单核细胞增生李斯特氏菌PCR-DHPLC检测新技术的建立

应用PCR结合变性高效液相色谱(DHPLC)技术建立食品中单核细胞增生李斯特氏菌fimY的快速检测方法。根据单核细胞增生李斯特氏菌prfA和hlyA基因序列的特点设计特异性引物,PCR扩增的产物经DHPLC技术进行快速检测。以单核细胞增生李斯特氏菌等61株参考菌株做特异性试验;单核细胞增生李斯特氏菌菌株稀释成不同梯度,进行灵敏度试验。试验结果表明该方法具有很好的特异性,灵敏度较高,检测低限可达到为181CFU/ml。可以快速、准确检测单核细胞增生李斯特氏菌,是食品中致病菌快速检测的新技术。     

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。     

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.     

乳制品中四种病原菌多重荧光PCR检测方法的建立与应用

本研究建立了可快速特异检测乳制品中阪崎肠杆菌、沙门氏菌、金黄色葡萄球菌和单核细胞增生李斯特氏菌的多重荧光PCR方法。通过设计阪崎肠杆菌、沙门氏菌、金黄色葡萄球菌和单核细胞增生李斯特氏菌的特异性引物和探针,建立了多重PCR反应体系,评价了该方法的特异性、灵敏度和精密度,并用该方法和国标法分别检测了150份乳制品中的4种致病菌,对检测结果进行对比。结果显示,检测目标菌为相应阳性,检测其他12种非目标供试菌为阴性;检测灵敏度分别为1 cfu/μL;其精密度的变异系数均小于5%;与国标检测方法相比其检测结果具有良好的一致性。     

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。     

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。     

单核细胞增生李斯特菌的检测技术进展

单核细胞增生李斯特菌（Listeria monocytogenes）是一类人畜共患的食源性致病菌。近年来其检测技术取得了迅猛的发展,本文对目前使用的基于培养、免疫学和分子生物学技术的三大类单核细胞增生李斯特菌检测方法进行了综述,同时对单核细胞增生李斯特菌检测的新策略进行了展望。     

2016-2018年辽宁省食品中单核细胞增生李斯特菌污染情况及毒力基因的检测

目的了解辽宁省食品中单核细胞增生李斯特菌毒力基因携带特点,对该省食品中单核细胞增生李斯特菌的污染情况进行调查。方法依据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》及采用PCR扩增技术检测的方法对2016-2018年采自该省15个监测点、收集的3 310份食品检出的47株单核细胞增生李斯特菌进行9种毒力基因检测。结果食品中单核细胞增生李斯特菌检出率为1.42%(47/3310),食品中单核细胞增生李斯特菌最少携带3种毒力基因,其中携带prfA、plcA、hly、mpl、plcB、inlA、inlB和iap八种毒力基因是该省食品中单核细胞增生李斯特菌的主要毒力基因型,达到检出菌总数的65.96%。结论研究结果证实辽宁省食品中存在单核细胞增生李斯特菌污染情况,应严格监控食品中单核细胞增生李斯特菌的携带情况。     

单增李斯特菌InternalinA的表达、纯化及多克隆抗体的制备

【目的】克隆表达单增李斯特菌膜表面蛋白InternalinA(InlA),经免疫家兔获得多克隆抗体,为建立其免疫磁珠富集快速检测方法奠定基础。【方法】利用生物软件设计单增李斯特菌inlA基因的引物,通过PCR扩增出inlA基因,并将其克隆至pET28a()原核表达载体,转化大肠杆菌BL21进行优化表达。镍柱纯化表达产物,质谱鉴定重组蛋白,ELISA分析其免疫原性。免疫家兔,制备其多克隆抗体。间接ELISA检测多抗的效价及交叉性,免疫荧光分析多抗与单增李斯特菌菌体结合的特异性。【结果】成功表达了InlA蛋白,融合表达产物分子量约为92 kD,质谱鉴定其为InlA蛋白;免疫家兔获得的抗血清效价为1:100 000,除与金黄色葡萄球菌约20%的交叉外,与副溶血弧菌等其它病源菌均无交叉;免疫荧光证实该多抗特异性结合于单增李斯特菌膜表面,与同种属的威尔斯李斯特菌不结合。【结论】成功制备了单增李斯特菌特异性的兔多克隆抗体,为单增李斯特菌免疫磁珠富集快速检测方法的建立奠定了基础。     

单核细胞增生李斯特菌检测技术研究进展

单核细胞增生李斯特菌(Listeria monocytogenes,LM)是常见的食源性致病菌之一。目前,在众多单增李斯特菌的检测方法中应用较广的是免疫学检测法、分子学检测法。免疫学检测时间短,操作简单,但该方法依赖高特异性的抗体,会出现假阳性,还需要进一步鉴定检测结果。分子学检测法克服了免疫学检测法不能在种的水平鉴定单增李斯特菌的缺点,省时省力,灵敏度高,但是分子学检测法需要丰富的操作经验,并且不适于现场大批量检测。新兴的代谢学检测法、光谱学检测法、生物传感器等也都有各自的优缺点。本文综合近年最新文献,就单增李斯特菌检测的最新方法、检测进展及未来发展趋势予以分析综述,以期为该菌的检测提供参考。     

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.     

结核分枝杆菌抗原检测研究进展

结核病仍然是一个严重的全球性公共卫生问题,有效控制结核病的障碍在于缺乏早期、准确的诊断方法。机体受到结核分枝杆菌感染后,体内首先出现的是结核分枝杆菌特异性抗原。因此,结核分枝杆菌抗原检测作为结核病早期诊断的方法可能具有很高的诊断价值。我们简要综述了结核分枝杆菌抗原检测的相关研究进展。     

生物传感器在食源性致病菌大肠杆菌O157∶H7检测中的应用进展

食源性致病菌是引发食物中毒的主要微生物，对人类的健康构成了重要威胁，也逐渐发展为公共卫生、食品工业部门高度重视的一个问题。近年来，因食源性致病菌产生的疾病在世界各地蔓延，其中，大肠杆菌O157∶H7是食源性致病菌种类中造成病死率较高的一种细菌，其感染剂量低、致病性强等特点引起研究人员和世界各国卫生组织的广泛关注。基于此，对大肠杆菌O157∶H7的检测方法及其演变进行了梳理和比较分析，并着重综述了生物传感器（biosensor）在其检测中的应用进展，以期为大肠杆菌O157∶H7快速、准确和可商业化的检测方法的研发提供参考。     

Detection and identification of Listeria monocytogenes in cooked sausage products and in milk by in vitro amplification of haemolysin gene fragments

Recent outbreaks of listeriosis have emphasized the urgent need for rapid and reliable detection methods for Listeria spp., especially in food. Haemolysin production is a major factor in the pathogenesis of listeriosis and the polymerase chain reaction (PCR) was used to amplify two specific DNA fragments of the alpha- and the beta-haemolysin genes. The amplification system specifically recognized L. monocytogenes strains. The detection limit determined with pure cultures was 10 bacteria when estimated with alpha-haemolysin primers. In the analysis of 50 samples of cooked sausage products, bacterial colonies suspected to be Listeria spp. were isolated by conventional methods from six samples. PCR analysis identified three of six as L. monocytogenes. Subsequent serotyping showed perfect agreement with the PCR results. Since enrichment is the most time consuming step in conventional methods a PCR procedure which allows the direct detection of L. monocytogenes in milk was developed. Pasteurized milk was artificially contaminated with various levels of L. monocytogenes. The detection limit was determined to be 10 bacteria/10 ml milk and direct detection and identification of L. monocytogenes took less than two working days. These results show that this haemolysin gene amplification system is very rapid and reliable and therefore avoids cumbersome and lengthy cultivation steps.     

Detection and identification of Listeria monocytogenes in cooked sausage products and in milk by in vitro amplification of haemolysin gene fragments

Recent outbreaks of listeriosis have emphasized the urgent need for rapid and reliable detection methods for Listeria spp., especially in food. Haemolysin production is a major factor in the pathogenesis of listeriosis and the polymerase chain reaction (PCR) was used to amplify two specific DNA fragments of the α- and the β-haemolysin genes. The amplification system specifically recognized L. monocytogenes strains. The detection limit determined with pure cultures was 10 bacteria when estimated with α-haemolysin primers. In the analysis of 50 samples of cooked sausage products, bacterial colonies suspected to be Listeria spp. were isolated by conventional methods from six samples. PCR analysis identified three of six as L. monocytogenes. Subsequent serotyping showed perfect agreement with the PCR results. Since enrichment is the most time consuming step in conventional methods a PCR procedure which allows the direct detection of L. monocytogenes in milk was developed. Pasteurized milk was artificially contaminated with various levels of L. monocytogenes. The detection limit was determined to be 10 bacteria/10 ml milk and direct detection and identification of L. monocytogenes took less than two working days. These results show that this haemolysin gene amplification system is very rapid and reliable and therefore avoids cumbersome and lengthy cultivation steps.     

数字PCR在食源性致病菌检测中的应用进展

食源性致病菌是食品安全的重大隐患,对人类健康造成极大危害,因此亟待研究和建立精准有效的食源性致病菌检测方法。随着单分子检测技术的快速发展,数字PCR技术因其具有超高的灵敏度、稳定性和低试剂消耗等优点而被广泛应用于食源性致病菌的检测。主要介绍了数字PCR的基本原理及其研究进展,深入探讨了其在检测大肠杆菌（Escherichia coli）、沙门氏菌（Salmonella）、金黄色葡萄球菌（Staphylococcus aureus）、空肠弯曲菌（Campylobacter jejuni）、志贺氏菌（Shigella）、克罗诺杆菌（Cronobacter）、副溶血性弧菌（Vibrio parahaemolyticus）、单核细胞增生李斯特氏菌（Listeria monocytogenes）和蜡状芽孢杆菌（Bacillus cereus）中的应用,为今后该技术在食源性致病菌检测中的研究与应用提供一定的技术性参考。     

重组酶介导扩增技术及其在病原微生物快速检测中的应用进展*

建立快速的病原学诊断方法对动物疫病的预防和控制、公共卫生安全等方面具有重要意义。重组酶介导扩增法(RAA)是一种新型恒温体外核酸扩增技术,在体外较低温度下就可以实现对DNA或RNA的快速扩增。RAA具有操作简便、快速、准确、节能、便捷等优点。重组酶、单链结合蛋白和DNA聚合酶是该技术的三大核心物质,利用这三种物质可替代传统PCR的热循环解链过程。该技术将会在病原微生物检测方面发挥重要作用。对RAA技术及其在病原检测方面的应用进行了综述,以期为相关领域的研究提供参考。     

Clinical applications of molecular biology for infectious diseases

Molecular biological methods for the detection and characterisation of microorganisms have revolutionised diagnostic microbiology and are now part of routine specimen processing. Polymerase chain reaction (PCR) techniques have led the way into this new era by allowing rapid detection of microorganisms that were previously difficult or impossible to detect by traditional microbiological methods. In addition to detection of fastidious microorganisms, more rapid detection by molecular methods is now possible for pathogens of public health importance. Molecular methods have now progressed beyond identification to detect antimicrobial resistance genes and provide public health information such as strain characterisation by genotyping. Treatment of certain microorganisms has been improved by viral resistance detection and viral load testing for the monitoring of responses to antiviral therapies. With the advent of multiplex PCR, real-time PCR and improvements in efficiency through automation, the costs of molecular methods are decreasing such that the role of molecular methods will further increase. This review will focus on the clinical utility of molecular methods performed in the clinical microbiology laboratory, illustrated with the many examples of how they have changed laboratory diagnosis and therefore the management of infectious diseases.