多重重组酶介导等温扩增技术检测食品中3种食源性致病菌

建立快速、灵敏的多重重组酶介导等温扩增方法（recombinase aided amplification,RAA）同时检测食品中的大肠埃希氏菌O157:H7、沙门氏菌和金黄色葡萄球菌。方法 根据大肠埃希氏菌O157:H7的rfbE基因、沙门氏菌的invA基因以及金黄色葡萄球菌的nuc基因的序列保守区域设计RAA特异性引物,筛选出最优的引物组合并优化各项实验条件;通过标准菌株验证方法特异性和灵敏度,并通过人工污染实验,验证方法在实际食品样本中的检测能力。结果 多重RAA方法最佳扩增温度为37℃,反应时间为50 min;方法特异性良好,仅对目标菌株存在特异性扩增条带,与其他常见食源性致病菌无交叉反应;方法对基因组DNA的检测灵敏度为0.10 ng/μL;方法对经过简单增菌的人工污染牛奶样本和牛肉干样本中目标菌株的检出限为100 CFU/mL。结论 本研究建立的多重RAA扩增方法具有特异性好、灵敏度高、检测通量高等优势,适用于多种场景下食品样本中3种食源性致病菌的快速检测。     

5种食源性致病菌PCR检测方法的建立

目的建立食品中的沙门氏菌、副溶血性弧菌、大肠埃希氏菌O157:H7、金黄色葡萄球菌和单核细胞增生李斯特氏菌的PCR快速检测方法。方法以缓冲蛋白胨水为增菌培养基,采用直接提取法提取DNA并测定其核酸浓度。对PCR的退火温度、模板浓度进行优化,把5种目标菌分为两组进行检测分析,并进行特异性和灵敏度实验。结果 5种目标菌培养后均表现出良好的生长趋势。PCR扩增最佳退火温度为59℃, 5种目标菌的引物特异性好,方法的检测灵敏度高。沙门氏菌、副溶血性弧菌、大肠埃希氏菌O157:H7、金黄色葡萄球菌和单核细胞增生李斯特氏菌最低检出核酸浓度分别为0.0202、0.158、0.187、2.30和1.05μg/mL。结论该方法简便、快速、灵敏度高,能满足食品安全检测要求。     

Escherichia coli O157:H7 facilitates the penetration of Staphylococcus aureus into table eggs

Abstract: Fresh eggshells collected from a local farm were subjected to different levels of surface contamination with feces containing different levels (3 to 5 log₁₀) of Escherichia coli O157:H7 or Staphylococcus aureus and incubated at 3 different temperatures (10, 25, and 32 °C). The penetration rates of contaminating bacteria were followed throughout the incubation period by tracing bacterial presence in shell, shell membranes, albumen, and yolk. The study revealed the ability of both E. coli O157:H7 and enterotoxigenic S. aureus to grow on shell in feces, penetrate the shell, and move and multiply within egg contents at different rates and periods depending on bacterial type and incubation conditions. High temperatures (25 and 32 °C) increased penetration rate, whereas storage at 10 °C decreased significantly the rate of penetration. High levels of contamination with E. coli O157:H7 also shortened the time needed for the penetration process. Results showed that when eggshells were contaminated with both organisms simultaneously, the penetration of E. coli O157:H7 preceded that of S. aureus and facilitated the invasion of the latter bacteria.     

多重荧光定量聚合酶链反应法检测肉制品中的3种食源性致病菌

摘要：目的 建立多重荧光定量聚合酶链反应法（quantitative polymerase chain reaction,qPCR）快速检测肉制品中沙门氏菌、单增李斯特菌、大肠杆菌O157:H7 3种食源性致病菌的方法。方法 根据沙门氏菌的invA基因、单增李斯特菌的hemolysin基因、大肠杆菌O157:H7的rfbE基因序列分别设计特异性引物及探针,通过优化反应体系,测定其灵敏度、特异性和重复性,建立了可同时检测上述3种食源性致病菌多重qPCR方法。结果 该方法只扩增3种靶细菌,对其他供试菌不扩增;沙门氏菌、单增李斯特菌、大肠杆菌O157:H7的检出限分别为101、102、102拷贝数/μL,并且拥有良好的重复性和特异性。人工污染的猪肉样品经SLE（Salmonella、Listeria monocytogenes and Escherichia coli 3种菌株的共增菌培养基）培养基富集8 h后,分别可以检测到初始菌液浓度为1.56×102 CFU/25 g的沙门氏菌,2.13×102 CFU/25 g的单增李斯特菌,2.32×102 CFU/25 g的大肠杆菌O157:H7。结论 所建立的多重qPCR灵敏度高、特异性强、重复性好,可同时检测肉制品中沙门氏菌、单增李斯特菌和大肠杆菌O157:H7 3种食源性致病菌,在提高食品安全和保护人类健康方面有重要意义。     

不同pH、温度、金属离子对溶菌酶抗菌稳定性的初步研究

本文选择金黄色葡萄球菌(Staphylococcus aureus)与大肠杆菌O157：H7(Escherichia coli O157：H7)为受试菌,以活菌抑制率为评价指标,评价不同pH、温度、金属离子条件对溶菌酶抗菌稳定性的影响。结果表明:溶菌酶对金黄色葡萄球菌与大肠杆菌O157：H7的最小抑菌浓度分别为1.3×10^-3 g/mL与2.6×10^-3 g/mL。溶菌酶在酸性条件(pH4~6),其对两种菌均能表现很好的抑菌效果。经过4~121 ℃处理后,溶菌酶的抗菌活性不受处理温度的影响。不同金属离子对溶菌酶抗菌活性影响较大,Zn²⁺、Mn²⁺与溶菌酶具有较强的协同抑菌效应,其中Zn²⁺效果最为明显,而Mg²⁺、Ca²⁺具有一定的拮抗抑菌效应,可降低溶菌酶的抑菌效果。     

Growth inhibition of foodborne pathogens by Oenococcus oeni

Abstract: To explore the possibility of using Oenococcus oeni to inhibit foodborne pathogens, and to characterize antimicrobial compounds produced by O. oeni, 24 strains of O. oeni were tested for their ability to inhibit growth of foodborne pathogens, Escherichia coli O157:H7, Salmonella enteritidis, and Listeria monocytogenes by using the spot‐on‐lawn method. Of the 24 strains, 17 strains were able to inhibit all 3 pathogens in this study. Proteases, catalase, and buffer solutions were used for determining the type of inhibitory compounds produced from 4 selected strains with stronger inhibitory activity. Antimicrobial activity of 2 strains against the pathogens was completely inactivated by buffer solution, and other 2 strains against E. coli O157:H7 were partially removed. The antimicrobial compound was not sensitive to selected proteases and catalase. Practical Application: There is little information available about using O. oeni for human pathogens control. The results of this study revealed such discovery and potential applications for pathogen control.     

Antimicrobial Effect of Sour Pomegranate Sauce on Escherichia coli O157 : H7 and Staphylococcus aureus

Pomegranate sauce is one of the most popular pomegranate products produced in Turkey. This study was conducted to determine the minimum inhibitory concentrations (MICs) of both traditional and commercial sour pomegranate sauce samples on Staphylococcus aureus (ATCC 25923) and Escherichia coli O157 : H7 (ATCC 43895). The initial microflora of the pomegranate sauce samples was determined by performing the enumerations of total aerobic mesophilic bacteria, yeast and mold, S. aureus, E. coli, and the determination of Salmonella spp. MIC tests were applied to the neutralized and the original (unneutralized) sour pomegranate sauce samples in order to put forth the inhibition effect depending on low pH value. It was found that inhibitory effect of the traditional and the commercial samples, except one sample, on pathogens was not only due to the acidity of the products. The results of MIC tests indicated that although both traditional and commercial samples showed a considerable inhibitory effect on test microorganisms, the traditional pomegranate sauce samples were more effective than the commercial ones.     

气体二氧化氯对苹果表面细菌杀菌规律研究

本实验研究了气体二氧化氯杀灭金黄色葡萄球菌、大肠杆菌、单核细胞增生李斯特氏菌以及腐生酵母菌四种苹果表面的危险致病菌的杀菌规律.在杀菌时间5min、杀菌温度25℃条件下,金黄色葡萄球菌、大肠杆菌、单核细胞增生李斯特氏菌和腐生酵母菌杀菌达到99.99%的杀菌率,所需的气体二氧化氯的浓度分别为23、21、6和11mg/L.在杀菌气体二氧化氯浓度10mg/L、杀菌温度25℃时,四种菌杀菌时间分别达到7、8、2和6.5min时,杀菌率均超过99.99%.在实验范围内,作用温度对杀菌效果影响不大.     

Kinetics of ultraviolet light inactivation of Escherichia coli O157:H7 in liquid foods

The effects of pH, depth of food medium and ultraviolet (UV) light dose on the inactivation of Escherichia coli O157:H7 in UV‐opaque products such as apple juice (pH 3.5) and egg white (pH 9.1) were investigated. The applied UV dose ranged from 0 to 6.5 mW min cm^?2, while the depths of the medium were 1, 3.5, 5 and 10 mm. The pH of the medium did not affect the inactivation of E coli O157:H7, since similar inactivation characteristics were obtained for both apple juice and liquid egg white. As expected, decreasing the depth of the medium increased the inactivation of E coli O157:H7. More than a 5‐log reduction was obtained when the fluid depth and UV dose were 1 mm and 6.5 mW min cm^?2 respectively. However, less than a 1‐log reduction was obtained when the fluid depth was 10 mm. A two‐phase kinetic model was used to model the inactivation of E coli O157:H7. This model indicated that at higher fluid depths the inactivation rate was controlled by the second, slower inactivation phase, resulting in a lower overall inactivation. The visual appearance of the treated apple juice and egg white did not show any discolouration changes during 4 weeks of storage at ambient temperature (25 °C). Copyright © 2003 Society of Chemical Industry     

生物传感器检测食源性大肠杆菌O157:H7研究新进展

近年来, 生物传感器因具有快速、简便、灵敏度高、低成本等优势被广泛应用到临床检测、环境监测等领域。该技术在食品安全领域也逐步得到重视, 尤其在病原微生物的快速检测方面。本文从免疫识别和核酸识别两方面简要介绍生物传感器技术检测食源性大肠杆菌O157:H7研究的最新进展, 对生物传感器技术存在的问题及未来的研究方向进行了总结及展望。     

Escherichia coli O157: H7 Growth in Laboratory Media as Affected by Phenolic Antioxidants

Five strains of Escherichia coli O157:H7 with ATCC 11775 E. coli were grown in brain heart infusion (BHI) broth (pH 5.8, adjusted with citric acid) and treated with butylated hydroxyanisole (BHA), butylated hy-droxytoluene (BHT), tertiary butylhydroquinone (TBHQ), and propyl gallate (PG) individually or combined. Additives ranged from 100–400 ppm with inocula levels between 5 and 104 CFU/mL in tissue culture plates or in flasks; samples were incubated at 4°C or 37°C for 24 hr. Additive antimicrobial efficacy varied with inoculum level and incubation temperature. BHA at <200 ppm was bactericidal on all strains. Poly-hydroxyl additives (TBHQ, PG) were less effective at 4°C. BHA-BHT combinations were synergistic at 4°C.     

Fate of Escherichia coliO157: H7 in Chinese-style sausage subjected to different packaging and storage conditions

In this study, Chinese-style sausages were subjected to air, vacuum or nitrogen packaging and stored at either 5 or 25°C. The survival characteristics of Escherichia coli O157: H7 during the storage period were determined. Results revealed that, when stored at 5°C, the number of viable E coli O157: H7 in sausages decreased slowly as the storage period extended, regardless of packaging methods. E coli O157: H7 in sausages decreased from an initial population of ca 5·97 log CFU g⁻¹ to ca 4·42–4·81 log CFU g⁻¹ after 40 days of storage at 5°C. It was also found that viable cells of E coli O157: H7 declined more rapidly in sausage stored at 25°C than at 5°C. No viable E coli O157: H7 was detected in either vacuum-packed or nitrogen-packed sausage after 40 days of storage at 25°C. On the other hand, the population of E coli O157: H7 reduced to non-detectable levels in air-packed sausages after 20 days of storage. Refrigerated storage and vacuum or nitrogen packaging provided conditions that slowed down the death rate of E coli O157: H7 in sausage. Furthermore, it was noted that, among the curing agents tested, NaCl exerted the most significant lethal effect on E coli O157: H7 in sausage during the storage period. © 1998 Society of Chemical Industry.     

副干酪乳杆菌Z17-壳聚糖复配对大肠杆菌O157:H7的抑菌效果及作用机制

目的：研究副干酪乳杆菌Z17-壳聚糖复配对草莓中大肠杆菌O157:H7抑菌活性及作用机制。方法：采用流式细胞术、傅里叶变换红外光谱、拉曼光谱及扫描电子显微镜技术分析副干酪乳杆菌Z17-壳聚糖对大肠杆菌O157:H7细胞膜的影响。结果：质量分数1.0%壳聚糖溶液与副干酪乳杆菌Z17复配处理能有效去除草莓上的大肠杆菌O157:H7,减菌率达99%;壳聚糖溶液与副干酪乳杆菌Z17共同作用3 h使大肠杆菌O157:H7 DNA胞外释放量达（381.00±3.53）ng/μL,细胞膜破损率为58.3%;细胞壁膜中脂肪酸、蛋白、肽聚糖、糖苷环、多糖结构成分被破坏;细胞膜局部位移变薄,大分子物质黏附于菌体细胞表面,细胞表面出现孔洞,胞内物质泄漏,最终导致菌体死亡。结论：副干酪乳杆菌Z17-壳聚糖能够有效地抑制草莓中大肠杆菌O157:H7,其抑菌作用靶点为大肠杆菌O157:H7的细胞膜,研究可为大肠杆菌O157:H7的生物防治提供参考。     

大肠杆菌O157:H7胶体金试纸条的研制

采用胶体金标记抗大肠杆菌O157:H7 单克隆抗体(鼠源),通过将大肠杆菌O157:H7 多克隆抗体和驴抗鼠抗体(二抗)喷涂于硝酸纤维素膜分别作为检测线和质控线,研制大肠杆菌O157:H7 胶体金快速检测试纸条。通过优化实验,确定最佳条件为标记量16.8μg/mL、标记pH8.0、封闭剂PEG20000、检测时样品最佳pH7.0～7.5。对26 株常见细菌交叉反应结果表明,该试纸条除与鼠伤寒沙门氏菌ATCC 13311 和金黄色葡萄球菌CMCC 26003 有轻微交叉反应外,与其他24 株菌均无交叉反应。该试纸条灵敏度为104CFU/mL。用该试纸条检测大肠杆菌O157:H7操作简便、快捷、灵敏度高、特异性强。     

Attachment of Escherichia coli O157:H7 to abiotic surfaces of cooking utensils

We examined the attachment of enterohemorrhagic Escherichia coli O157:H7 to abiotic surfaces of cooking utensils. When the cell suspension in 0.85% NaCl (about 100 cells/mL, 10 mL) was contacted with various abiotic surfaces (square pieces, 25 cm2) at 25 °C for 20 min, the number of attached cells varied depending on the types of abiotic materials. The pathogen well attached to stainless steel (about 50 cells/25 cm2), pure titanium (35 to 45 cells/25 cm2), and glass (about 20 cells/25 cm2), but little attached to aluminum foil and plastics, irrespective of strains used. Fewer cells (below 10 cells/25 cm2) attached to stainless steel, pure titanium, and glass surfaces conditioned with aseptically sliced beef (sirloin) and autoclaved beef tallow at 25 °C for 20 min, but bovine serum albumin did not reduce the number of attached cells. The cells grown at 15 °C to the stationary phase (OD660 = about 2.8) less attached to the abiotic surfaces than those grown at 25 °C and 37 °C. When we pretreated the cells at 37 °C for 2 h with 50 μM N-hexanoyl-L-homoserine lactone (HHL), the number of cells attached to stainless steel was reduced by 70%. The number of cells attached to cooking utensils seemed to change depending on types of abiotic materials, adhesion of beef tallow to abiotic surfaces, growth temperature of the pathogen, and HHL-producing bacteria.     

肠出血性大肠杆菌O157:H7的分子生物学检测及PCR检测

大肠杆菌一些特殊的血清型具有致病性,肠出血性大肠杆菌是大肠杆菌的一个亚型,主要致病菌株为O157:H7,可引起感染性腹泻,因能引起人类的出血性肠炎而得名。本文综述了分子生物学检测肠出血性大肠杆菌O157:H7的研究进展。分子生物学检测是利用抗原抗体特异性结合反应检测各种物质的分析方法,主要包括酶联免疫吸附法(ELISA)、胶体免疫金层析法以及免疫磁珠分离法(IMS)。PCR技术检测肠出血性大肠杆菌O157:H7,主要包括常规PCR检测、多重PCR检测以及实时荧光定量PCR检测。这两种方法灵敏度高、特异性强、操作简便、结果准确等优点,是检测肠出血性大肠杆菌O157:H7的常用方法。     

食源性致病菌大肠杆菌O157:H7检测方法的研究进展

食源性致病菌是引起食物中毒的重要病原微生物,严重威胁人类健康,对食源性致病菌进行快速、准确的鉴定检测,是预防和控制致病菌的有效方法,而大肠杆菌O157:H7因其感染剂量低,致病性强,引起公众的广泛关注。本文综述了目前用于检测食源性致病菌大肠杆菌O157:H7的主要方法,包括细菌分离法,免疫学检测方法,分子生物学检测方法,并简单介绍了这些方法的优缺点,以期为检测大肠杆菌O157:H7时提供参考。     

Survival of Escherichia coli O157:H7 in dry sausage fermented by probiotic lactic acid bacteria

Probiotic Lactobacillus rhamnosus GG, L rhamnosus E‐97800, L rhamnosus LC‐705 and commercial Pediococcus pentosaceus were studied for their ability to inhibit the growth of Escherichia coli O157:H7 in dry sausage. The strains were able to produce technologically high‐quality dry sausage. The number of E coli O157:H7 decreased from approximately 5 to approximately 2 log cfu g⁻¹ It was concluded that the above‐mentioned strains and the commercial culture were equally effective in inhibiting E coli O157:H7. © 2000 Society of Chemical Industry     

Survival of Escherichia coli O157:H7 in meat product brines containing antimicrobials

Brine solution injection of beef contaminated with Escherichia coli O157:H7 on its surface may lead to internalization of pathogen cells and/or cross-contamination of the brine, which when recirculated, may serve as a source of new product contamination. This study evaluated survival of E. coli O157:H7 in brines formulated without or with antimicrobials. The brines were formulated in sterile distilled water (simulating the composition of freshly prepared brines) or in a nonsterile 3% meat homogenate (simulating the composition of recirculating brines) at concentrations used to moisture-enhance meat to 110% of initial weight, as follows: sodium chloride (NaCl, 5.5%) + sodium tripolyphosphate (STP, 2.75%), NaCl + sodium pyrophosphate (2.75%), or NaCl + STP combined with potassium lactate (PL, 22%), sodium diacetate (SD, 1.65%), PL + SD, lactic acid (3.3%), acetic acid (3.3%), citric acid (3.3%), nisin (0.0165%) + ethylenediamine tetraacetic acid (EDTA, 200 mM), pediocin (11000 AU/mL) + EDTA, sodium metasilicate (2.2%), cetylpyridinium chloride (CPC, 5.5%), or hops beta acids (0.0055%). The brines were inoculated (3 to 4 log CFU/mL) with rifampicin-resistant E. coli O157:H7 (8-strain composite) and stored at 4 or 15 °C (24 to 48 h). Immediate (0 h) pathogen reductions (P < 0.05) of 1.8 to ≥ 2.4 log CFU/mL were observed in brines containing CPC or sodium metasilicate. Furthermore, brines formulated with lactic acid, acetic acid, citric acid, nisin + EDTA, pediocin + EDTA, CPC, sodium metasilicate, or hops beta acids had reductions (P < 0.05) in pathogen levels during storage; however, the extent of pathogen reduction (0.4 to > 2.4 log CFU/mL) depended on the antimicrobial, brine type, and storage temperature and time. These data should be useful in development or improvement of brine formulations for control of E. coli O157:H7 in moisture-enhanced meat products. PRACTICAL APPLICATION: Results of this study should be useful to the meat industry for developing or modifying brine formulations to reduce the risk of E. coli O157:H7 in moisture-enhanced meat products.