单增李斯特氏菌MALDI-TOF-MS鉴定与分型研究

为建立单增李斯特氏菌的基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)快速鉴定与分型方法,实验收集37株单增李斯特氏菌分离株,应用MALDI-TOF-MS采集图谱,获取独特的蛋白质指纹图谱,汇总成标准图谱,建立单增李斯特氏菌鉴定数据库。采用单增李斯特氏菌标准菌株进行验证,表明鉴定结果的可信度很高。在数据库信息的基础上,对37株单增李斯特氏菌分离株进行聚类分型。分型结果表明,在蛋白质水平上,MALDI-TOF-MS可把37株单增李斯特氏菌分成9个型别。     

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.     

单核细胞增生李斯特氏菌国家标准检验方法的验证与探讨

本文旨在验证、分析和探索食品中单核细胞增生李斯特氏菌的检验方法。方法:依据GB4789.30-2016,首先将实验样品预增菌24 h后,之后,用李斯特氏菌显色培养基和PALCAM琼脂分离培养,将菌株进行染色显微镜观察、动力试验、生化鉴定、溶血试验、协同溶血试验、李斯特氏菌属鉴定试剂盒、全自动微生物生化鉴定仪等鉴别菌种的方法。结果:实验样品和单核细胞增生李斯特氏菌以及伊氏李斯特氏菌菌落均在李斯特氏菌显色培养基上呈蓝绿色,周围环绕着白色晕圈,实验样本及4种李斯特氏菌标准菌株均是革兰氏阳性,短杆状,不产芽孢和荚膜;生化反应中与葡萄糖、MRVP、麦芽糖、七叶苷、甘露醇等发酵反应结果一致。除了伊氏李斯特氏菌在木糖发酵管中反应呈黄色,为阳性外,其他李斯特氏菌菌株都呈绿色,为阴性。在溶血反应中,英诺克李斯特氏菌在血平板上没有溶血,为阴性,而其他菌株产生透明溶血圈,为阳性;由全自动微生物生化鉴定仪鉴定菌株均为李斯特氏菌属。结论:通过对国家标准测试方法—单增李斯特氏菌的验证,建议通过选择性培养基进行分离后,用木糖、鼠李糖生化反应区分单增李斯特氏菌与其他李斯特氏菌,最后用单增李斯特氏菌生化鉴定试剂盒验证,使检测效率提高同时也提升了检测的准确度。     

云南淡水鱼中单增李斯特氏菌污染状况及耐药性分析

目的了解云南淡水鱼中单增李斯特氏菌污染状况及耐药情况。方法从云南省昆明市、曲靖市、大理州、红河州4个淡水鱼主产区采集淡水鱼样品110件,参照GB 4789.30—2016《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》分离鉴定单增李斯特氏菌,并进一步开展16S rRNA序列鉴定和同源性分析,对鉴定菌株采用微量肉汤稀释法分析抗生素敏感性。结果110件样品检出2株单增李斯特氏菌(DZ-054、DZ-101),检出率为1.82%。2株单增李斯特氏菌之间相似度达到99.64%。DZ-101菌株对氨苄西林、青霉素敏感(S),DZ-054菌株对抗生素不敏感。结论云南省淡水鱼中单增李斯特菌的污染率较低,但仍有一定的安全隐患,相关部门应加强监管。     

单增李斯特菌检测技术研究进展

单增李斯特菌(Listeria monocytogenes)是一种人畜共患食源性致病菌,可使人畜患脑膜炎、心肌炎、败血症、早产等疾病,危害较大。有效控制食品中的单增李斯特菌,是食品安全的重要课题之一。本文对单增李斯特菌的主要检测技术,如传统分离鉴定、免疫法、分子生物学法、全自动微生物分析系统、生物传感器检测作了简要叙述,为深入研究提供参考。并对我国单增李斯特菌的检测监控进行了展望。     

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结果为29.85 μg/mL。该质粒DNA参考物质量值可靠,均匀性和稳定性良好,可以在-20 ℃下保存1年以上。质粒DNA参考品在实时荧光PCR检测中的适用性证明,可以保证实时定量PCR检测结果与单增李斯特菌基因组DNA参考品具有可比性。 结论 研制的质粒DNA标准物质可以替代单增李斯特菌基因组DNA对单增李斯特菌进行质量监控,满足单增李斯特菌检测在实验室质量的有效控制和试验方法验证的需要。 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PCR检测中的适用性。结果 质粒DNA参考物质的最终定值结果为29.85 μg/mL。该质粒DNA参考物质量值可靠,均匀性和稳定性良好,可以在-20 ℃下保存1年以上。质粒DNA参考品在实时荧光PCR检测中的适用性证明,可以保证实时定量PCR检测结果与单增李斯特菌基因组DNA参考品具有可比性。结论 研制的质粒DNA标准物质可以替代单增李斯特菌基因组DNA对单增李斯特菌进行质量监控,满足单增李斯特菌检测在实验室质量的有效控制和试验方法验证的需要。     

基质辅助激光解吸电离飞行时间质谱在 李斯特菌检测和鉴定中的应用

为建立食品中各种李斯特菌的快速检测和鉴定方法,应用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)对单增李斯特菌、绵羊李斯特菌、英诺克李斯特菌、威尔斯李斯特菌和格氏李斯特菌5种李斯特菌进行检测,并与传统的生化鉴定方法(VITEK 2)和聚合酶链式反应(PCR)方法检测结果进行比较;用不同培养基和不同培养时间培养的单增李斯特菌对该方法进行检测,还对60份人工感染5种李斯特菌的食品样品分别进行MALDI-TOF-MS、PCR以及传统生化方法的检测和鉴定。结果表明：MALDI-TOF-MS能够快速、可靠地区分上述5种李斯特菌,而且具有很好的稳定性和重复性,在检测时间、重复性和准确性方面的总体表现优于传统生化鉴定方法。MALDI-TOF-MS技术适用于对李斯特菌等食源性病原菌进行高通量、低成本的快速鉴定。     

食品中单核细胞增生李斯特菌株的分离及聚合酶链式反应鉴定

目的 建立单核细胞增生李斯特菌的聚合酶链式反应(PCR)检测方法,了解市售食品中单核细胞增生李斯特菌的污染情况.方法 采集成都市市售生畜肉、生禽肉、熟肉制品、水产品、生食蔬菜以及其他熟食等食品样品共135份,采用李氏增菌肉汤(LB1,LB2)进行初增菌,应用选择性分离培养基PALCAM和在TSA-YE平板上进行分离,利用单增李斯特显色平板进行鉴定;根据李斯特菌的特异性基因iap基因设计引物,采用PCR方法检测所有分离的李斯特菌株;根据单增李斯特菌的特异性基因hly基因和prfA基因设计引物检测单核细胞增生李斯特菌株.结果 135份样品中共检出李斯特菌17株,检出率为12.6％;其中单核细胞增生李斯特菌4株,检出率为3.0％.结论 本研究建立的PCR方法具有特异性,本市市售食品不同程度受到李斯特菌的污染.     

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。     

LAMP法检测单核细胞增生性李斯特菌的研究

单核细胞增生性李斯特氏菌(Listeria monocytogenes)简称单增李斯特菌,属李斯特菌属,是食源性致病菌中的一种,它能够引起人畜共患病,在食源性细菌中毒中占主要地位。为了鉴定该菌,以单增李斯特菌的内化素基因(inlA)为靶基因,通过LAMP法对该基因扩增,并优化其反应条件。过夜培养的单增李斯特菌的菌液,取1 mL提取细菌基因组DNA,提取完成后测得其DNA含量为227 ng/μL,然后依次将其进行10倍梯度稀释,并以此作为LAMP实验模板,最终实验结果表明,LAMP检测单增李斯特菌纯菌培养物的检测限为2.27 fg/μL。     

Application of a multiplex polymerase chain reaction assay for the simultaneous confirmation of Listeria monocytogenes and other Listeria species in turkey sample surveillance

A multiplex polymerase chain reaction was developed to simultaneously identify Listeria monocytogenes and species of the genus Listeria. Two sets of primers were used, with the first amplifying a 938-bp region of the 16S rRNA gene that is highly conserved in all Listeria species and the second amplifying a 174-bp region of the listeriolysin (hlyA) gene of L. monocytogenes. Thus, isolates of Listeria spp. yield a single 938-bp product, whereas L. monocytogenes isolates yield both the 938-bp product and a 174-bp product. The specificity of the assay was verified with all six Listeria species and 11 serotypes of L. monocytogenes, as well as nonrelated bacteria. The multiplex PCR assay was used to determine the incidence of Listeria spp., especially L. monocytogenes, in mechanically separated turkey samples (n = 150 samples). L. monocytogenes strains were selected by using the University of Vermont two-step enrichment protocol and plating to selective Palcam agar. The multiplex PCR assay was used for verification of presumptive Listeria colonies. Approximately 38% of mechanically separated turkey samples (57 of 150) yielded L. monocytogenes; an additional 18% of these samples (27 of 150) harbored other Listeria spp. Fifty-one percent (29 of 57) of the L. monocytogenes isolates were of serogroup 1, 44% (25 of 57) were of serogroup 4, and 2% (1 of 57) were assigned to serogroups other than 1 and 4.     

Comparison of multiplex PCR with conventional biochemical methods for the identification of Listeria spp. isolates from food and clinical samples in Queensland, Australia

Listeria monocytogenes is an important foodborne pathogen with high mortality. L. monocytogenes and five other Listeria species can frequently be found in the same sample. To identify Listeria isolates found in foods to the species level, two multiplex PCRs were designed. The PCR and conventional biochemical methods were compared for the identification of 456 Listeria isolates collected from routine food quality monitoring schemes between June 2004 and February 2006 and for 62 L. monocytogenes isolates from patients between 1999 and 2005. The results showed that the PCR and biochemical methods had 100% agreement in Listeria identification. The distribution of Listeria species from foods was as follows: L. monocytogenes, 50.4%; L. innocua, 33.8%; L. welshimeri, 14.9%; L. seeligeri, 0.7%; L. grayi, 0.2%; and L. ivanovii, 0.0%. Additional analyses were performed to identify the major serotypes (1/2a, 1/2b, 1/2c, and 4b) and the three lineages of L. monocytogenes isolates from foods and patients, with 1/2a (69.6%) and 1/2b (21.7%) dominating the food isolates and 1/2b (54.8%) and 4b (30.7%) dominating the patient isolates. The lineage results showed that isolates of 1/2a and 1/2c belonged to lineage II and that isolates of 1/2b and 4b belonged to lineage I. The multiplex PCRs for Listeria identification that have been established provide an accurate and rapid method for food quality control. This study has provided the basic knowledge of distribution of Listeria species and L. monocytogenes serotypes in Queensland, Australia, which is useful for epidemiological investigations of listeriosis.     

链置换恒温扩增快速检测单核细胞增生李斯特氏菌

目的开发单核细胞增生李斯特氏菌快速、简便、准确的链置换恒温扩增(strand displacement amplification,SDA)检测方法。方法根据单核增生李斯特菌特异性基因prfA基因片段,设计链置换扩增特异性引物,建立单核细胞增生李斯特菌SDA快速检测方法,同时对设计的引物的特异性和灵敏度进行研究。结果所建立的单核细胞增生李斯特菌SDA扩增方法灵敏度达到了7.0×10~2 CFU/mL,检验技术可以特异性地扩增单核增生李斯特菌。结论本试验所建立的单核细胞增生李斯特菌SDA检验技术具有快速、简便、特异性强的特点,具有在进出口实验室推广应用前景。     

生肉中单核细胞增生性李斯特菌的分离与鉴定

目的了解哈尔滨市售生肉中单核细胞增生性李斯特菌的污染状况。方法将显色培养基法、API试剂条法和PCR法联合应用对哈尔滨市售128份生肉进行单核细胞增生性李斯特菌的分离鉴定。结果共分离出单核细胞增生性李斯特菌18株,检出率为14.06%。其中生猪肉检出率最高,达20.00%。结论哈尔滨市售生肉中单核细胞增生性李斯特菌污染状况比较严重,应引起有关部门的重视,加强管理和监测。     

单增李斯特菌在生食鱼片中生长模型的建立

为研究生食鱼片中单增李斯特菌的生长规律,将单增李斯特菌接种到经冷杀菌后的3 种生食鱼片（三文鱼片、金枪鱼片、鲷鱼片）中,分别置于4、8、15、25、35 ℃环境下培养,间隔适当时间取出计数。用5 种常用的一级模型（Gompertz模型、Baranyi模型、Logistic模型、Richards模型和MMF模型）对实验数据进行拟合,通过比较相关系数R2和均方误差（mean square error,MSE）,确定最适一级模型为Gompertz模型。建立单增李斯特菌生长动力学参数（最大比生长速率μm和迟滞期λ）关于温度、pH值和水分活度的二级平方根扩展模型,并应用相关系数R2、偏差值Bf和准确值Af进行验证。结果表明,构建的二级模型能够很好地描述生食鱼片中单增李斯特菌的生长情况。     

食品中3种致病李氏菌MPCR-DHPLC检测方法的建立

应用复合PCR(multiplex PCR,MPCR)结合变性高效液相色谱(denaturing high-performance liquid chromatography,DHPLC)技术建立食品中单核细胞增生李斯特菌、绵羊李斯特菌和英诺克李斯特菌的快速高通量检测方法。根据单核细胞增生李斯特菌、绵羊李斯特菌和英诺克李斯特菌的特异基因序列分别设计引物,MPCR扩增的产物经DHPLC技术进行快速检测。以94株参考菌株做特异性实验,并开展了重现性检测实验。MPCR-DHPLC方法同步检测到单核细胞增生李斯特菌、绵羊李斯特菌和英诺克李斯特菌的特异性阳性吸收峰,未检测到李斯特菌属其他近源种和非近源种参考菌株的阳性吸收峰,且重现性良好。该方法具有很好的特异性,可以快速、准确、高通量地检测食品中单核细胞增生李斯特菌、绵羊李斯特菌和英诺克李斯特菌,是食品微生物快速检测的新技术。     

The occurrence of Listeria species in milk and dairy products: a national survey in England and Wales

A total of 4172 samples of milk, cheese and other dairy products were examined over a 1-year period for the presence of Listeria species. Strains of Listeria were found most frequently in soft, ripened cows milk cheese; 63 out of 769 (8.2%) samples contained Listeria monocytogenes, 25 samples contained species other than L. monocytogenes, and 18 samples contained both L. monocytogenes and other Listeria spp. Eleven samples of pasteurized cows milk (1.1%) from four dairies contained L. monocytogenes, and other Listeria spp. were isolated from a further five samples. Goats and ewes milk and their products, yogurt, cream and ice cream also occasionally contained Listeria spp. Levels of Listeria were usually low, but 20 samples of cheese contained more than 1000 cfu/g. Most strains of L. monocytogenes belonged to serotype 1/2 (58%) or serotype 4b (33%).     

Peptide nucleic acid fluorescence in situ hybridization for identification of Listeria genus, Listeria monocytogenes and Listeria ivanovii

A fluorescent in situ hybridization (FISH) method in conjunction with fluorescin-labeled peptide nucleic acid (PNA) probes (PNA-FISH) for detection of Listeria species was developed. In silico analysis showed that three PNA probes Lis-16S-1, Lm-16S-2 and Liv-16S-5 were suitable for specific identification of Listeria genus, Listeria monocytogenes and Listeria ivanovii, respectively. These probes were experimentally verified by their reactivity against 19 strains of six Listeria species (excluding newly described species Listeria marthii and Listeria rocourtiae) and eight other bacterial species. The PNA-FISH method was optimized as 30 min of hybridization with 0.2% Triton X-100 in the solution and used to identify 85 Listeria strains from individual putative Listeria colonies on PALCAM agar plates streaked from selectively enriched cultures of 780 food or food-related samples. Of the 85 Listeria strains, thirty-seven were identified as L. monocytogenes with the probe Lm-16S-2 and two as L. ivanovii with the probe Liv-16S-5 which was in agreement with the results obtained by the API LISTERIA method. Thus, the PNA-FISH protocol has the potential for identification of pathogenic Listeria spp. from food or food-related samples.     

Evaluation and interlaboratory validation of a selective agar for phosphatidylinositol-specific phospholipase C activity using a chromogenic substrate to detect Listeria monocytogenes from foods

Phosphatidylinositol-specific phospholipase C (PI-PLC) activity is a potential virulence factor and is exhibited only by the Listeria species Listeria monocytogenes and Listeria ivanovii. A chromogenic substrate for the direct detection of PI-PLC activity is available in a new medium (BCM L. monocytogenes plating agar). The use of a chromogenic substrate offers a mechanism with which to directly screen for L. monocytogenes and L. ivanovii other than the esculin used in Oxford (OXF) and Palcam (PAL) agars, which screen for all Listeria species. The specificity levels of BCM plating agar and of BCM confirmation and rhamnose agars were evaluated with 107 Listeria and 10 Bacillus species isolates. In addition, BCM L. monocytogenes plating agar was compared with standard Listeria selective agars (OXF and PAL agars) with regard to the recovery of L. monocytogenes from 2,000 food and environmental samples obtained from eight participating laboratories. A Listeria species was isolated from at least one of the agars in 209 analyses, and L. monocytogenes was isolated in 135 of these analyses. In 27 of the analyses in which L. monocytogenes was isolated, one or more of the selective differential agars used failed to isolate L. monocytogenes, and therefore the results of these analyses were discrepant. Relative to a reference method involving the use of all three agars (OXF, PAL, and BCM agars), the OXF-BCM, PAL-BCM, and OXF-PAL combinations had sensitivities of 99.3, 99.2, and 90.2%, respectively. In statistical analyses of the different combinations of agars, the OXF-BCM and BCM-PAL combinations were found to be superior to the OXF-PAL combination for the detection of L. monocytogenes.     

乳粉中单核细胞增生李斯特氏菌能力验证结果分析

摘要：目的 对本实验室单核细胞增生李斯特氏菌检测能力进行验证,以提高或保证实验室能力验证工作中对单核细胞增生李斯特氏菌检测结果的准确性。方法 按照样品的作业指导书开启样品,按照GB 4789.30-2016《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》进行检验,同时用MALEI-TOF-MS质谱仪对可疑菌进行鉴定,对2种方法结果进行比对。结果 FC02220009样品检出单核细胞增生李斯特氏菌;FC02220080样品未检出单核细胞增生李斯特氏菌。国标方法和MALDI-TOF-MS方法鉴定结果一致。结论 为保证检验结果准确可靠,避免假阴性出现,可多种鉴定手段同时进行。