加工食品中若干动物成分的PCR检测技术应用研究

本研究应用PCR技术检测了12种加工食品中的牛、羊、鸡、猪等动物源性成分，在此基础上还开发了真核生物所共有的18S核糖体DNA（18S rDNA）与食品中牛、羊、鸡、猪等多种动物物种特异性基因片段之间的二重PCR检测方法，还分析了牛、羊源性成分单PCR检测的灵敏度，以及18S rDNA与牛、羊之间的二重PCR检测方法的灵敏度。     

基于DNA测序方法检测生鲜肉中5种动物源性成分

目的建立生鲜肉中猪、牛、羊、鸡、鸭源性成分的DNA测序鉴别方法,并对采集自北京各地区生鲜肉品进行检测验证。方法合成动物线粒体上12S rRNA、cytochrome b、cytochrome c oxidase基因引物,建立猪、牛、羊、鸡、鸭源性成分的DNA测序鉴别方法,并对50份猪、牛、羊、鸡、鸭肉品进行检测。结果使用线粒体上12S rRNA、cytochrome b、cytochrome c oxidase基因引物对猪、牛、羊、鸡、鸭肉品进行扩增,分别获得456、400和710 bp大小的DNA片段。只有12S rRNA基因引物均可对此5种动物源性成分扩增且效果良好。扩增产物进行序列测定。根据序列构建的系统进化树发现,此方法可有效区分样品中猪、牛、羊、鸡、鸭源性成分。结论此方法快速、简便,可准确检测50种生鲜肉样品中的动物源性成分,作为肉类鉴别的新方法。     

食用植物油中动物源性成分PCR检测方法的建立

本文建立了应用PCR技术快速鉴别食用植物油中动物源性成分的方法。以食用植物油中掺入的动物源性基因为靶标,自行设计16S rRNA基因通用引物,同时选用动物物种特异性引物进行PCR扩增,分别得到相应的目的片段。通过三对16S rRNA基因通用引物,建立了从食用植物油中快速检测是否含有动物源性成分的方法;利用所选的动物物种特异性引物,进一步建立了从食用植物油中鉴定猪、牛、羊、鸡、鱼5种动物源性成分的方法,此方法能检测出含有0.1%(m/m)动物源性成分的植物油。     

基于动物基因信息分析的食用油鉴别检测方法研究

本研究通过选用4种特定的动物引物探索一种和多种动物油脂在食用油中的检出限和检出状况,建立了一套可行的应用PCR技术鉴别检测食用油掺杂动物油脂的方法。以牛、羊、猪、鸡4种动物组织提取出的DNA分别验证其各自引物的特异性;试验将单一动物油脂(猪油、牛油、羊油和鸡油)按不同比例与食用油混掺,用吸附柱法提取油中DNA,检测出食用油中0.1%(m/m)单一动物源性成分;再将两种、三种、四种动物油脂分别与食用油混掺,提取DNA后进行PCR扩增,辨析每种动物基因信息的检出状况;最终利用选取的特异性动物引物,从包含一种或多种动物信息的食用油中鉴别检测出猪、牛、羊、鸡4种动物源性成分,此法可实现检出含有0.1%(m/m)动物源性成分的食用油掺假,为食用油掺假鉴别提供了新的参考方法。     

种属特异性PCR法鉴别罐头食品中猪、牛、羊、鸡、鸭源性成分

建立适用于肉类罐头等长时间高温加工食品中猪、牛、羊、鸡、鸭5种动物源成分种属特异性PCR鉴别方法。通过使用猪、牛、羊、鸡、鸭的种属特异性引物,对5种动物的总DNA模板进行PCR扩增,得到分别为212、147、202、131、201 bp的扩增产物,将测序结果在美国国家生物技术信息中心(US National Center for Biotechnology Information,NCBI)进行BLAST比对确认实验的准确性,并对市售罐头样品进行检测。该方法5种动物引物种属特异性良好,对猪、牛、羊、鸭源性成分的检测灵敏度可达1%,对鸡源性成分的检测灵敏度为2.5%。在对市售肉类罐头样品的检测中,6.6%样品与标签标注结果不符。该方法操作简便,成本低,结果准确可靠,可广泛用于肉类罐头食品和长时间高温加工食品中猪、牛、羊、鸡、鸭5种动物源成分的鉴别,具有十分广泛的实际应用价值。     

饲料中六种动物源性成分多重PCR快速检测方法

采用试剂盒对含有牛、羊、猪等6种动物源性成分饲料中的DNA进行提取,根据动物线粒体DNA序列设计引物.以18 s rRNA作为内源基因对照,对DNA提取产物进行6种动物源性成分多重PCR扩增,同时对6种动物源性成分进行特异性鉴定.结果分别扩增出6种动物的特异性条带,证明该多重PER方法具有较高的特异性和灵敏度.     

硫酸软骨素中掺假动物成分的检测研究

研究建立硫酸软骨素中牛、羊、猪、鸡、鸭、鹅等动物源性成分的PCR检测方法,该方法特异、灵敏,检测限为0.1%.运用建立的方法对市场上的38个不同动物成分来源的硫酸软骨素样品进行检测,在27个硫酸软骨素样品中检出其它动物成分.该检测方法能够用于硫酸软骨素中牛、羊、猪、鸡、鸭和鹅源性成分的原料成分鉴别.     

高效快速检测食物中猪成分的PCR和RT—PCR方法

依据猪线粒体DNA中的细胞色素b基因序列设计一对特异性引物,用于检测食物中的猪源成分DNA扩增片段(130 bp)可以从猪的基因组中检出而对其它物种(牛、羊、鸡等)样品的检测为阴性,且在各种组织中稳定存在,亦可应用于实时荧光PCR从加工的熟食和混合食物中灵敏地栓出痕量猪DNA.     

基于单拷贝核基因的数字PCR定量检测肉制品中羊源性成分

GeneBank搜索牛、绵羊、山羊、猪、马、驴、鸡、鸭、鹅、火鸡、狗、猫、鼠、兔、貂等15种动物的单拷贝核基因组序列信息,应用生物信息学分析筛选15种动物共有的种间保守区域,设计一对可同时扩增15种动物源性DNA的内参基因引物和探针;同时分析筛选绵羊和山羊共有且和其余动物种内特异性区域,设计一对只能扩增羊源性DNA的特异性基因引物和探针。基于微滴数字PCR技术,引入内参基因校正羊种属特异性基因测定方法,建立科学准确的肉制品中羊源性成分量化判定方法。结果表明,所建立方法具有良好的特异性和通用性,内参基因和羊种属特异性基因的最低检出限分别为32和26 copies/μL,模拟添加样品的正确度偏差均值为8.66%,符合数字PCR方法制定指南要求不得大于25%,说明量化判定方法结果具有较高的准确性。     

GNM C7-8实时荧光PCR同时快速检测8种动物源性成分

目的基于八模块设计的GNMC7-8实时荧光PCR系统实现对8种动物源性成分同时快速检测。方法基于猪朊蛋白基因、牛生长激素基因、羊生长激素基因、驴线粒体ATPase 6基因、马线粒体ATPase 6基因、鸭生长激素基因、水貂线粒体细胞色素b基因、狐狸线粒体细胞色素氧化酶Ⅰ亚基基因引物设计了8种动物源性成分实时荧光PCR检测方法。应用GNMC7-8实时荧光PCR系统,对8种动物源性成分启动同时检测和每隔5min顺序启动不同时检测,并将检测结果和ABI7500荧光PCR分别检测结果进行比较。结果 GNM C7-8实时荧光PCR系统同时检测猪、牛、羊、驴、马、鸭、水貂、狐狸8种动物源性成分,起峰周期数分别为30、27、21、21、24、17、15、17,与其不同时检测结果一样,与ABI7500荧光PCR系统检测结果相比,猪、羊、马、鸭、水貂5种成分的起峰快1～2个周期数,其余3种成分检测结果一致,检测完成时间少16～20 min。结论 GNM C7-8实时荧光PCR系统8个模块独立运行,互不干扰,无交叉污染,能够同时快速检测8种动物源性成分,为快速筛查动物源性成分提供强有力的设备技术支持。     

一起食物中毒事件中产独特肠毒素表型的金黄色葡萄球菌病原学分析

目的对一起疑似为金黄色葡萄球菌所导致的食物中毒事件进行葡萄球菌肠毒素检测,结合金黄色葡萄球菌病原学分析,为明确食物中毒诊断提供依据。方法根据流行病学调查,采用ELISA方法对可疑食物进行葡萄球菌肠毒素检测,同时对可疑食物和患者呕吐物进行金黄色葡萄球菌分离,运用Vitek2 Compact全自动细菌鉴定仪和血浆凝固酶试验鉴定为金黄色葡萄球菌,采用脉冲场凝胶电泳(PFGE)对病原菌进行同源性分析,以ELISA方法对检出的金黄色葡萄球菌菌株进行肠毒素检测,用PCR方法对肠毒素基因进行分型。结果食物和患者样品中分别分离出2和11株金黄色葡萄球菌,PCR方法及ELISA方法对肠毒素分型结果显示,其中12株同时存在SEA、SEB、SED、SEE 4种肠毒素及相关基因,PFGE聚类分析显示,其中12株产肠毒素金黄色葡萄球菌具有高度同源性。结论本起食物中毒事件为具有独特肠毒素表型的金黄色葡萄球菌导致,在金黄色葡萄球菌中毒实验室调查过程中,肠毒素检测结合病原菌溯源分析可以为相关公共卫生事件提供科学依据。     

Comparison of antibiogram, staphylococcal enterotoxin productivity, and coagulase genotypes among Staphylococcus aureus isolated from animal and vegetable sources in Korea

Staphylococcal food poisoning is caused by enterotoxin-producing Staphylococcus aureus. We investigated the prevalence of such organisms in samples of bovine mastitic milk (n = 714), raw meat (n = 139), and vegetables (n = 616). We determined the degrees of relatedness of isolates as indicated by antibiogram, staphylococcal enterotoxin (SE) productivity, and coagulase gene restriction fragment length polymorphism analysis. We examined 297 S. aureus isolates and found SE production in 57 (31.8%), 4 (7.8%), and 49 (73.1%) isolates from raw milk, raw meat, and vegetables, respectively. A high proportion of the isolates obtained from milk produced more than two types of toxins (mainly SEA, SEB, and/or SEC), whereas isolates from raw meat and vegetables primarily produced SEA alone. Most isolates were sensitive to cephalothin (97.6%), gentamicin (80.8%), erythromycin (79.5%), and tetracycline (72.7%), but were resistant to penicillin (90.2%) and ampicillin (88.9%). The proportion of antibiotic-resistant isolates differed according the source of the bacteria; the milk and vegetable isolates were more resistant to penicillin and ampicillin than were the meat isolates (P < 0.05), whereas tetracycline resistance was limited to the milk and vegetables isolates. The coagulase genotypes (I to XII) varied with the source of the organism, and only a few genotypes prevailed in each source: II (42.4%) and IV (24%) types in isolates from milk, IX (35.3%) and XI (45%) from raw meat, and III (40.3%) and XII (32.8%) from vegetables. These findings suggest that remarkable differences exist in antibiogram, SE productivity, and coagulase genotypes, resulting in limited clonal transmission of S. aureus into various food sources. As enterotoxin production only occurs when S. aureus grows to high numbers, staphylococcal food poisoning can be prevented by proper refrigeration.     

Influence of Staphylococcus aureus strain-type on mammary quarter milk somatic cell count and N-acetyl-beta-D-glucosaminidase activity in cattle from eight dairies

The hypothesis tested was that there are differences in pathogenicity between strains of Staphylococcus aureus that cause bovine mastitis. Mammary quarter milk somatic cell count (SCC) and N-acetyl-beta-D-glucosaminidase (NAGase) activity were used as indicators of the pathogenicity of different strains of S. aureus that infect the bovine udder. Eight commercial dairy herds with a history of S. aureus in bulk tank milk cultures were studied. Initially, composite foremilk samples were collected from all lactating cattle in each herd and cultured for staphylococci. Subsequently, all cows with a coagulase-positive staphylococcal intramammary infection (IMI) at the initial sampling that were still present in the herd of origin had individual mammary quarter foremilk samples collected. Coagulase-positive staphylococcal isolates were confirmed as S. aureus using a commercial biotyping system. Staphylococcus aureus isolates were strain-typed using pulsed-field gel electrophoresis. Mammary quarter milk SCC and N-acetyl-beta-D-glucosaminidase activity were determined for each cow. The difference in mean somatic cell count and mean NAGase activity for mammary quarters infected with the same strain of S. aureus and for uninfected quarters on the same cow was calculated. One-way analysis of variance was used to assess differences between strains within a herd. Overall, no significant differences were found between strains, suggesting that the degree of udder parenchymal injury induced by S. aureus IMI is in general significantly affected by factors other than strain type.     

The significance of some potentially pathogenic microorganisms in occurrence of food toxicosis. Part 1. S. aureus and staphylococcal enterotoxins

The data on the nomenclature, classification and properties of staphylococci and staphylococcal enterotoxins produced by them are presented. The analysis of cultural and biochemical properties of 137 strains of staphylococci isolated from raw milk and "Russian" cheese was performed. The high degree of correlation between the ability of S. aureus produce enterotoxins and the presence of enzymes coagulase, thermostable DNase, and other factors of pathogenicity is established.     

An outbreak of food poisoning due to egg yolk reaction-negative Staphylococcus aureus

An outbreak of staphylococcal food poisoning due to an egg yolk (EY) reaction-negative strain occurred in Japan. Twenty-one of 53 dam construction workers who ate boxed lunches prepared at their company cafeteria became ill, and eight required hospital treatment. The outbreak showed a typical incubation time (1.5-4 h with a median time of 2.7 h) and symptoms (vomiting and diarrhea) of staphylococcal food poisoning. Staphylococcus aureus, which produces staphylococcal enterotoxin (SE) A, was isolated from four fecal specimens of eight patients tested. Scrambled egg in the boxed lunches contained 20-40 ng/g of SEA, and 3.0 x 10(9)/g of viable S. aureus cells that produced this toxin. All isolates from patients and the food were EY reaction-negative, coagulase type II, and showed the same restriction fragment length polymorphism (RFLP) pattern. We concluded that the outbreak was caused by scrambled egg contaminated with EY reaction-negative S. aureus. In Japan, outbreaks of staphylococcal food poisoning are mainly caused by EY reaction-positive S. aureus, and EY reaction-negative colonies grown on agar plates containing EY are usually not analyzed further for detection of S. aureus. The present outbreak suggested that EY reaction-negative isolates should be subjected to further analysis to detect the causative agents of staphylococcal food poisoning.     

The prevalence,genetic diversity and antibiotic resistance of Staphylococcus aureus in milk,whey, and cheese from artisan farm dairies

In this study, coagulase positive staphylococci were detected in 45% of the 69 bovine milk, whey and cheese samples taken from five farm dairies, and all raw milk samples were contaminated. Genetic diversity, staphylococcal enterotoxin genes and antimicrobial susceptibility in putative Staphylococcus aureus isolates were investigated. Sixty-one percent of the 72 isolates analysed belonged to the same pulsed field gel electrophoresis group. The spa-typing revealed seven different spa types, t2678 being the most prevalent, but t127 and t197 were also detected. Sixteen different toxin gene profiles were identified in 87.5% of the isolates with sec and tst being the most frequent (52.5%), followed by seg and seh. All isolates were methicillin-sensitive S. aureus, and sensitive to the 12 antibiotics tested. The prevalence of S. aureus and the high diversity of isolates carrying enterotoxin genes constitute grounds for food safety concern in artisanal cheese making, whether pasteurised or not.     

Identification and antagonistic activity of lactic acid bacteria occurring in porcine blood from industrial slaughterhouses--a preliminary study

Ninety-seven lactic acid bacteria (LAB) were isolated from slaughterhouse porcine blood in order to select autochthonous LAB strains for use as biopreservatives of this by-product. They were identified by 16S rDNA sequencing; and their inhibition capacity was determined against four bacterial species frequently found in contaminated blood, i.e. Staphylococcus aureus, Escherichia coli, Pseudomonas fluorescens and Bacillus spp. The taxonomic study showed an unexpected low diversity of LAB in blood, i.e. only 8 different species were found, from which just 4, i.e. Enterococcus raffinosus, Lactobacillus murinus, Lactobacillus reuteri and Lactococcus garvieae, amounted to more than 90% of all isolates. Inhibition tests in solid culture media proved that S. aureus and Bacillus spp. were inhibited by most LAB strains obtained from porcine blood. E. coli was the indicator less affected by the isolated LAB species. Several isolates efficiently inhibited the growth of all tested indicators.     

Biotyping and enterotoxigenicity of Staphylococci isolated from fresh and frozen meat marketed in Jordan

A total of 286 fresh and processed meat samples marketed in Jordan were collected for isolation and typing of Staphylococci. Devriese’s system was followed for biotyping of the isolated Staphylococcus aureus subsp. aureus and enterotoxigenicity of the isolates was also determined. S. aureus subsp. aureus was found in 80.8% of the samples. Means counts for meats of the various sources ranged from 5.3 × 10² to 4.3 × 10⁴ cfu/g. However, only 33.6% of the samples had coagulase positive Staphylococci. Biotyping of S. aureus subsp. aureus isolates revealed that 12%, 24%, 8%, and 27% are human, bovine, ovine and non-host specific biovars, respectively. Only 28% of these isolates, of unknown type, were isolated from goat’s meat. Enterotoxigenic S. aureus subsp. aureus isolates accounted for 13.5% of a total of 231 Staphylococci. The non-S. aureus enterotoxigenic isolates were identified as Staphylococcus epidermidis, Staphylococcus sciuri and Staphylococcus intermedius. Staphylococci enterotoxin types A, C, and B accounted for 57%, 36% and 7% of the isolated S. aureus subsp. aureus, respectively. This study showed that the enterotoxigenic Staphylococci represent a potential hazard in meat and the Devriese’s biotyping system needs to be extended to accommodate the isolates of unknown type.     

Characteristics of enterotoxin H-producing Staphylococcus aureus isolated from clinical cases and properties of the enterotoxin productivity

Staphylococcal enterotoxin H (SEH) is predicted to be involved in staphylococcal food poisoning. To characterize SEH-producing Staphylococcus aureus isolates from staphylococcal food poisoning cases in Japan, we investigated the relationship between SEH production and coagulase serotype, which is an epidemiological marker, and compared the properties of SEH production with those of staphylococcal enterotoxins A (SEA) and B (SEB). SEH production was determined by a newly developed sandwich enzyme-linked immunosorbent assay. Eighty-six (59.7%) of 144 isolates from staphylococcal food poisoning cases produced SEH. Seventy-one of the SEH-producing isolates simultaneously produced SEA, SEB, or both. All SEH-producing isolates belonged to coagulase type VII, which was the predominant type, representing 99 (68.8%) of 144 isolates. The amount of SEH produced in brain heart infusion was almost the same as the amount of SEA and approximately 10-fold lower than that of SEB. SEH and SEA were produced mainly during the late exponential phase of growth, whereas SEB was produced mostly during the stationary phase. The production levels of SEH and SEA were gradually affected by decreases in water activity, but the production of SEB was greatly reduced under conditions of low water activity. These findings indicate that SEH-producing S. aureus isolates are of high prevalence in staphylococcal food poisoning cases. Given the unique epidemiological characteristic of these isolates, SEH and SEA probably are responsible for food poisoning.     

Comparison of virulence and antibiotic resistance genes of food poisoning outbreak isolates of Staphylococcus aureus with isolates obtained from bovine mastitis milk and pig carcasses

Staphylococcus aureus is the etiological agent in a variety of infections in humans and livestock and produces enterotoxins leading to staphylococcal food poisoning (SFP), one of the most prevalent foodborne intoxication diseases worldwide. Pork and bovine milk are considered possible sources of SFP because pig skin is often colonized by S. aureus and bovine mastitis caused by S. aureus is common, but conclusive data are limited. The objective of the present study was to compare S. aureus isolates associated with cases of SFP with isolates obtained from bovine mastitis milk and pig carcasses. DNA microarray analysis and spa gene typing were performed with 100 S. aureus isolates: 20 isolates related to outbreaks of SFP in humans, 39 isolates obtained from pig carcasses, and 41 isolates collected from bovine mastitis milk. No overlap in spa types was observed for SFP isolates (t008, t015, t018, t024, t056, t084, t279, t377, t383, t648, t733, t912, t1239, t1270, t4802, and t6969) and isolates gathered from milk or pork. The porcine isolates were assigned to t034, t208, t337, t524, t899, t1939, t2922, t2971, t4475, and t7006, and the bovine isolates belonged to t267, t524, t529, t1403, t2953, t7007, t7008, and t7013. Comparison of microarray profiles revealed similar virulence gene patterns for isolates collected from the same host (pigs or cattle) but few similarities between SFP isolate profiles and the profiles of isolates obtained from bovine mastitis milk and pig carcasses. Although only some bovine and porcine isolates possessed the β-lactamase gene blaZ (milk, 24%; pork, 28%), significantly higher numbers of SFP isolates contained blaZ (90%). Investigations of these isolates provided no evidence that pork or bovine mastitis milk represent common sources of SFP.