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。     

沙门氏菌、金黄色葡萄球菌、志贺氏菌、单增李斯特菌选择性共增菌培养基的研制

目的：研制一种对沙门氏菌（Salmonella）、金黄色葡萄球菌（Staphylococcus aureus）、志贺氏菌（Shigella）和单增李斯特菌（Listeria monocytogens）的选择性共增菌培养基（SSSL培养基）。方法：挑选添加成分进行单因素试验,确定SSSL培养基的成分及配比,采用平板计数法验证SSSL培养基的增菌效果。结果：确立了SSSL培养基配方,目标菌在SSSL增菌培养基中培养8 h后,菌体浓度都达到了105~106CFU/mL,而且抑制非目标菌的生长。结论：SSSL培养基能用于沙门氏菌、金黄色葡萄球菌、志贺氏菌和单增李斯特菌选择性共增菌,可望与多种检测方法联用,以提高检测率和准确性。     

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。     

一种能同时富集沙门氏菌、金黄色葡萄球菌和单增李斯特菌的培乔基

[目的]设计制备一种能够同时富集沙门氏菌、金黄色葡萄球菌及单增李斯特菌的复合增菌肉汤.[方法]挑选合适的添加剂进行单因素实验,确定增菌肉汤的成分及配比,采用平板计数法及三重荧光PCR技术验证肉汤的增菌效果.[结果]结果得到一种能同时富集沙门氏菌、金黄色葡萄球菌及单增李斯特菌的选择性增菌肉汤(SSL),经验证SSL可使得3种目标菌以相对一致的速度进行富集,经过37℃ 150 r/min振荡培养24 h后,菌体浓度到达10~7～10~8 CFU/mL,非目标菌生长受到抑制.应用荧光PCR扩增样品,可同时得到3种目标菌的扩增曲线.在710份实际样品检测中,无假阳性及假阴性报告.[结论]研究结果表明,SSL肉汤可用于沙门氏菌、金黄色葡萄球菌及单增李斯特菌的共增菌,可用于多重PCR检测的前增菌.     

一种能同时富集沙门氏菌、金黄色葡萄球菌和单增李斯特菌的培养基

摘要：【目的】设计制备一种能够同时富集沙门氏菌、金黄色葡萄球菌及单增李斯特菌的复合增菌肉汤。【方法】挑选合适的添加剂进行单因素实验,确定增菌肉汤的成分及配比,采用平板计数法及三重荧光PCR技术验证肉汤的增菌效果。【结果】结果得到一种能同时富集沙门氏菌、金黄色葡萄球菌及单增李斯特菌的选择性增菌肉汤（SSL）,经验证SSL可使得3种目标菌以相对一致的速度进行富集,经过37℃ 150r/min 振荡培养24h后,菌体浓度到达107~108CFU/mL,非目标菌生长受到抑制。应用荧光PCR扩增样品,可同时得到3种     

食品中单核细胞增生李斯特氏菌的测量不确定度评定

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》第二法平板计数法和JJF 1059.1—2012《测量不确定度评定与表示》,对单增李斯特氏菌平板计数法测量不确定度的来源进行分析,以建立食品中单核细胞增生李斯特氏菌平板计数法的测量不确定度评定方法。结果发现:通过建立数学模型T=AB/Cd,分析不确定度来自样品制备、样品稀释及重复测量的过程,样品的存储采集、样品的均质、培养基配制的时间、培养基灭菌温度、培养箱温度及培养时间对总不确定度的贡献较小。单核细胞增生李斯特氏菌平板计数法的检测结果为(2.9±1.2)×10~6CFU/g,对同一样品测定10次的扩展不确定度为1.2×10~6。     

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。[结论] 建立的荧光RAA法可用于口岸和其他场所进行单增李斯特菌的快速检测与监控。     

选择性富集沙门氏菌、副溶血弧菌和霍乱弧菌的共增菌培养基SVV

本研究通过单因素试验和响应面分析试验建立了能够选择性富集沙门氏菌、副溶血弧菌和霍乱弧菌的共增菌培养基SVV,采用平板计数法及三重荧光PCR技术验证了SVV的增菌效果。结果表明:SVV能同时富集以不同浓度比例混合的3种目标菌,37oC振荡培养18h后,菌体浓度达到105~108CFU/mL;SVV强烈抑制大部分的非目标菌;用荧光PCR方法检测经过37oC振荡培养18h的10份人工接种样品和608份实际样品,结果表明目标菌在SVV中增殖18h后菌量达到检测限以上,SVV联合荧光PCR检测方法的检出率为4.06%,比传统检测方法(3.78%)高,无假阴性和假阳性。SVV可望应用于水产品中沙门氏菌、副溶血弧菌和霍乱弧菌检测前的增菌处理,可简化检测过程,有效克服漏检,提高检出率。     

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。     

餐饮厨房中单增李斯特菌污染的分布特征及风险分析

为了解餐饮厨房中自制即食食品单增李斯特菌污染状况及厨房内各媒介单增李斯特菌污染的分布特征,并据此分析餐饮厨房中导致即食食品发生单增李斯特菌污染的可能高风险点,在我国3个省份的29个餐饮厨房中采集环境样本及厨房自制餐饮即食食品1 455份进行单增李斯特菌检验。检测结果进行统计学分析,探寻餐饮厨房自制即食食品污染与环境暴露之间的关系。结果显示:餐饮厨房中单增李斯特菌的污染广泛存在,自制即食食品、食品原料与环境样品中单增李斯特菌的检出无差异。针对单增李斯特菌的监测,应关注餐饮环节的污染,凉菜间的冰箱可能成为导致餐饮厨房自制即食食品被单增李斯特菌污染的危险因素。     

Specific detection of cytopathogenic Listeria monocytogenes using a two-step method of immunoseparation and cytotoxicity analysis

The development of rapid methods for detection of viable Listeria monocytogenes is crucial to prevent listeriosis and product recalls. While immunomagnetic separation has been used for isolating Listeria spp., lack of specificity and pathogenicity determination render this method unsatisfactory. A two-step method using Protein A agarose beads (Immunobeads) coated with a more specific antibody, monoclonal antibody (MAb)-C11E9 for L. monocytogenes was developed. Immunobeads were allowed to capture Listeria cells from a variety of samples and tested for cytopathogenic action on a murine hybridoma B-lymphocyte, Ped-2E9 cell line by Trypan blue staining, and by an alkaline phosphatase (AP)-based cytotoxicity assay. The two-step method was used to test uninoculated hotdogs, bologna, and raw beef, chicken, and pork samples, following selective enrichment in half-Fraser broth. Pure culture studies proved the assay to be specific for L. monocytogenes, while a similar assay with Dynal Anti-Listeria immunomagnetic beads was positive for L. monocytogenes, L. ivanovii, and L. seeligeri. Detection and confirmation of cytopathogenicity of Listeria cells from food samples after 24-h selective enrichment were completed in 2-4 h. Isolates were further analyzed by the CAMP test for hemolytic activity and RiboPrinter for genomic patterns. Using immunoseparation and cytotoxicity as a two-step rapid method, viable L. monocytogenes could be isolated, detected, and confirmed as cytopathogenic in 28 h or less.     

Detection of Listeria monocytogenes in cheese with the magnetic immuno-polymerase chain reaction assay.

A new detection system, the magnetic immuno-polymerase chain reaction (PCR) assay (MIPA) has been developed to detect Listeria monocytogenes in food. This method separates Listeria cells from PCR-inhibitory factors present in enrichment broths containing food samples by using magnetic beads coated with specific monoclonal antibodies (MAbs). The separated bacteria were lysed, and the supernatant containing the bacterial DNA was subjected to the PCR. Detection of L. monocytogenes in three naturally contaminated cheese samples with two different MAbs and PCR primers specific for the gene encoding the delayed-hypersensitivity factor showed that with MAb 55 all three samples were positive whereas with MAb A two samples were positive. A further improvement of the method was obtained by using a PCR step based on the listeriolysin O gene. A MIPA employing MAb 55 and the listeriolysin O gene primer set detected L. monocytogenes after 24 h of culture in Listeria Enrichment Broth samples from Port Salut artificially contaminated with 40 CFU/25 g. We could detect 1 CFU of L. monocytogenes per g of cheese after a second enrichment for 24 h in Fraser broth. The analysis time including both enrichments is approximately 55 h.     

Direct detection of Listeria monocytogenes in 25 milliliters of raw milk by a two-step PCR with nested primers.

A method for direct detection of Listeria monocytogenes in 25 ml of raw milk is presented. The detection limit can be situated between 10 and 5 CFU. The detection method is based on chemical extraction of the milk components and PCR amplification with two nested pairs of primers specific for Listeria monocytogenes.     

A comparison of immunomagnetic and surface adhesion immunofluorescent techniques for the rapid detection of Listeria monocytogenes and Listeria innocua in meat

an immunomagnetic immunofluorescent method was investigated for the rapid detection of Listeria monocytogenes and Listeria innouca . This technique involved enrichment of the suspect sample at 30°C overnight. Listeria monocytogenes cells were isolated from the enriched sample using immunomagnetic separation and Listeria were subsequently visualized using an immunofluorescent microscopy technique. This technique was used in the detection of Listeria cells from pure culture, inoculated beef mince samples and naturally contaminated retail beef mince samples. A detection level of approximately 1×10³ cfu ml⁻¹ was achieved. When compared with traditional detection methods no false negatives or positives were recorded for L. monocytogenes or L. innocua . The immunomagnetic immunofluorescent technique had a detection level similar to a previously described surface adhesion immunofluorescent technique. Isolation of the Listeria cells by surface adhesion involved dipping a membrane attached to a microscope slide into the enriched sample for 10 min. This was quicker and simpler to perform than the immunomagnetic separation technique which took 2 h to carry out.     

基于免疫磁分离的三重荧光定量PCR检测食品中沙门氏菌、志贺氏菌和金黄色葡萄球菌

【目的】开发一种同时对食品中沙门氏菌、志贺氏菌和金黄色葡萄球菌快速、灵敏、准确的检测方法。【方法】利用特异性免疫磁球,在37°C条件下从250 m L猪肉增菌液体系中边富集边循环捕获目标菌。快速提取DNA后,利用特异性的引物与探针,对3种食源性致病菌进行三重荧光定量PCR检测。【结果】针对沙门氏菌、志贺氏菌和金黄色葡萄球菌的检测限分别达到2.0、6.8和9.6 CFU/g。方法总体灵敏度、特异性和准确度达到99.2%、100%及99.5%。对151份实际样品进行检测,与国标(GB/T 4789.4-2010、GB 4789.5-2012和GB/T4789.10-2010)方法的检测结果相比,金黄色葡萄球菌有一例阴性偏差。【结论】开发的基于免疫磁分离的三重荧光定量PCR方法,能够在8 h内完成对食品中3种致病菌检测,并且灵敏度高、特异性好、检测准确,可以作为快速应对此类食品安全突发事件的检测手段。     

A CHEMILUMINESCENCE BIOSENSOR COUPLED WITH IMMUNOMAGNETIC SEPARATION FOR RAPID DETECTION OF SALMONELLA TYPHIMURIUM

A chemiluminescence biosensor, using a fiber-optic-linked photometer and a data acquisition unit connected to a PC, was developed in conjunction with immunomagnetic separation for rapid detection of Salmonella Typhimurium. Magnetic microbeads coated with Anti-Salmonella antibodies and anti-Salmonella antibodies conjugated with horseradish peroxidase (HRP) were added to artificially-inoculated samples, and the immuno-reaction was completed in 60 min resulting in a sandwich complex. A magnetic field was applied to collect magnetic beads and the addition of luminol to HRP-conjugated antibodies resulted in a chemiluminescence reaction. The signal was collected through a fiber optic light guide, measured with a photometer, and recorded in the data acquisition unit. The minimum detection limit of the chemiluminescence biosensor for S. Typhimurium was 1.97 × 10³ CFU/mL and the range of the detectable signal was from 8.6 to 350 mV for cell numbers from 1.97 × 10³ to 1.97 × 10⁶ CFU/mL. Signal values for 10⁶ CFU/mL of S. Typhimurium were at least 97 and 394% higher than the corresponding values for S. enteritidis and four times the signal values for others including S. montevideo, S. california, S. heidlberg, and S. seftenberg, respectively. The biosensor response showed a significant difference (P < 0.05) between 10³ CFU/mL S. Typhimurium and 10⁶ CFU/mL of commonly-occurring bacteria in foods including Listeria monocytogenes, Pseudomonas aeruginosa, Citrobacter freundii, Campylobacter jejuni, Escherichia coli O157, and generic Escherichia coli. A regression equation, V = 0.0262 N ^5.7713, with R²= 0.9713 was obtained for the calibration curve over the detection range for S. Typhimurium. The whole procedure could be completed within 90 min.     

Sensitive and specific detection of Listeria monocytogenes in milk and ground beef with the polymerase chain reaction.

A sensitive and specific method for detection of Listeria monocytogenes in milk and ground-beef samples is described. It consists of culturing samples in listeria enrichment broth (LEB) and subculturing them from LEB to listeria plating media, followed by DNA extraction and species-specific detection of the organism by using the polymerase chain reaction (PCR). In developing the L. monocytogenes PCR assay, five oligonucleotide primers complementary to the nucleotide sequence of the listeriolysin O gene were synthesized and used in amplification experiments. PCR products of the predicted size, based on nucleotide sequence information, were generated with DNA from all of 72 L. monocytogenes strains with five different primer pairs. DNA from Listeria ivanovii, Listeria innocua, Listeria seeligeri, Listeria welshimeri, Listeria grayi, and Listeia murrayi strains and a panel of 47 bacterial strains representing 17 genera did not generate PCR products with the primer pairs employed. As little as 1 pg of L. monocytogenes DNA could be detected with the assay. To determine the most sensitive culture protocol to use in conjunction with the PCR assay, milk (10 ml) and ground-beef (25 g) samples were inoculated with L. monocytogenes at concentrations ranging from 0 to 10(5) CFU ml-1 or g-1, as appropriate for the sample. PCR assays on DNA extracted from growth on listeria plating media, inoculated with 24-h LEB samples cultures, were most sensitive, allowing detection of as little as 0.1 CFU of L. monocytogenes ml-1 or g-1 of milk and ground beef, respectively.     

Sensitive and specific detection of Listeria monocytogenes in milk and ground beef with the polymerase chain reaction.

A sensitive and specific method for detection of Listeria monocytogenes in milk and ground-beef samples is described. It consists of culturing samples in listeria enrichment broth (LEB) and subculturing them from LEB to listeria plating media, followed by DNA extraction and species-specific detection of the organism by using the polymerase chain reaction (PCR). In developing the L. monocytogenes PCR assay, five oligonucleotide primers complementary to the nucleotide sequence of the listeriolysin O gene were synthesized and used in amplification experiments. PCR products of the predicted size, based on nucleotide sequence information, were generated with DNA from all of 72 L. monocytogenes strains with five different primer pairs. DNA from Listeria ivanovii, Listeria innocua, Listeria seeligeri, Listeria welshimeri, Listeria grayi, and Listeia murrayi strains and a panel of 47 bacterial strains representing 17 genera did not generate PCR products with the primer pairs employed. As little as 1 pg of L. monocytogenes DNA could be detected with the assay. To determine the most sensitive culture protocol to use in conjunction with the PCR assay, milk (10 ml) and ground-beef (25 g) samples were inoculated with L. monocytogenes at concentrations ranging from 0 to 10(5) CFU ml-1 or g-1, as appropriate for the sample. PCR assays on DNA extracted from growth on listeria plating media, inoculated with 24-h LEB samples cultures, were most sensitive, allowing detection of as little as 0.1 CFU of L. monocytogenes ml-1 or g-1 of milk and ground beef, respectively.     

RAPID DETECTION OF SALMONELLA TYPHIMURIUM IN FOOD SAMPLES USING A BIENZYME ELECTROCHEMICAL BIOSENSOR WITH FLOW INJECTION

A bienzyme (tyrosinase and horseradish peroxidase) electrochemical biosensor was developed for detection of Salmonella typhimurium, and evaluated for application in a flow injection system coupled with immunomagnetic separation for food samples. Parameters for immunomagnetic separation, enzymatic reaction, flow injection and electrochemical detection were determined using pure culture samples. The selectivity was tested in the presence of Listeria monocytogenes, Campylobacter jejuni and E. coli 0157:H7. The results showed a linear relationship for logarithmic values between peak current ratio and the cell number of S. typhimurium in the range of 10³ 10⁵ cfu/mL, with R²= 0.99. The detection limit of this method was 1.09 × 10³ cfu/mL for S. typhimurium and the detection time was 2.5 h. Samples of chicken carcass wash water and ground beef were used to evaluate the biosensor. The results demonstrated that this biosensor has a potential for rapid detection of different pathogens in various food samples.