3 种食源性致病菌Tem-PCR检测方法的建立

应用靶序列富集多重聚合酶链式反应（target-enriched multiplex-polymerase chain reaction,Tem-PCR）技术建立金黄色葡萄球菌、沙门氏菌和志贺氏菌3 种食源性致病菌的快速检测方法。分别以金黄色葡萄球菌femA基因、沙门氏菌invA基因和志贺氏菌ipaH基因为靶基因,设计3 对特异性引物,并与通用引物连接组成Tem-PCR引物,通过反应条件优化,建立了Tem-PCR检测方法。结果显示,建立的Tem-PCR方法特异性强,对沙门氏菌、金黄色葡萄球菌和志贺氏菌的检测灵敏度分别为1.1×103、2×103 CFU/mL和1.2×103 CFU/mL。Tem-PCR检测方法有效地解决了传统多重PCR引物扩增效率不均衡的问题。     

3种食源性致病菌多重PCR检测体系的建立

目的:探究用PCR方法检测食品中金黄色葡萄球菌(Staphylococcus aureus)、沙门氏菌(Salmonella spp.)及志贺氏菌(Shigella spp.)的检测灵敏度,建立快速检测3种食源性致病菌的多重PCR方法。方法:分别依据金黄色葡萄球菌的fem A、沙门氏菌的hil A基因及志贺氏菌的ipa H基因设计3对特异性引物,对3对引物进行多重PCR反应体系构建与优化。结果:建立的多重PCR方法具有灵敏度高、特异性强、方便快捷的优点。结论:研究结果为上述3种食源性致病菌快速检测试剂盒的研发及在食品安全检测工作中的应用提供了重要技术保障。     

二重PCR法快速检测3种食源性致病菌

目的建立快速检测食品中金黄色葡萄球菌(Staphylococcus aureus)、沙门氏菌属(Salmonella spp.)及志贺氏菌属(Shigella spp.)的二重PCR方法。方法分别针对金黄色葡萄球菌.femA和nuc、沙门氏菌属invA和hilA及志贺氏菌属ipaB和ipaH设计6对特异性引物,检测每对引物的特异性及灵敏度,组合优化各自二重PCR反应体系。结果初步建立了针对这3类致病菌的二重PCR检测方法,整个检测过程不超过18 h,检测灵敏度均可达10 pg DNA/reaction。结论所建立的针对3类致病菌的二重PCR检测方法具有灵敏度高、特异性强和方便高效等优点,具有良好的市场应用前景。     

多重实时荧光定量PCR同时检测冷冻蔬菜中4种食源性致病菌

目的 建立一种基于TaqMan探针的多重实时荧光定量PCR(multiplex quantitative real-time PCR, multiplex qPCR)同时检测冷冻蔬菜中4种食源性致病菌的方法。方法 针对金黄色葡萄球菌nuc基因、痢疾志贺菌rfc基因、沙门氏菌invA基因、单增李斯特氏菌hly基因, 设计4对引物和探针, 优化反应体系, 建立稳定的多重qPCR反应体系。通过阳性菌株污染的方法验证体系的特异性, 并确定了冷冻蔬菜样品在细菌水平的检出限。结果 各对引物和探针对目标菌有较强的特异性, 对其他非目标菌进行检测均未检出, 人工污染冷冻蔬菜中志贺菌的检出限为102 CFU/g, 沙门氏菌和单增李斯特氏菌的检出限均为103 CFU/g, 金黄色葡萄球菌的检出限为104 CFU/g。结论 本研究可以实现冷冻蔬菜样品中4种致病菌qPCR高效检测。     

2010年山西省食品中食源性致病菌监测分析

目的 了解山西省食源性致病菌的污染现状.方法 按照2010年度《全国食源性致病菌监测工作手册》进行.结果 1 576份样品中共检出阳性菌株149株,检出率为9.45％,其中单核细胞增生李斯特氏菌、副溶血性弧菌污染比较严重,总检出率为9.50％和8.22％,金黄色葡萄球菌、阪崎肠杆菌和沙门氏菌的总检出率分别为3.02％、1.11％和0.87％.不同类别食品中致病菌的检出率差异较大,生肉中致病菌的检出率高居榜首为49.33％,主要污染菌为沙门氏菌、单核细胞增生李斯特氏菌;沙拉中致病菌的检出率为17.39％,主要污染菌为单核细胞增生李斯特氏菌、金黄色葡萄球菌;生食水产品中致病菌的检出率为16.94％,主要污染菌为单核细胞增生李斯特氏菌、副溶血性孤菌;熟肉制品中致病菌的检出率为9.84％,主要污染菌为单核细胞增生李斯特氏菌、金黄色葡萄球菌、沙门氏菌;中式凉拌菜中致病菌的检出率为8.89％,主要污染菌为金黄色葡萄球菌;鲜榨果汁中致病菌的检出率为6.67％,主要污染菌为金黄色葡萄球菌;鲜冻水产品中致病菌的检出率为5.00％,主要污染菌为副溶血性弧菌;婴幼儿配方粉/米粉/谷粉/豆奶粉中阪崎杆菌的检出率为1.11％.结论 山西省多种食品均存在食源性致病菌不同程度的污染,应加大对散装食品、即食食品和生食水产品的监管.     

食品中3种致病菌的Taqman多重荧光定量PCR检测

利用实时荧光定量PCR(RTi-PCR)技术探索病原微生物的快速高通量检测方法已是食品微生物学的研究热点之一。首先,通过调整混合培养基的配比摸索食品中金黄色葡萄球菌、沙门氏菌和志贺氏菌的快速共增菌条件,并选择稳定高效的细菌基因组DNA提取方法,然后根据3种目标致病菌的保守基因分别设计RTi-PCR引物和双标记Taqman探针,分别使用FAM、TAMRA及CY5作为报告基团,对致病菌进行多重RTi-PCR检测,并设计、构建了阳性内参用于检测反应体系。结果表明,方法针对42种已知目标菌株和21种已知菌株组合特异性良好,检测限可达101 cfu/PCR反应,且反应体系各组分间未见交叉干扰。阳性内参Ct值保持稳定,有效地保证了检测的可靠性。对人工污染的食品盲样检测验证了方法的实用性。总之,建立了一种食品中金黄色葡萄球菌、沙门氏菌和志贺氏菌同时快速检测的多重RTi-PCR方法,为食源性致病菌的检测提供了新的方法学依据。     

2010~2016年云南省肉类制品致病菌污染状况调查

目的 了解云南省2010~2016年肉类制品中单核细胞增生李斯特氏菌、金黄色葡萄球菌和沙门氏菌的污染状况, 为预防和控制食源性疾病提供基础数据和科学依据。方法 2010~2016年, 在云南省采集3022件肉类制品样品, 对单核细胞增生李斯特氏菌、金黄色葡萄球菌和沙门氏菌污染状况进行监测。结果 3022件肉类制品中共检出阳性致病菌312株, 总检出率为10.32%, 金黄色葡萄球菌、单核细胞增生李斯特氏菌、沙门氏菌的检出率依次为4.96%、3.97%、1.39%; 散装样品致病菌检出率(11.60%)高于预包装样品致病菌检出率(7.06%); 餐饮环节致病菌检出率(11.85%)与流通环节致病菌检出率(10.08%)无显著性差别。结论 2010~2016年云南省肉类制品中李斯特氏菌、金黄色葡萄球菌污染较沙门氏菌严重, 应加强肉类制品在贮存、运输过程中的监督管理。     

杨梅果实提取物抑菌特性的研究

对杨梅果实乙醇提取物进行了抑菌特性的研究,测试了提取物对几种常见菌株的最低抑菌浓度,研究了温度、pH、盐、糖等对抑菌活性的影响。实验结果表明:杨梅果实提取物对志贺氏痢疾杆菌、伤寒沙门氏菌、溶血性链球菌和金黄色葡萄球菌具有一定的抑制作用,对大肠杆菌和黑曲霉几乎没有抑菌作用;提取物对不同菌株的最低抑菌浓度(MIC)分别为:0.031g/mL(金黄色葡萄球菌)、0.125g/mL(志贺氏菌)、0.125g/mL(沙门氏菌)、0.062g/mL(链球菌);提取物对温度具有良好的稳定性,在pH值5～8的范围内具有良好的抑菌作用,另外,提取物与盐、糖具有良好的协同抑菌作用。     

壳聚糖对包装猪肉中接种的四种致病菌生长的影响

将人工接种了沙门氏菌(Salmonella)、金黄色葡萄球菌(Staphylococcus aureus),李斯特单核增生菌(listeria monocrtogenes)和耶尔森氏菌(Yersinia)的鲜猪肉,分别浸入1%壳聚糖—2%醋酸混合液(简称CA液)、2%醋酸溶液(简称A液)和无菌蒸馏水中约10秒钟,取出沥干2～3分钟,然后用聚乙烯袋和真空袋包装,贮藏在30℃条件下,观察CA液和A液对鲜肉上接种的这四种致病菌的生长影响,并且比较聚乙烯包装和真空包装对CA液及A液抑菌作用的影响.结果表明:CA液或A液对李斯特单核增生菌没有杀灭能力,但CA液对沙门氏菌、金黄色葡萄菌和耶尔森氏菌具有一定的杀灭能力,A液也具有一定的杀灭金黄色葡萄球菌力.在保藏过程中,A液对鲜肉上的李斯特单核增生菌、沙门氏菌的生长没有抑制作用,但CA液均可抑制鲜肉上接种的这四种致病菌的生长,CA液的抑菌作用明显地比A液的强.虽然聚乙烯包装或真空包装不会影响CA液对鲜肉上李斯特单核增生菌和沙门氏菌的抑制,但真空包装增强了CA液和A液对鲜肉上金黄色葡萄球菌的抑制作用.同相应包装中空白样品相比,CA液或A派均不能减少鲜肉上这四种致病菌的最大菌数值.     

3种食源性致病菌的实时荧光PCR快速检测

DNA提取方法采用先钢珠机械破坏细菌细胞壁,再添加丙酮、蛋白酶K溶解去除杂质,大大提高了食源性致病菌基因组DNA的得率和纯度。继而针对金黄色葡萄球菌egc基因、沙门氏菌NA(not availabale)基因、志贺氏菌ipaH基因为靶基因设计特异性引物对样品建立实时荧光定量PCR检测方法。结果试验设计的引物对3种细菌具有很强特异性,除目标细菌外,对单核细胞增生李斯特氏菌、副溶血性弧菌、蜡样芽孢杆菌3种对照菌均未检测到荧光信号。对金黄色葡萄球菌、沙门氏菌和志贺氏菌的检测灵敏度分别达到10~(-6)、10~(-6)、10~(-7) ng/μL。     

Effect of accelerated natural lactic fermentation of infant food ingredients on some pathogenic microorganisms

Accelerated natural lactic fermentation in mixtures of water and ground sorghum, millet and pigeon pea was obtained by gradual selection of lactic acid bacteria, through inoculum recycling. Weaning food prepared from ingredients fermented this way, contained approx. 0.7% lactic and 0.05% acetic acids and had a pH of about 3.8. In porridges, a pH of less than or equal to 4.0 was required to cause death of Salmonella typhimurium and Staphylococcus aureus. Several intestinal pathogenic bacteria were inoculated into sour porridge. The most resistant Salmonella sp. died at a rate of 1.2 log cycle/h; the most resistant Shigella sp. at 0.9 log cycle/h; and the most resistant Escherichia coli strain at 0.6 log cycle/h. A yeast, Candida albicans, could grow well in the sour product, whereas a bacteriophage (MS-2) was inactivated at a rate of 0.1 log cycle/h. In the acid-sensitive bacterial cultures, no gradual adaptation to acid environments could be observed. The survival studies were carried out at 30 degrees C.     

食源性致病菌高光谱识别模型构建

针对食源性致病菌检测过程耗时费力的问题,应用高光谱成像技术获取了12,18,24h3个生长期的5类致病菌[金黄色葡萄球菌(S.aureus)、单增李斯特菌(LM)、致泻大肠埃希氏菌(DEC)、肠炎沙门氏菌(SE)和福氏志贺菌(S.flexneri)]的高光谱图像,利用连续投影算法(SPA)结合相关分析(CA)提取不同生长期致病菌敏感波段并建立了最小二乘支持向量机(LS-SVM)判别模型。结果表明,5类致病菌对不同波长光的反射能力不同。SPA-CA筛选出的5个敏感波段(462,498,649,853,979nm)的反射率可较好地反映5类致病菌在不同生长期的波谱特性。基于该敏感波段反射率构建的LS-SVM模型能有效地判别出S.aureus、LM和DEC 3类致病菌,而SE和S.flexneri两种属容易被互相误判,SE被误判成S.flexneri的概率为11.2%,S.flexneri被误判成SE的概率为19.9%。LS-SVM模型对5类致病菌的总体识别正确率为90.9%。综上,高光谱成像技术结合化学计量学方法具有快速诊断食源性致病菌的能力。     

干酪乳杆菌发酵荔枝汁在模拟胃肠道中耐受能力及其抑菌性的研究

以干酪乳杆菌发酵荔枝汁为研究对象,在人工胃液、人工肠液环境下对干酪乳杆菌的存活性能进行研究;同时,采用琼脂扩散法,研究其对六种常见食源性致病菌(肠炎沙门氏菌、宋氏志贺氏菌、大肠埃希氏菌O157:H7、单增李斯特氏菌、金黄色葡萄球菌和阪崎肠杆菌)的抑菌性,以及探究酸和酶等因素对发酵液抑菌性的影响。结果表明:在模拟胃肠消化中,干酪乳杆菌在pH 1.5、pH 2.0、pH 3.0和pH 4.0的人工胃液中培养4 h后,活菌数分别达到了260 CFU/mL、8.63×107 CFU/m L、9.57×107 CFU/m L和1.15×108CFU/m L;在人工肠液中消化12 h后,活菌数均在107 CFU/m L以上。在抑菌试验中,经去酸、去过氧化氢和酶解等不同处理的发酵液对肠炎沙门氏菌等五种常见食源性致病菌具有明显的抑制效果,而对阪崎肠杆菌均未表现出抑制作用,其中胞外多糖、H2O2和蛋白肽类物质对五种常见的致病菌是主要的抑菌物质。研究结果为干酪乳杆菌发酵荔枝汁能否在胃肠道中发挥有益作用提供体外实验的理论依据以及在当地食品工业的开发应用提供科学依据。     

自然发酵腐与人工接种发酵腐乳的微生物比对研究

目的分析自然发酵腐乳与人工接种发酵腐乳两者微生物种类及可能产生的安全隐患,并对腐乳的生产环节中易产生的杂菌提出改进方案。方法利用传统的微生物分离鉴定方法,依据SB/T 10170-2007行业标准及食品安全国家标准食品微生物学检验等方法对影响腐乳中微生物的几个安全性指标和致病菌进行检测。结果通过2种方式培育的毛霉腐乳,其微生物含量和种类对比性很强,自然发酵腐乳的方式其细菌含量高,致病菌中金黄色葡萄球菌和沙门氏菌也均有检出,其他致病菌也存在很大的风险。选用人工接种发酵的腐乳其可培养出的微生物种类少,致病菌也未检出。结论选用人工接种发酵的腐乳较自然发酵的腐乳安全性高,更符合市场需求。     

应用基因芯片技术检测肉及肉制品中5种致病菌

建立一种运用多重聚合酶链式反应(PCR)结合基因芯片技术检测大肠埃希氏菌、沙门氏菌、金黄色葡萄球菌、志贺氏菌和单核细胞增生李斯特菌5种食源性致病菌的快速、准确、灵敏的方法。分别选取编码大肠埃希氏菌的slt基因、沙门氏菌invA基因、金黄色葡萄球菌nuc基因、志贺氏菌ipaH基因和单核细胞增生李斯特菌inlA基因,并以细菌16S rDNA基因作为阳性对照,设计引物和探针,进行多重PCR扩增,产物与含特异性探针的芯片杂交。结果表明：该基因芯片可同时特异性地检测5种致病菌,多重PCR检测灵敏度为20pg,而DNA芯片检测灵敏度可达2pg;用所制备的基因芯片检测实际肉及肉制品样品,准确率高于传统培养法。所建立的基因芯片检测方法特异性好、灵敏度高,可为食源性致病菌的检测提供理想手段。     

Effect of thermophilic lactic acid bacteria on the viability of Salmonella serovar Typhimurium PT8 during milk fermentation and preparation of buffalo's yogurt

Viability of dairy-borne Salmonella enterica ssp. enterica serovar Typhimurium PT8 was studied during the fermentation of skim milk by thermophilic lactic acid bacteria (LAB). Longer generation times of Salmonella were found in mixed cultures of skim milk containing Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus or a mixture of them (1:1), as compared with single cultures of the pathogen. Salmonella was less able to survive in mixed cultures with these LAB during prolonged incubation at 41°C and also during cold storage of the fermented milk. L actobacillus ssp. bulgaricus and its mixture with S. thermophilus were more inhibitory to the growth and survival of Salmonella than was S. thermophilus . This was associated with higher ability of L . ssp. bulgaricus and the mixture to develop acidity in milk than S. thermophilus . Examining the antibacterial activity of these LAB towards Salmonella showed that other factors including heat-resistant and heat-labile compounds were involved in inhibiting the pathogen by these cultures. The viability of the same Salmonella strain during the preparation and cold storage of buffalo's yogurt was also examined. Salmonella was found to survive longer in yogurt made with starter containing probiotic bacteria than in that prepared with the traditional starter. This was ascribed to the development of lower pH by the traditional starter.     

Growth and survival of foodborne pathogens in beer

This work aimed to assess the growth and survival of four foodborne pathogens (Escherichia coli O157:H7, Salmonella Typhimurium, Listeria monocytogenes, and Staphylococcus aureus) in beer. The effects of ethanol, pH, and storage temperature were investigated for the gram-negative pathogens (E. coli O157:H7 and Salmonella Typhimurium), whereas the presence of hops ensured that the gram-positive pathogens (L. monocytogenes and S. aureus) were rapidly inactivated in alcohol-free beer. The pathogens E. coli O157:H7 and Salmonella Typhimurium could not grow in the mid-strength or full-strength beers, although they could survive for more than 30 days in the mid-strength beer when held at 4°C. These pathogens grew rapidly in the alcohol-free beer; however, growth was prevented when the pH of the alcohol-free beer was lowered from the "as received" value of 4.3 to 4.0. Pathogen survival in all beers was prolonged at lowered storage temperatures.     

Effect of environmental conditions during soya-bean fermentation on the growth of Staphylococcus aureus and production and thermal stability of enterotoxins A and B

During tempe manufacture, Staphylococcus aureus was able to survive in newly started soya-bean soaks (final pH 4.7), whereas it died during soaks with accelerated souring (final pH 4.0). No S. aureus were detected after subsequent heat treatment of soaked beans. S. aureus strain S6 could produce enterotoxins A and B if inoculated on cooked soya-beans. Its growth and enterotoxin production were not influenced by inoculation rate nor by the presence of actively growing Rhizopus oligosporus, the fungus responsible for tempe formation. Enterotoxin levels were highest after 48 h fermentation. Presence of lactobacilli (L. brevis or L. plantarum) during the tempe fermentation did not influence staphylococcal growth, but enterotoxin levels were reduced. Reduced oxygen pressure did not affect staphylococcal growth, but resulted in lower enterotoxin levels after 48 h incubation. The thermal stability of enterotoxin A in tempe was significantly lower than in Brain Heart Infusion broth of comparable pH. Heating of enterotoxin-containing tempe mash reduced enterotoxin A by 99.7% as measured with ELISA and animal feeding methods.