基于环介导等温扩增技术的产呕吐毒素 蜡样芽胞杆菌特异性检测

本研究旨在建立食品中产呕吐毒素蜡样芽胞杆菌的快速检测方法。基于产呕吐毒素蜡样芽胞杆菌合成酶基因cesB靶基因,设计4条特异性引物(2条内引物、2条外引物),建立环介导等温扩增技术(LAMP)。然后采用2株产呕吐毒素蜡样芽胞杆菌、19株蜡样芽胞杆菌和41株非蜡样芽胞杆菌验证了该LAMP具有很好的特异性。LAMP检测的灵敏度在DNA水平上达到1.49 pg/μL,纯菌的灵敏度为5×10~3 cfu/mL。人工污染米饭样品,当起始污染量为2 cfu/g时,37℃增菌培养6 h,用试剂盒法和煮沸法提取的DNA,都可以检出产呕吐毒素蜡样芽胞杆菌。采用此LAMP方法进行了72份实际样品的检测,检出2份产呕吐毒素蜡样芽胞杆菌阳性样品,与传统方法一致。因此,本研究建立的LAMP检测方法可应用于食品中产呕吐毒素蜡样芽胞杆菌的快速特异检测。     

3种常见食源性致病菌多重重组酶聚合酶扩增检测方法研究

目的建立了食品中沙门菌、单核细胞增生李斯特菌和蜡样芽胞杆菌多重重组酶聚合酶扩增（recombinase polymerase amplification,RPA）快速检测方法。方法选择沙门菌invA基因、单增李斯特菌hlyA基因和蜡样芽孢杆菌16S RNA序列为目标基因进行扩增,建立并优化多重RPA扩增体系和扩增条件;评价反应体系的特异性和灵敏度,并对人工污染食品样品和实际样品进行检测。结果多重RPA反应体系能够在20 min完成三种目标基因的扩增,特异性强;对沙门菌、单增李斯特菌和蜡样芽孢杆菌的灵敏度分别为2.70×105、1.30×105、1.44×104 CFU/mL;能够用于人工污染样品和实际样品的检测。结论本研究建立的多重RPA等温扩增方法特异性强,操作快速、简单,为食源性致病菌的快速检测提供新方向     

环介导等温扩增技术快速检测椰毒假单胞菌的研究

建立环介导等温扩增技术(LAMP)快速检测椰毒假单胞菌的方法.根据公布的椰毒假单胞菌16S～23SrRNA基因序列设计引物,建立了LAMP反应体系,在此基础上检测了蜡样芽孢杆菌菌液和人工污染蜡样芽孢杆菌的银耳样品,并将LAMP法与PCR法进行比较.结果表明,LAMP检测方法具有较高的特异性和敏感性,椰毒假单胞菌的检出限为5.4CFU/mL,是PCR方法检测灵敏度的1000倍,人工污染银耳样品中的椰毒假单胞菌的检出限为76CFU/g,样品中椰毒假单胞菌的检测过程(包括DNA提取、LAMP和电泳)可在2h内完成,因此LAMP可以简便快速有效地检测食品中的椰毒假单胞菌.     

食品中蜡样芽孢杆菌的检测方法研究进展

蜡样芽孢杆菌是一种食源性致病菌,可引起恶心、呕吐、腹胀、腹痛及腹泻等疾病,因此,建立食品中蜡样芽孢杆菌的快速检测方法至关重要。总结近年来食品中蜡样芽孢杆菌的检测方法,主要包括以基因检测为基础的聚合酶链式反应及实时荧光定量PCR技术,这两种方法可快速对菌种进行定性和定量,是食品中致病菌检测的常用方法,但是有些致病相关毒素是预先形成的,即使菌体被破坏,毒素依然存在于食品中,所以,该方法不能全面评估致病菌污染的食品引起食物中毒的风险。     

免疫磁珠技术分离检测熟制食品中的蜡样芽孢杆菌

目的 建立一种免疫磁珠技术分离富集熟制食品中的蜡样芽孢杆菌。方法 在一定量的蜡样芽孢杆菌体系中优化免疫磁珠与抗体用量以及最佳反应时间。在最佳实验条件下, 对免疫磁珠分离蜡样芽孢杆菌的敏感性和特异性进行实验, 并对实际熟制食品的蜡样芽孢杆菌进行分离。结果 最终确定了蜡样芽孢杆菌抗体添加量50 μg/mL、免疫磁珠添加量100 μL, 免疫磁珠与菌液最佳作用时间为15 min, 当菌液稀释度达到10?7, 免疫磁珠法仍然可以检出, 相应的细菌数为4 CFU/mL。结论 本实验制备的免疫磁珠技术灵敏性高, 特异性好, 节省了检测时间和费用, 适用于食品中的蜡样芽孢杆菌的检测。     

三类传统发酵食品中蜡样芽孢杆菌的污染状况研究

为探究辣椒酱、酸汤、毛豆腐三类传统发酵食品的蜡样芽孢杆菌（Bacillus cereus）污染状况,采用国标GB 4789.14—2014《食品微生物学检验蜡样芽孢杆菌检验》方法测定样品的污染菌数及分离菌株的生理生化特征,并通过聚合酶链式反应（PCR）方法探究分离菌株的毒力基因谱和多样性。结果表明,5个辣椒酱及4个酸汤样品的蜡样芽孢杆菌检出率均为100%,菌数在3.6～460 MPN/g之间。而7个毛豆腐的蜡样芽孢杆菌检出率为28.57%,其中2个夏季样品的蜡样芽孢杆菌菌数>7 lg(CFU/g),5个秋季样品未检出蜡样芽孢杆菌,污染差异可能与气温变化、运输方式有关,需要实地采样并进行季节性分析。从三类样品中分离的94个菌株均符合蜡样芽孢杆菌生理生化特征,毒力基因均为腹泻型,且菌株多样性与样品差异性有关。综上,蜡样芽孢杆菌的污染特性具有明显的样品差异性,可能与发酵食品的原料、发酵工艺相关。     

食品中蜡样芽孢杆菌的研究进展

蜡样芽孢杆菌是常见食品污染菌,其产生的毒素可引起食物中毒.文中概述了蜡样芽孢杆菌的生物学特性、致病性、安全性、检测方法、食品中的限量标准及其控制方法.有效地控制该菌在食品中的污染,保护公众健康.     

实时荧光环介导等温扩增技术检测牛乳中的蜡样芽孢杆菌

建立牛乳中蜡样芽孢杆菌便捷可靠的检测方法,根据已公布的蜡样芽孢杆菌hblA基因序列设计内外引物,并向反应体系中加入荧光染料SYBRGreen Ⅰ,利用实时荧光监测仪,建立实时荧光环介导等温扩增技术（real-timefluorescence loop-mediated isothermal amplification,RealAmp）检测蜡样芽孢杆菌的方法,扩增产物经电泳和酶切鉴定。通过21 株致病菌验证RealAmp特异性,并比较了RealAmp与普通环介导等温扩增技术的敏感性,对人工污染的检出限进行了测定。结果表明对21 株致病菌进行特异性实验,4 株蜡样芽胞杆菌呈阳性结果,17 株非蜡样芽胞杆菌均呈阴性结果。RealAmp检测纯菌的灵敏度为8.2 CFU/mL,比普通环介导等温扩增技术的灵敏度高10 倍,人工污染牛乳RealAmp的检出限为8.2 CFU/mL。并且在20 min左右即可判定结果。该方法快速、准确、灵敏度高、操作便捷、可实时监控检测蜡样芽孢杆菌,有望成为快速检测蜡样芽孢杆菌的有效方法。     

传统发酵豆制品中3种致病菌的三重荧光PCR快速检测方法的建立

为了建立传统发酵豆制品中单增李斯特菌、蜡样芽孢杆菌和金黄色葡萄球菌的三重荧光PCR快速检测方法。以单增李斯特菌hly A基因、蜡样芽孢杆菌Cereolysin AB基因和金黄色葡萄球菌nuc基因为靶基因设计引物与TaqMan探针,通过优化PCR反应体系,建立了可同时检测单增李斯特菌、蜡样芽孢杆菌和金黄色葡萄球菌的三重荧光定量PCR体系,并进行了特异性和敏感性试验。结果显示,该方法灵敏度高,特异性强,重复性好。对26株非目标菌进行检测,结果均为阴性,而定量检测批内和批间的变异系数均小于2%。单增李斯特菌、蜡样芽孢杆菌和金黄色葡萄球菌敏感性试验结果表明,这三种细菌的最低检测浓度分别为3×103cfu/mL、2×104cfu/mL、2×104cfu/mL。应用该方法可在8h内完成对样品中单增李斯特菌、蜡样芽孢杆菌和金黄色葡萄球菌的同步检测。     

食源性蜡样芽孢杆菌的危害及其检测方法研究进展

蜡样芽孢杆菌是一种常见的食源性致病菌,可通过污染乳制品、米饭、散装熟肉制品和豆制品等食品引起婴幼儿及成人食物中毒。采用准确、高效的蜡样芽孢杆菌检测方法,是预防食源性蜡样芽孢杆菌病及食品安全质量控制的关键。蜡样芽孢杆菌检测方法主要包括细菌培养分离鉴定法、免疫学检测方法和核酸检测方法等。本文总结了各类检测方法的核心技术特征和应用实例,为食源性蜡样芽孢杆菌的快速检测方法的研发和使用提供思路。     

Development of rapid real-time PCR and most-probable-number real-time PCR assays to quantify enterotoxigenic strains of the species in the Bacillus cereus group

Members of the Bacillus cereus group may produce diarrheal enterotoxins and could be potential hazards if they enter the food chain. Therefore, a method capable of detecting all the species in the B. cereus group rather than B. cereus alone is important. We selected nhe as the target and developed a real-time PCR assay to quantify enterotoxigenic strains of the B. cereus group. The real-time PCR assay was evaluated with 60 B. cereus group strains and 28 others. The assay was also used to construct calibration curves for different food matrices and feces. The assay has an excellent quantification capacity, as proved by its linearity (R2 > 0.993), wide dynamic quantification range (10(2) to 10(7) CFU/g for cooked rice and chicken, 10(3) to 10(7) CFU/ml for milk, and 10(4) to 10(7) CFU/g for feces), and adequate relative accuracy (85.5 to 101.1%). For the low-level contaminations, a most-probable-number real-time PCR assay was developed that could detect as low as 10(0) CFU/ml. Both assays were tested with real food samples and shown to beconsiderably appropriate for B. cereus group detection and quantification.     

A PCR assay based on a sequence-characterized amplified region marker for detection of emetic Bacillus cereus

A PCR assay for the detection of Bacillus cereus strains able to produce an emetic toxin (cereulide) was developed in this study based on a sequence-characterized amplified region (SCAR) derived from a random amplified polymorphic DNA (RAPD) fragment. One of the RAPD fragments generated was selected, cloned, and sequenced. A set of PCR primers was newly designed from the SCAR obtained (the sequence of the cloned RAPD fragment) and used in this assay. To determine the specificity of the assay, 30 different B. cereus strains, 8 other Bacillus strains (of six species), and 16 other non-Bacillus strains (from 16 genera) were tested. Results were positive for every emetic B. cereus strain and for only one nonemetic B. cereus strain. For all other bacterial strains, results were negative. Bacterial DNA for PCR was prepared by a simple procedure using Chelex 100 resin from the bacterial colony on the agar plate or from culture after growth in brain heart infusion medium. This PCR assay enabled us to detect the bacteria of emetic B. cereus grown on agar plates but not the bacteria of nonemetic B. cereus. To test this PCR assay for the monitoring of the emetic bacteria, 10 to 70 CFU of B. cereus DSM 4312 (emetic) per g of food was inoculated into several foods as an indicator, followed by a 7-h enrichment culture step. Because this PCR assay based on the SCAR derived from the RAPD fragment was able to detect bacterial cells, this assay should be useful for rapid and specific detection of emetic B. cereus.     

The microbiological quality of cooked rice from restaurants and take-away premises in the United Kingdom.

The microbiological quality of 4,162 samples of cooked rice from restaurants and take-away premises in the United Kingdom was examined, including ready-to-eat rice purchased at point-of-sale and rice that was stored precooked for reheating on demand. The majority of point-of-sale cooked rice samples (1,855 of 1,972; 94%) were of acceptable microbiological quality, but 15 (1%) samples were of unacceptable quality (Bacillus spp. and B. cereus, > or = 10(5) CFU/g; Escherichia coli, > or = 10(4) CFU/g), indicating a potential risk to health. The prevalence of Bacillus spp., B. cereus, and E. coli was significantly greater in precooked stored rice than in point-of-sale cooked rice (P < 0.005 to 0.0005). Bacillus spp. (> or = 10(4) CFU/g), B. cereus (> or = 10(4) CFU/g), and E. coli (> or = 10(2) CFU/g) were present in 7%, 2%, and 9% of precooked stored samples, respectively, compared to 2%, 0.5%, and 1%, respectively in point-of-sale samples. Although final heating at the point of sale reduces the levels of microorganisms present in rice it will not inactivate the B. cereus emetic toxin if present. Rice from Indian premises was of poorer microbiological quality than that from Chinese and other premises. Although most point-of-sale cooked rice samples (94%) were of an acceptable microbiological quality, evidence from this study indicates that the microbiological quality of cooked rice sold from certain outlets in the UK is of concern.     

Parallel analysis of 7 food-borne pathogens using capillary electrophoresis-based single-strand conformation polymorphism

Capillary electrophoresis-based single-strand conformation polymorphism (CE-SSCP) coupled with multiplex polymerase chain reaction (PCR) method was used for the detection of 7 pathogens associated with foodborne illness, including Salmonella enterica, Clostridium perfringens, Bacillus cereus, Listeria monocytogenes, Yersinia enterocolitica, Vibrio parahaemolyticus, and Escherichia coli O157:H7. The method was applied to model food systems, both of culture medium and cooked rice. The detection limit of individual microbes was in the range of 10¹–10³ CFU/g, and that of the mixture of 7 microbes was 10³ CFU/g in the cooked rice sample. This method allowed the detection and identification of all 7 food-borne pathogens within 5 hr without the requirement for enrichment steps.     

Production of Bacillus cereus emetic toxin (cereulide) in various foods

To determine the role of Bacillus cereus as a potential pathogen in food poisoning, the production of an emetic toxin (cereulide) by B. cereus was quantified in various food sources. The amount of emetic toxin in 13 of 14 food samples implicated in vomiting-type food poisoning cases ranged from 0.01 to 1.28 microg/g. A vomiting-type strain, B. cereus NC7401, was inoculated into various foods and incubated for 24 h at 20, 30, and 35 degrees C. In boiled rice, B. cereus rapidly increased to 10(7)-10(8) cfu/g and produced emetic toxin at both 30 and 35 degrees C. In farinaceous foods, the production of emetic toxin was as high as that in the food samples implicated in food poisoning. Low levels of emetic toxin were detectable in egg and meat and their products and a small quantity of toxin was detectable in liquid foods such as milk and soymilk when not aerated. Bacterial growth and toxin production was inhibited in foods cooked with vinegar, mayonnaise, and catsup, supposedly by the decreased pH of acetic acid. This is the first report that has quantified emetic toxin of B. cereus in various foods.     

Prevalence and genetic diversity of Bacillus cereus in dried red pepper in Korea

Bacillus cereus is a foodborne spore-forming bacterial pathogen that is ubiquitous in the natural environment. Infections with this pathogen manifest as diarrheal or emetic types of food poisoning. In this study, 140 samples of dried red pepper purchased in Korea were assayed for the presence of B. cereus according to the U.S. Food and Drug Administration standard culture method. A multiplex PCR assay was developed for the rapid confirmation of B. cereus as an alternative to conventional biochemical confirmation tests. The genetic diversity of B. cereus isolates was investigated using a random amplified polymorphic DNA (RAPD) assay. B. cereus was found in 84.3% of the dried red pepper samples, with an average concentration of 1.9 x 10(4) CFU/g. B. cereus could be detected and distinguished from B. thuringiensis in the multiplex PCR assay by using the BCFW1 plus BCrevnew and the K3 plus K5 primer sets designed to detect the gyrB gene of B. cereus and B. thuringiensis and the cry gene of B. thuringiensis. A RAPD assay using the OPG 16 and MUP 3 primers was used to successfully distinguish among isolates, thus elucidating the genetic diversity of B. cereus isolates. The discriminating ability of the OPG 16 primer (142 types) was about threefold higher than that of MUP 3 (52 types) in the RAPD assay.     

恒温隔绝式PCR快速检测蜡样芽孢杆菌方法的建立及应用

该研究以编码旋转酶B亚基的gyrB基因为靶基因,利用恒温热隔绝式PCR（Insulatedisothermal PCR,IiPCR）,建立一种快速检测蜡样芽孢杆菌的方法。同时,将建立的方法与聚合酶链式反应（Polymerase Chain Reaction,PCR）和实时荧光定量PCR（SYBER Green Ⅰ荧光染料法和TaqMan探针法）检测方法相比较,并应用于不同类型零售食品中的蜡样芽孢杆菌检测。结果表明建立的检测方法能在40 min内迅速判定出结果（+、-、？）;与大肠杆菌、金黄色葡萄球菌、单增李斯特菌、肠炎沙门氏菌等均无交叉反应,显示出良好的特异性;方法的最低检出限为1.5×102 CFU/mL,优于普通PCR,与实时荧光定量PCR检出限相当;对42份零售食品进行检测,共发现45.23%的样本被蜡样芽胞杆菌污染（19/42）。这一结果与常规PCR检测结果一致,但检测时间至少节省了4 h以上。该研究为蜡样芽孢杆菌提供了一种快速、便捷、特异性强、灵敏度高、适用于现场实时检测的检测方法。     

Bacterial contamination of ready-to-eat foods and fresh products in retail shops and food factories.

Raw vegetables cut for salad, cooked salad, cooked rice, boiled noodles, bean curd, and cooked Japanese foods were purchased in 27 retail shops in Tokyo. Intact vegetables before being processed and ready-to-eat fresh salad products were obtained from two food factories located in the suburbs of Tokyo. Two hundred thirty-eight retail samples, 137 samples of intact vegetables, and 159 samples of fresh products were examined for aerobic plate count (APC), coliforms, Escherichia coli, Listeria spp., Staphylococcus aureus, and Bacillus cereus. The APC of retail foods were 2.1 to 5.7 log CFU/g, and the range for the coliforms was 0.1 to 2.3 log CFU/g. The APC and coliform values showed that the raw vegetables cut for salad were the most heavily contaminated among the six kinds of ready-to-eat foods examined. Although L. monocytogenes was not detected, two samples of raw vegetables and five kinds of cooked foods yielded Listeria spp. S. aureus was detected in one sample of Japanese cooked food. The APC of the intact vegetables were 2.9 to 7.3 log CFU/g upon arrival and 2.2 to 7.2 log CFU/g after 3 days storage at 10 degrees C. The APC of the fresh products were 3.4 to 7.6 log CFU/g upon arrival and 4.7 to 8.7 log CFU/g after 3 days storage at 10 degrees C. The isolation rates for coliforms were 6.1 to 50% for intact vegetables and 50 to 66.7% for fresh products. E. coli was detected only in the fresh products. B. cereus was isolated from 20.1% (17 of 81) of the intact vegetables and 9.2% (8 of 87) of the fresh products.     

Survival and growth of foodborne pathogens during cooking and storage of oriental-style rice cakes

Fresh cooked rice cakes for retail sale are typically held at room temperature because refrigeration dramatically reduces their quality. Room temperature, high water activity, and a pH of > 4.6 provided an environment conducive to pathogen growth. To date, no studies have been published regarding survival and growth of foodborne pathogens in fresh cooked rice cakes. This study was undertaken to investigate the effect of steam cooking on foodborne pathogens and their subsequent growth in five varieties of rice cakes made from flours of regular rice, sweet rice, white rice, tapioca, and mung bean. Bacillus cereus spores were detected in white rice, tapioca, and mung bean samples. The rice cake flours were inoculated with non-spore-forming foodborne pathogens (Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium, Listeria monocytogenes, and Staphylococcus aureus) or spore-forming bacteria (Bacillus cereus) and steam cooked (100 degrees C) for 30 min. Steam cooking significantly reduced (> 6 log CFU/g) non-spore-forming foodborne pathogens in all samples and inactivated spores of B. cereus by 1 to 2 log CFU/g. Although spores of B. cereus survived steam cooking and germinated during 3 days of storage at room temperature, populations in most rice cakes remained below 106 CFU/g, which is the threshold for producing toxin. Rice cakes made from mung bean flour supported growth and germination of B. cereus spores above that critical level. In mung bean rice cakes, enterotoxin production was detected by the second day, when B cereus cell populations reached about 6.9 log CFU/g. The toxin concentration increased with storage time. However, our results suggest that rapid growth of total mesophilic microorganisms by more than 7 to 8 log CFU/ml during the first day of storage produced off flavors and spoilage before B. cereus was able to grow enough to produce toxins. Therefore, steam-cooked rice cakes made from a variety of flours including mung bean flour are safe for sale for up to 1 day after storage at room temperature and are free of B. cereus toxins.     

多重PCR检测4种常见食源性致病菌

目的 建立一种多重聚合酶链式反应法(multiplex polymerase chain reaction, MPCR)快速检测肉制品中金黄色葡萄球菌、沙门氏菌、志贺氏菌和单增李斯特氏菌的分析方法。方法 选取金黄色葡萄球菌nuc基因、沙门氏菌SipB基因、志贺氏菌ipaH基因、单增李斯特菌inlA基因作为目标基因, 设计4对PCR引物, 建立并优化多重PCR反应体系, 评价该体系的特异性和灵敏度, 并对人工污染的熟肉样品进行检测。结果 构建的多重PCR方法特异性强、灵敏度高, 人工污染熟肉匀浆中4种致病菌的检出限为103 CFU/mL。结论 构建的多重PCR检测方法能够快速、准确、高效地检测肉制品中金黄色葡萄球菌、沙门氏菌、志贺氏菌和单增李斯特氏菌, 为食源性疾病菌的快速检测提供参考依据。