effective against Gram-positive organisms such as L. monocytogenes, and because many LAB are granted Generally Regarded as Safe (GRAS) status by global food safety authorities. Similarly, bacteriophages are also considered a safe form of biocontrol since they have high specificity for their target bacterium. Both bacteriocins and bacteriophages have shown success in reducing L. monocytogenes populations in cheeses in the short term, but regrowth of surviving cells can commonly occur in the finished cheeses. Competition with native microbiota, not mediated by bacteriocin production, has also shown potential to inhibit the growth of L. monocytogenes in cheeses, but the mechanisms are still unclear. Here, we have reviewed the current knowledge on the growth of L. monocytogenes in fresh and surface-ripened soft and semisoft cheeses, as well as the various methods used for biocontrol of this common foodborne pathogen.     

一种快速鉴定单增李斯特菌的方法

通过单增李斯特菌的生化特性对李斯特菌进行鉴定,并根据单增李斯特菌的actA基因序列,以及其他李斯特菌的特异性靶序列设计引物,用PCR技术快速鉴定单增李斯特菌.结果表明,生化实验时间较长且误差较大,而通过PCR能成功扩增出827bp的特征性条带,同时用CTAB/NaCI法和热裂解法分别提取基因组后进行PCR和电泳,结果两种方法都可以很好的扩增出特征性条带,而热裂解法由于更简便快速,实验确定了热裂解法是一种快速有效的鉴定单增李斯特菌的方法.     

Identification of Listeria innocua surrogates for Listeria monocytogenes in hamburger patties

ABSTRACT:  Listeria innocua M1 has been used by many researchers as a nonpathogenic thermal processing surrogate for Listeria monocytogenes . However, L. innocua M1 has been criticized because its thermal survivability characteristics are not as closely parallel to L. monocytogenes as some would like in a variety of foods and processing conditions. The present study was conducted to compare multiple L. innocua and L. monocytogenes strains to validate L. innocua M1 as the ideal surrogate under high-temperature thermal processing conditions for L. monocytogenes . The D - and z -values of L. innocua M1, L. innocua strain SLCC 5639 serotype (6a), SLCC 5640 (6b), SLCC 2745 (4ab), and L. monocytogenes F4243 (4b) were calculated for raw hamburger patties. Hamburger patties were inoculated with 10^7–8 CFU/g of L. monocytogenes or L. innocua . Samples were heat treated at 4 temperatures (62.5 to 70 °C). At each temperature, the decimal reduction time ( D -value) was obtained by linear regression of survival curves. The D - and z -values were determined for each bacterium. The D -values of L. innocua and L. monocytogenes serotypes ranged from 3.17 to 0.13 min at 62.5 to 70 °C, and the z -values of L. innocua and L. monocytogenes were 7.44 to 7.73 °C. Two of the 4 L. innocua serotypes used in this experiment have the potential for use as surrogates in hamburger meat with varying margins of safety. L. innocua M1 should serve as the primary nonpathogenic surrogate with the greatest margin of safety in verifying a new thermal process to destroy L. monocytogenes .     

单核细胞增生李斯特氏菌的PCR检测方法

研究比较了裂解法、热煮沸法、试验盒法提取单增李斯特氏菌DNA的效果 ,认为Promega试剂盒法提取的DNA ,得率高、纯度好。同时根据单增李斯特氏菌的毒力相关基因的 5对引物 ,进行了引物的特异性研究 ,结果发现inlA引物、inlB引物、plcA引物特异性好 ,可以用于食品中单增李斯特氏菌的检测     

食源性单增李斯特菌检测技术研究进展

由单核细胞增生李斯特氏菌(简称单增李斯特菌)(Listeria monocytogenes,L.monocytogenes)引起的李斯特菌病,被认为是世界范围内主要的食源性疾病之一,致死率可高达20％～30％.单增李斯特菌普遍存在于各类食品中,其在低温、有氧或无氧条件下、以及较宽的pH和渗透压范围内均可生长繁殖.因此,...     

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。     

李斯特菌噬菌体的分离鉴定及其裂解特性

以单核细胞增生性李斯特菌(Lm)为宿主菌,从畜禽养殖区污水中分离(Lm)特异的噬菌体,分析其生物学特性、裂解特性及其在食品中的灭菌效果。用双层平板法从污水样中分离Lm噬菌体,PEG/NaCl法沉淀纯化噬菌体,负染色后电镜观察。分析噬菌体对宿主菌的裂解特性及其对温度和pH值的敏感性,同时对该噬菌体进行宿主谱系分析。将噬菌体运用于即食性食品中,分析其潜在的灭菌效果。结果表明:分离的噬菌体为裂解性噬菌体,属长尾噬菌体科,命名为LipG2-5。体外能够高效裂解宿主菌,对温度及pH值亦有良好的耐受性。基因组酶切分析表明,LipG2-5为双链DNA噬菌体。噬菌体谱系分析表明,该噬菌体为1株宽宿谱噬菌体,在固态及液态即食性食品中能够高效杀灭Lm。     

一株食源性多重耐药单核细胞增生李斯特菌的耐药性研究

本研究对食品样本中分离的一株多重耐药单核细胞增生李斯特菌进行耐药机制的探讨,以期对食源性单核细胞增生李斯特菌多重耐药现象的控制提供理论依据。本文通过聚合酶链式反应筛选耐药决定因子,质粒消除及自然转化实验对耐药决定因子进行定位及传播能力的探讨,最后通过传代实验验证该菌株多重耐药性传播的稳定性。结果表明,对检测到的多重耐药菌株LM78(耐受氯霉素、红霉素、链霉素、四环素、复方新诺明)进行相关耐药基因检测,检测到cat、erm B、tet S 3个耐药基因。质粒消除后MIC值下降到敏感范围,且该质粒可通过自然转化在不同菌属间传递,说明这些耐药基因存在于质粒上。该质粒在无抗生素选择压力下连续传代,仍具有较高稳定性。食源性致病菌多重耐药性有可能通过不同细菌种属间转移,进而由食物链向人类传播,对人类健康造成潜在的威胁。     

单核细胞增生李斯特菌质粒DNA参考物质的研制

目的 研制一种能够用于快速鉴定单核细胞增生李斯特菌(以下简称单增李斯特菌)的质粒DNA标准物质.方法 设计一种质粒DNA包含目前常用于单增李斯特菌检测的hlyA、plcB、inlA基因,并对其稳定性、均匀性和量值可追溯性进行评价.评估其在实时定量聚合酶链式反应(PCR)检测中的适用性.结果 质粒DNA参考物质的最终定值...     

盐水鸭中单核细胞增生李斯特菌生长预测模型的建立

为研究盐水鸭中单核细胞增生李斯特菌(Listeriamonocytogenes,Lm)的生长规律,通过测定4、10、16、25℃条件下的生长数据,选用4种常用的一级模型(Gompertz、Logistic、Richards及MMF模型)对数据进行拟合,比较各模型决定系数R^2和均方误差(MSE),确定最适一级模型,根据一级模型得到的最大比生长速率(μmax)和迟滞期(λ)建立与温度相关的二级模型。结果表明:Gompertz模型拟合的生长曲线R^2均达到0.99以上,为最适一级模型,在25℃条件下,Lm经0.78 h后即进入对数期,从4℃提高到10℃时,生长速率从0.02 1g(cfu/g)·h^-1增至0.05 1g(cfu/g)·h^-1,说明温度对盐水鸭中Lm的生长影响较大。选用Ratkowsky平方根模型建立的温度与μmax关系的二级模型R^2为0.98,偏差因子(Bf)、准确因子(Af)分别为0.99、1.01,二次多项式模型建立的温度与λ关系的R^2为0.99,Bf、Af分别为1.01、1.08,表明所建两种模型均能较好地描述盐水鸭中Lm的生长情况。本研究建立的生长模型可为监控盐水鸭的食品安全和风险评估提供参考。     

Growth of Listeria monocytogenes on vacuum-packaged horsemeat for human consumption

In order to investigate the likelihood of Listeria monocytogenes (serotype 4b, ATCC 19115) growth on vacuum-packaged horsemeat at refrigeration temperature, fourteen horsemeat surface/volume homogeneous 150 g weight pieces were superficially inoculated with serotype 4b L. monocytogenes and vacuum packaged. The samples were stored at 4 ± 1 °C. Two pieces (one for pH determination and one for L. monocytogenes counts) were examined at days 0, 7, 14, 21, 28, 35 and 42. Surface pH did not show significant variations during the experiment. The average L. monocytogenes initial contamination level was 1.77log₁₀ CFU/g. A lag phase of 7 days was recorded. The exponential growth rate between day 7 to day 35 was 0.125log₁₀ CFU/day, corresponding to 3.51log₁₀ CFU/g in 28 days. At the end of the experiment the mean L. monocytogenes log₁₀ CFU/g was 5.78.     

单核细胞增生性李斯特菌自溶素研究进展

自溶素是由细菌产生的可以降解细菌细胞壁的肽聚糖水解酶,参与细菌的分裂、细胞壁更新、细菌自溶等生理过程。自溶素也是细菌的重要毒力因子之一,参与细菌的黏附与侵袭,与细菌的致病性密切相关。本文对单核细胞增生性李斯特菌的胞壁水解酶p60、酰胺酶Ami、自溶素IspC、自溶素Auto与胞壁水解酶MurA等自溶素的蛋白结构、生理功能及致病性进行了简要介绍。     

肉中单核细胞增生李斯特菌PCR快速检测方法建立

目的:建立单核细胞增生李斯特菌(Listeria monocytogenes,LM)快速、敏感、特异的PCR诊断方法。方法:采用聚合酶链式反应技术(PCR)特异性扩增单核细胞增生李斯特菌内化素基因(ivlA),并评价该方法的特异性与敏感性。结果:在445bp处出现inlA基因的目的片断,只有单增李斯特菌的目的片段获得扩增,其他菌种扩增均呈阴性;该方法可以检测到DNA的检测限。结论:PCR方法比传统细菌检测方法更特异、快速、灵敏和简便;为肉中单增李斯特菌的快速检测提供了新的手段。     

Control of Listeria monocytogenes in Vacuum-Packaged Pre-Peeled Potatoes

Growth of Listeria monocytogenes on the surface of fresh peeled potatoes, treated with sulfite or a commercial browning inhibitor (CBI), packaged under vacuum and stored at 4,15 and 28°C was determined. At 4°C, L. monocytogenes did not grow in all treated potatoes even after 21 days. At 15°C, L. monocytogenes grew to 7 log₁₀ CFU/g within 12 days in the potatoes treated with sulfite or CBI. At 28°C, L. monocytogenes population was greater than 3 log₁₀ CFU/g by 24 h in all samples regardless of treatment. Sulfites or a CBI appeared to provide a measure of safety in pre-peeled potatoes packaged under vacuum when kept at proper refrigeration temperatures.     

免疫磁分离技术在食源性单增李斯特菌检测中应用的研究进展

食源性单增李斯特菌是李斯特菌属中的唯一能引起人类疾病的病原菌,致死率30%~70%,并严重威胁着人类健康。早期快速准确地检测出食品中可能污染的单增李斯特菌对于减少死亡率非常重要,因此亟需建立一些快速、灵敏和高特异性的检测方法。现有单增李斯特菌的检测方法对未经前增菌的食品样本检测灵敏度较低,限制了这些方法直接用于食品样本中单增李斯特菌的快速检测。免疫磁分离是一种可以短时间内高效富集样本中目的菌的技术,与常用的检测方法结合,可以缩短检测周期,提高检测灵敏度。本文综述了免疫磁分离技术在食源性单核细胞增生性李斯特检测中应用的研究进展。      

基于双启动引物特异性检测单核细胞增生李斯特氏菌的PCR方法

以单核细胞增生李斯特氏菌iap基因为靶基因,利用一新型PCR引物设计方法--双启动引物（Dual-priming oligonucleotide,DPO）,建立了特异性检测单核细胞增生李斯特氏菌的DPO-PCR方法,测试了DPO-PCR方法退火温度不敏感性、特异性及灵敏度,并在实践检测中进行了初步应用。结果显示:该方法检测单核细胞增生李斯特氏菌的灵敏度为1.51×102CFU/mL;退火温度不敏感性测试中,与常规PCR引物相比,DPO引物在48⁶8℃退火温度范围内均能够高效率地扩增靶基因;特异性测试中,DPO-PCR方法能特异地检测出目标菌,与其他菌株无非特异性扩增反应,比常规PCR方法显示出更强的特异性。实践应用证明,利用DPO-PCR方法对130份样本进行检测,共计检出9份单核细胞增生李斯特氏菌阳性样本,经国标法（GB/T 4789.30-2008）复检,两者检测结果一致,显示出良好的实用性,为单核细胞增生李斯特氏菌的快速准确检测提供了新方法。