五种食源性致病菌多重PCR的条件优化和检出限分析

对沙门菌、金黄色葡萄球菌、大肠杆菌O157∶H7、单核增生李斯特菌和福氏志贺菌5种食源性致病菌的多重PCR反应条件进行优化并分析方法的DNA检测限。方法 根据沙门菌的invA基因、金黄色葡萄球菌的16S rDNA基因、大肠杆菌O157∶H7的eaeA基因、单核增生李斯特菌的hlyA基因和福氏志贺菌的ipaH基因设计5对特异性引物进行多重PCR,对反应条件包括镁离子浓度、引物浓度和退火温度进行优化试验,并确定该检测方法的检出限。结果 最佳反应条件为金黄色葡萄球菌、沙门菌和福氏志贺菌引物浓度为0.25μmol/L,单核增生李斯特菌引物浓度为0.4μmol/L,大肠杆菌O157∶H7引物浓度为0.3μmol/L,Mg²⁺浓度为2.25mmol/L,退火温度为60℃;在此条件下金黄色葡萄球菌、肠炎沙门菌、大肠杆菌O157∶H7、单核增生李斯特菌、福氏志贺菌的多重PCR的DNA检出限分别为6.4、32、32、800和160pg。结论 通过对5种引物的PCR条件进行优化和检出限的分析,为食品中该5种致病菌的快速检测奠定基础,对实际应用具有指导意义。     

多重PCR法检测鲜切哈密瓜中3种食源性致病菌

建立可同时检测鲜切哈密瓜中的单增李斯特菌、鼠伤寒沙门氏菌和大肠杆菌O157:H7的多重聚合酶链式反应(polymerase chain reaction,PCR)检测方法。根据单增李斯特菌inl A基因、鼠伤寒沙门氏菌inv A基因、大肠杆菌O157:H7的wzy基因设计3对特异性引物。对多重PCR反应体系中的引物浓度、退火温度、Mg2+浓度、d NTP浓度进行了优化,并确定适宜的多重PCR反应体系及反应条件。结果表明:25μL反应体系,10×PCR buffer为2.5μL,Mg Cl2(25 mmol/L)为3.5μL,d NTPs(2.5 mmol/L)为2μL,inl A基因上下游引物(5μmol/L)为1μL,inv A基因上下游引物(5μmol/L)为1μL,wzy基因上下游引物(5μmol/L)为2μL,单增李斯特菌DNA模板为1μL,鼠伤寒沙门氏菌DNA模板为1μL,大肠杆菌O157:H7 DNA模板为1μL,ex Taq DNA聚合酶(5 U/L)为0.3μL,加dd H2O补足25μL。反应条件为95℃预变性3 min;94℃变性30 s,53.9℃退火30 s,72℃延伸30 s,32个循环;72℃延伸10 min。鲜切哈密瓜中人工接种的目标菌的灵敏度为单增李斯特菌2.7×104 CFU/g,大肠杆菌O157:H7 3.3×104 CFU/g以及鼠伤寒沙门氏菌3.8×104 CFU/g。该技术可为快速检测鲜切哈密瓜中病原菌污染度及其控制提供参考依据。     

多重PCR同时检测食品中4 种细菌与常见霉菌

建立一种同时检测食品中金黄色葡萄球菌、单核细胞增生李斯特氏菌、沙门氏菌、大肠杆菌O157:H7以及霉菌的多重聚合酶链式反应（polymerase chain reaction,PCR）检测方法。根据金黄色葡萄球菌的耐热核酸酶基因（nuc）、沙门氏菌的侵袭正调节蛋白基因（hilA）、大肠杆菌O157:H7鞭毛基因（flic）、单核细胞增生李斯特氏菌的毒力调控蛋白基因（prfA）及霉菌18S?rRNA基因V5区分别设计5?对特异性引物,并对PCR扩增反应体系和扩增条件进行优化,确定获得特异性良好的PCR扩增产物。而后在37?℃对人工污染致病菌的香肠、面包和豆腐进行增菌培养,5?种致病微生物在20?h内的检出限均可达到100?CFU/25?g。本实验建立的多重PCR检测方法适用于食品中4?种细菌与常见霉菌的同时检测,相较于传统检测方法,具有快速、简便、特异性高的优点。     

多重PCR检测食源性致病菌的研究

建立多重PCR方法来同时检测和鉴定食品中单增李斯特氏菌、金黄色葡萄球菌和肠出血性大肠杆菌三种致病菌。试验以单增李斯特菌蛋白转录调控基因(hlyA)、金黄色葡萄球菌耐热核酸酶基因(nuc)和肠出血性大肠杆菌O157︰H7的O157抗原特异基因(rfbE)为靶基因设计引物,并对其反应体系进行优化,确定其灵敏度及检出限。多重PCR反应的灵敏度为102cfu/mL,检出限为103cfu/mL。该研究建立的多重PCR检测方法简单、快速、灵敏度高具有很好的应用前景。     

食源性致病菌多重PCR检测技术建立及其应用

针对6种食源性致病菌特异性基因(金黄色葡萄球菌nuc基因、副溶血性弧菌tdh基因、单核细胞增生李斯特菌hly基因、阪崎克罗诺杆菌ompA基因、鼠伤寒沙门氏菌invA基因和大肠杆菌O157:H7 rfbE基因)建立了多重PCR检测技术,分析了其特异性和敏感性,并评估了其在人工染菌牛奶中的应用可行性.结果表明:该检测技术可...     

食品中5种致病菌多重PCR快速检测技术的建立与应用

目的建立一种能同时检测食品中沙门氏菌、大肠杆菌O157∶H7、金黄色葡萄球菌、单核细胞增生李斯特菌和蜡样芽胞杆菌的多重PCR快速检测方法。方法分别针对沙门氏菌侵袭基因invA、大肠杆菌O157∶H7肠溶血素A基因HlyA、金黄色葡萄球菌耐热性核酸酶基因nuc、单核细胞增生李斯特菌李氏溶血素O基因hlyA和蜡样芽孢杆菌肠毒素FM基因entFM序列设计引物,进行PCR扩增及电泳检测。同时优化反应体系,测定特异性和灵敏性。结果初步建立的多重PCR方法可简便、快速、灵敏地实现对5种致病菌的同时检测,整个检测过程少于30h,且检测灵敏度均可达102CFU/ml。结论这种方法是对传统检测方法的有效改进,为食源性致病菌的快速高通量检测提供了理想手段,有良好的应用前景。     

LAMP法检测牛奶中金黄色葡萄球菌、鼠伤寒沙门菌和大肠杆菌O517:H

目的建立一种能快速准确检测牛奶中金黄色葡萄球菌(Staphylococcus aureus)、鼠伤寒沙门菌(Salmonella typhimurium)和大肠杆菌O517:H7(Escherichia coli O517:H7)的环介导恒温扩增方法(loop mediated isothermal amplification,LAMP)。方法以牛奶为研究对象,分别利用金黄色葡萄球菌nuc,鼠伤寒沙门菌invA和大肠杆菌O517:H7的rfbE保守基因设计LAMP特异引物,通过优化反应温度、时间等条件,建立检测牛奶中金黄色葡萄球菌、鼠伤寒沙门菌和大肠杆菌O517:H7的LAMP反应体系。并验证了本方法的特异性和灵敏度。结果 LAMP法对牛奶中金黄色葡萄球菌、鼠伤寒沙门菌和大肠杆菌O517:H7的灵敏度均为1.0×10~2拷贝/μL,比常规PCR灵敏度提高了10倍。金黄色葡萄球菌和大肠杆菌O517:H7的LAMP反应条件为60℃、30 min,鼠伤寒沙门菌为60℃、60 min。利用SYBR Green I染料在紫外光下可直接观察检测结果,金黄色葡萄球菌、鼠伤寒沙门菌和大肠杆菌O517:H7阳性样品均呈绿色;进一步利用荧光定量PCR仪可检测到对应的扩增曲线。通过牛奶样品模拟掺杂实验,验证了方法的实用性和可靠性。结论本研究建立的检测牛奶中金黄色葡萄球菌、鼠伤寒沙门菌和大肠杆菌O517:H7的LAMP方法具有特异性强、灵敏度高、操作简便等特点,可用于牛奶中相关致病菌的检测,对预防和控制3种致病菌的传染有重要意义。     

2009年嘉兴市食源性病原菌监测分析

目的监测2009年嘉兴市食品中致病菌污染状况。方法共采集275份生熟食品样品,分离沙门菌、单核细胞增生李斯特菌、大肠杆菌O157:H7、金黄色葡萄球菌和副溶血性弧菌。结果 75份生肉、水产品食源性病原菌总污染率45.3%,其中检出沙门菌6株,单核细胞增生李斯特菌12株,金黄色葡萄球菌4株,副溶血性弧菌18株。未检出大肠杆菌O157:H7。200份即食食品总污染率3.0%,以金黄色葡萄球菌污染为主。结论 2009年嘉兴市主要污染食品品种是冷藏冷冻生肉,污染的食源性致病菌以单核细胞增生李斯特菌和副溶血性弧菌为主。     

肉中4种致病菌的PCR快速检测方法的建立

目的:建立一种能同时检测肉中金黄色葡萄球菌、志贺氏菌、沙门氏菌和单核增生李斯特菌的多重PCR检测方法。方法:根据金黄色葡萄球菌的耐热核酸酶基因(nuc)、沙门氏菌的侵袭蛋白基因(invA)、志贺氏菌的侵袭性质粒抗原基因(ipaH)和单核细胞增生性李斯特菌的内化素基因(inlA)设计引物,通过优化好的反应体系进行多重聚合酶链反应(PCR)扩增目的基因。结果:特异性实验结果表明4种菌均能在相应位置扩增出特异性条带。对污染4种菌的猪肉进行检测,确定出金黄色葡萄球菌和沙门氏菌的检出限是102CFU/mL,志贺氏菌和单核增生李斯特菌的检出限是101CFU/mL。结论:本实验建立的多重PCR方法比传统细菌检测方法更特异、快速、灵敏,适用于肉中金黄色葡萄球菌、志贺氏菌、沙门菌和单核增生李斯特菌的快速检测。     

多重PCR快速检测两种致泻性大肠杆菌方法的建立

本研究以肠出血性大肠杆菌O157∶H7及肠侵袭性大肠杆菌为目标菌,针对大肠杆菌共同基因uid A、肠出血性大肠杆菌O157∶H7的特异基因O-antigen、肠侵袭性大肠杆菌的特异基因ipa H设计3对引物,建立并优化了检测两种菌的多重PCR检测方法,进行了特异性验证和灵敏度分析,并应用于人工接种新鲜莴苣的检测中。实验结果表明,本研究建立的三重PCR快速检测两种致病菌的检出限为6.3×103CFU/m L,具有较好的灵敏度和特异性。利用所建立的多重PCR方法对人工接种肠出血性大肠杆菌O157∶H7和肠侵袭性大肠杆菌的新鲜莴苣进行检测,检出限为7.8×104CFU/m L。此方法能够对肠出血性大肠杆菌O157∶H7和肠侵袭性大肠杆菌两种食源性致病菌进行快速检测。     

冰鲜鸡肉中3 种主要致病菌的共修复- 增菌条件研究

研究冰鲜鸡肉中鼠伤寒沙门氏菌(S. typhimurium)、单增李斯特菌(L.monocytogenes)和大肠杆菌(E. coli) O157:H7的共修复-增菌条件。通过传统技术和PCR技术相结合的方法,研究肉鸡屠宰浸烫脱毛工艺对3种目标菌的热激损伤及共修复-增菌条件,并利用共修复-增菌结果,对生产线和超市的冰鲜鸡肉样品中3种目标菌的污染状况进行调查。结果表明：肉鸡屠宰浸烫脱毛工艺条件能使部分S. typhimurium、L.monocytogenes和E. coli O157:H7处于亚致死状态;TSB-YE培养基对热损伤S. typhimurium、L.monocytogenes和E. coli O157:H7的修复效果最好,且对前两种菌的修复时间为2h,对后一种菌的修复时间为4h;TSB-YE培养基在16h对3种致病菌的共增菌效果最好;运用共修复-增菌条件,对80份实际样品中的3种致病菌检出率分别为：S. typhimurium 22.5% (18/80)、L.monocytogenes 11.3% (9/80)和E. coli O157:H7 18.8% (15/80)。     

主要食源性致病菌的PCR检测方法优化及在冷鲜猪肉病原污染调查中的应用

为优化针对主要食源性致病菌的PCR鉴定体系,以及了解猪肉制品中主要食源性致病菌的污染状况,对文献中报道的沙门氏菌属(鼠伤寒沙门氏菌及肠炎沙门氏菌)的5套PCR体系及大肠杆菌O157:H7的6套PCR体系进行比较分析,结果筛选出2套最优体系。基于这两套体系以及本实验室研究人员建立的单增李斯特菌的特异性检测方法,对从杭州市场上采集的50份常温鲜猪肉及150份冷鲜猪肉中单增李斯特菌、沙门氏菌及大肠杆菌O157:H7的污染状况进行调查。调查结果显示,200份样品中致病菌总检出率为21%。冷鲜猪肉中的致病菌检出率(24.7%)明显高于常温鲜猪肉(10%)。单增李斯特菌在常温鲜猪肉中的检出率为2%,而在冷鲜猪肉中检出率达14.7%;沙门氏菌在冷鲜猪肉中的检出率(11.3%)亦高于常温鲜猪肉(8.0%);大肠杆菌O157:H7均未检出。在检出的沙门氏菌中,有4株为鼠伤寒沙门氏菌,均来自冷鲜猪肉。本研究结果提示,市场上冷鲜猪肉存在微生物性安全隐患,这可能是由于其较为复杂的生产流通环节受到污染所致。     

Survival of Escherichia coli O157:H7, Salmonella enteritidis, Staphylococcus aureus, and Listeria monocytogenes in Kimchi

The survival of gram-positive and gram-negative foodborne pathogens in both commercial and laboratory-prepared kimchi (a traditional fermented food widely consumed in Japan) was investigated. It was found that Escherichia coli O157:H7, Salmonella Enteritidis, Staphylococcus aureus, and Listeria monocytogenes could survive in both commercial and laboratory-prepared kimchi inoculated with these pathogens and incubated at 10 degrees C for 7 days. However, when incubation was prolonged, the S. aureus level decreased rapidly from the initial inoculum level to the minimum detectable level within 12 days, whereas Salmonella Enteritidis and L. monocytogenes took 16 days to reach similar levels in commercial kimchi. On the other hand, E. coli O157:H7 remained at high levels throughout the incubation period. For laboratory-prepared kimchi, the S. aureus level decreased rapidly from the initial inoculum level to the minimum detectable level within 12 days, and L. monocytogenes took 20 days to reach a similar level. E. coli O157:H7 and Salmonella Enteritidis remained at high levels throughout the incubation period. The results of this study suggest that the contamination of kimchi with E. coli O157:H7, Salmonella Enteritidis, S. aureus, or L. monocytogenes at any stage of production or marketing could pose a potential risk.     

Comparative survival of Salmonella typhimurium DT 104, Listeria monocytogenes, and Escherichia coli O157:H7 in preservative-free apple cider and simulated gastric fluid

This study compared the survival of three-strain mixtures (ca. 10(7) CFU ml(-1) each) of Salmonella typhimurium DT104, Listeria monocytogenes, and Escherichia coli O157:H7 in pasteurized and unpasteurized preservative-free apple cider (pH 3.3-3.5) during storage at 4 and 10 degrees C for up to 21 days. S. typhimurium DT104 populations decreased by <4.5 log10 CFU ml(-1) during 14 days storage at 4 and 10 degrees C in pasteurized cider, and by > or =5.5 log10 CFU ml(-1) during 14 days in unpasteurized cider stored at these temperatures. However, after 7 days at 4 degrees C, the S. typhimurium DT104 populations had decreased by only about 2.5 log10 CFU ml(-1) in both pasteurized and unpasteurized cider. Listeria monocytogenes populations decreased below the plating detection limit (10 CFU ml(-1)) within 2 days under all conditions tested. Survival of E. coli O157:H7 was similar to that of S. typhimurium DT104 in pasteurized cider at both 4 and 10 degrees C over the 21-days storage period, but E. coli O157:H7 survived better (ca. 5.0 log10 CFU ml(-1) decrease) than S. typhimurium DT104 (> 7.0 log10 CFU ml(-1) decrease) after 14 days at 4 degrees C in unpasteurized cider. In related experiments, when incubated in simulated gastric fluid (pH 1.5) at 37 degrees C, S. typhimurium DT104 and L. monocytogenes were eliminated (5.5-6.0 log10 CFU ml(-1) decrease) within 5 and 30 min, respectively, whereas E. coli O157:H7 concentrations decreased only 1.60-2.80 log10 CFU ml(-1) within 2 h.     

Effects of cetylpyridinium chloride, acidified sodium chlorite, and potassium sorbate on populations of Escherichia coli O157:H7, Listeria monocytogenes, and Staphylococcus aureus on fresh beef

The effects of selected food-grade antimicrobial agents at decreasing the number of pathogenic bacteria on fresh beef were determined. Beef cubes inoculated with Escherichia coli O157:H7, Listeria monocytogenes, or Staphylococcus aureus were sprayed with 0.5% cetylpyridinium chloride (CPC), 0.12% acidified sodium chlorite (ASC), 0.1% potassium sorbate (PS), or an equal mix of any two solutions. The beef samples were placed on absorbent tray pads sprayed with each single or mixed solution, wrapped with polyvinyl chloride film, heat sealed, and stored at 4 degrees C for 2 weeks. Surface sanitization using CPC, ASC, or an equal mix of these two agents effectively reduced microbial numbers on the beef during storage. At day 0, ASC and the CPC-ASC mix reduced the number of E. coli O157:H7 by 2.50 and 1.58 log CFU/cm2, respectively. CPC demonstrated a 3.25-log reduction of L. monocytogenes and a 4.70-log reduction of S. aureus at 14 days. The CPC-PS mix reduced E. coli O157:H7 numbers by 1.46, L. monocytogenes by 2.95, and S. aureus by 4.41 log CFU/cm2 at 14 days. PS alone and the mixed solutions, CPC-ASC, CPC-PS, or ASC-PS, were not as effective as ASC or CPC alone. To effectively reduce E. coli O157:H7, L. monocytogenes, or S. aureus numbers, higher (> 0.1%) concentrations of PS were necessary. Loss of redness and light color of beef surfaces consistently coincided with decreases in pH for ASC-treated beef samples.     

Simultaneous detection of Escherichia coli O157:H7, Listeria monocytogenes and Salmonella strains by real-time PCR

A protocol enabling simultaneous detection of Escherichia coli O157:H7, Listeria monocytogenes and Salmonella strains was devised and evaluated using artificially contaminated fresh produce. Association of Official Analytical Chemists (AOAC)-approved polymerase chain reaction (PCR) detection methods for three human pathogens were modified to enable simultaneous and real-time detection with high throughput capability. The method includes a melting-curve analysis of PCR products, which serves as confirmatory test. The modified protocol successfully detected all three pathogens when fresh produce was washed with artificially contaminated water containing E. coli O157:H7 and S. typhimurium down to the predicted level of 1 to 10 cells/ml and L. monocytogenes at 1000 cells/ml. The ability to monitor several pathogens simultaneously will save time and increase our ability to assure food safety.     

Optimization of rapid detection of Escherichia coli O157:H7 and Listeria monocytogenes by PCR and application to field test

For rapid detection of Escherichia coli O157:H7 and Listeria monocytogenes, simple methods for sample preparation and PCR were established and applied to a field test. To improve specificity, primer sets LP43-LP44 and C(+)-D(-) were selected for E. coli O157:H7 and L. monocytogenes, respectively. Through centrifugation and partial heat treatment after enrichment, E. coli O157:H7 and L. monocytogenes were detected at 1 initial CFU without genomic DNA extraction in the culture and with artificially inoculated food samples including milk, chicken, ham, and pork. Based on the optimized PCR method, a feasibility test was carried out using randomly collected field samples. To remove false positives and false negatives, a PCR method using several primer sets, including the optimized primer set, and a standard culture method were used. With the PCR detection and standard culture methods, two pork samples were positive for L. monocytogenes after enrichment, indications that the PCR assay could be effectively used for rapid, sensitive, and species-specific detection of foodborne pathogens.     

Inhibition of Escherichia coli O157:H7, Listeria monocytogenes and Staphylococcus aureus on sliced roast beef by cetylpyridinium chloride and acidified sodium chlorite

The effects of 0.5% cetylpyridinium chloride (CPC), 0.12% acidified sodium chlorite (ASC) and a mix of equal volume of the two (0.25% CPC-0.06% ASC) on Escherichia coli O157:H7, Listeria monocytogenes and Staphylococcus aureus were evaluated on inoculated sliced roast beef. The antimicrobial agents were, respectively, sprayed on the beef surfaces and tray absorbent pads, and samples were stored at 4 degrees C for 10 days (d). At 0 d, L. monocytogenes and S. aureus were reduced to undetectable levels in 2 h after spraying with CPC. CPC-ASC treatment reduced E. coli O157:H7, L. monocytogenes and S. aureus by 4.07, 6.37 and 4.32 log cfu/cm2, respectively, at 0 d. ASC treatment reduced the population of E. coli O157:H7 by 6.09 log cfu/cm2 at 10 d. CPC treatment caused a slight discoloration and ASC-treated beef surfaces demonstrated the lowest redness and highest lightness. The grey colour and off-odour were significant in the ASC-treated beef samples, while CPC-treated samples demonstrated less off-odor and brown colour from 0 to 4 d. Based on our results, it appears that the application of CPC on sliced roast beef can extend the shelf-life of the product without impairing its quality.