2005—2012年宁波市食源性金黄色葡萄球菌肠毒素基因和多位点序列分型研究

目的了解宁波市食源性金黄色葡萄球菌(S.aureus)肠毒素基因分布和分子分型特征。方法收集2005—2012年宁波市食品中金黄色葡萄球菌菌株,利用聚合酶链式反应(PCR)检测肠毒素A、B、C和D基因(sea,seb,sec和sed),对肠毒素基因阳性菌株利用多位点序列分型(MLST)进行分子分型。结果 2005—2012年共分离菌株190株,肠毒素基因阳性菌株为41株,阳性率为21.58%。4种肠毒素基因阳性率分别为7.37%(14/190)、5.26%(10/190)、8.95%(17/190)和5.79%(11/190),其中13株菌株具有两个及两个以上肠毒素基因。41株菌株可分为12个序列型(ST),以ST5、ST6、ST188和ST1为主,共占75.61%(31/41),在进化树上主要形成4个分支。结论 2005—2012年宁波市食品中金黄色葡萄球菌携带肠毒素基因频率较高,需加强监测。肠毒素基因阳性菌株与中国其他地区食品株在分子分型上存在较大差异。     

噬菌体编码的金黄色葡萄球菌肠毒素A的 研究进展

金黄色葡萄球菌(Staphylococcus aureus)是一种人畜共患的食源性致病菌,并且能够产生不同种类的毒素,其中金黄色葡萄球菌肠毒素(staphylococcal enterotoxin,SE)是引起食物中毒的一类主要毒素。金黄色葡萄球菌肠毒素A(staphylococcal enterotoxin A,SEA)在食物中毒事件中的检出率最高,毒性最强,成为目前研究的热点。编码金黄色葡萄球菌肠毒素A的基因sea是由噬菌体携带并在菌体之间传播的。本研究主要综述了编码SEA的噬菌体与SEA之间的关系、SEA的分类、在不同的食品基质中环境因子对SEA产生量、sea基因相对表达量的影响及SEA新型的检测方法等,并介绍了目前金黄色葡萄球菌肠毒素研究的难点及研究方向的分析。     

利用简并引物检测金黄色葡萄球菌肠毒素A、B基因

通过设计简并引物建立一种PCR技术同步快速检测金黄色葡萄球菌肠毒素A和B基因的方法。根据金黄色葡萄球菌肠毒素A、B基因编码序列,设计一对特异性简并引物SEAB来扩增靶基因片段,长度分别为105bp和135bp,通过对金黄色葡萄球菌肠毒素A、B菌株和4株对照菌株进行PCR检测,评价该引物的特异性;对金黄色葡萄球菌肠毒素B的DNA系列10倍稀释,对其灵敏性进行PCR检测。结果显示,金黄色葡萄球菌肠毒素A和B菌株的DNA检测结果呈阳性,4株对照菌株的检测结果呈阴性,通过基因测序证实了PCR产物的特异性,SEB的DNA最低检测浓度为3.58ng,整个检测过程不超过20h。建立了一个特异、快速灵敏的在同一条件下,金黄色葡萄球菌肠毒素A、B基因的PCR检测方法。     

PCR检测食品中金黄色葡萄球菌肠毒素基因

目的:检测食品中金黄色葡萄球菌肠毒素基因谱携带情况,探讨与食物中毒发病相关的金黄色葡萄球菌基因类型。方法:取食品中检出的金黄色葡萄球菌进行分离培养鉴定,通过PCR技术检测样本18种不同型的肠毒素基因的携带情况,并进行统计学分析。结果:食品中检出的金黄色葡萄球菌肠毒素基因有较高的阳性检出率,主要检出sea、seb、sei、seg、sel、sem、sen等基因,其中sea基因检出率最高,达到77.27%;sei、seg、sem、sen基因具有相关性,检出率为9.09%;seb、sel基因的检出率为4.55%。结论:在食品中主要检出金黄色葡萄球菌肠毒素sea、sei、seg、sem、sen、seb、sel等基因,其中,sei、seg、sem、sen基因的相关性还有待进一步深入研究。     

食源性金黄色葡萄球菌肠毒素基因型分布研究

目的 采用PCR法扩增食源性金黄色葡萄球菌中肠毒素基因以了解该菌肠毒素基因携带情况,比较食物中毒和食品监测来源菌株中肠毒素基因检出率差异.方法 合成sea、seb、sec、sed和see五种肠毒素基因特异性引物,用常规PCR方法扩增食物中毒和食品监测来源菌株中各自肠毒素基因,同时采用mini-VIDAS检测食物中毒来源菌株中肠毒素.结果 110株菌株中有30株检出肠毒素基因,检出率为27.3％,肠毒素基因阳性菌株均只检出1种肠毒素基因.其中来自2起食物中毒的14株菌株均检出seb型肠毒素和相关基因,检出率为100％.来源于食品监测样本的96株菌株中有16株检出肠毒素基因,检出率为16.7％,包括sea型4株、seb型2株、sec型4株、sed型6株.结论 在宁波市食品监测中所分离的金黄色葡萄球菌所携带的肠毒素基因主要有sea、seb、sec和sed四型,而seb型肠毒素是引起金黄色葡萄球菌肠毒素所致食物中毒的主要因素.     

广州市白云口岸航空食品中金黄色葡萄球菌凝固酶基因分型及肠毒素基因研究

目的对2013—2015年从广州市白云口岸航空食品中分离的金黄色葡萄球菌进行基因分型研究,为食源性金黄色葡萄球菌分子溯源提供基础数据。方法以血浆凝固酶和肠毒素为目标基因,采用聚合酶链式反应(PCR)方法对9株金黄色葡萄球菌进行基因分型,其中6株为航空食品分离株,1株为配餐车间大门手拭分离株,2株为标准菌株。肠毒素基因检测包括5种传统肠毒素基因(sea、seb、sec、sed、see)和6种新型肠毒素基因(ser、seg、seh、sei、sej、sep)。结果 6株航空食品分离株的血浆凝固酶基因扩增分型结果为2个PCR型,酶切后得3种亚型;肠毒素基因检测结果显示有2株航空食品分离株含有肠毒素基因,检出率为33.3%(2/6),检出的基因为2种传统肠毒素基因(sec、sed)和4种新型肠毒素基因(ser、seg、sei、sej),均同时携带2种以上肠毒素基因。结论血浆凝固酶基因扩增分型结果显示,不同时间、不同采集地点存在相同的基因型,提示金黄色葡萄球菌存在交叉污染的可能性;航空食品分离株共检出6种肠毒素基因,提示金黄色葡萄球菌基因型多样性,应加强其他新型肠毒素基因检测。     

食源性金黄色葡萄球菌肠毒素基因及其表达检测

金黄色葡萄球菌作为一种常见人类病原菌,可通过各种途径污染食品,引发食物中毒。其中,金黄色葡萄球菌肠毒素(SE)的分泌会大大增加其侵袭性和致病力。通过对比肠毒素基因的携带及其表达情况,有助于进一步分析肠毒素表达的环境条件,为产肠毒素金黄色葡萄球菌的防范和控制提供理论和数据支持。本研究采用PCR和3M^TMTecra^TM微孔板法分别检测33株食源性金黄色葡萄球菌中5种传统肠毒素SEA^SEE基因的携带及表达情况,结果显示,该批金黄色葡萄球菌中SE基因的检出率为100%,携带率最高和最低的分别为seb(48.48%,16/33)和see(9.09%,3/33)。而其中SE蛋白得到表达的菌株仅为63.64%(21/33),这表明不是有SE基因携带就一定可以有相应毒素蛋白的表达,可能还需相应的系统调控和适宜的环境因素。     

速冻面米制品中金黄色葡萄球菌和肠毒素A基因三重PMA-qPCR快检方法的建立

为建立准确定量速冻面米制品中金黄色葡萄球菌活菌,筛查肠毒素sea基因并指示PCR反应假阴性的三重PMA-qPCR检测方法。针对金黄色葡萄球菌特异靶基因RpiR和肠毒素基因sea序列保守区,设计引物、探针和构建扩增内标,优化建立三重qPCR检测体系,建立PMA食品前处理方法去除死菌DNA,构建纯培养物和速冻面米制品中污染金黄色葡萄球菌的定量标准曲线,应用三重PMA-qPCR方法检测人工污染金黄色葡萄球菌的速冻面米制品。采用GB4789.10-2016中规定的选择平板计数法,评价三重PMA-qPCR方法的检测性能。结果表明,三重PMA-qPCR检测体系扩增效率高,特异性好。当食品中金黄色葡萄球菌污染量大于10~3CFU/g时,靶基因RpiR可获得有效扩增,污染量在10~3～10~7CFU/g之间时,扩增曲线线性良好(R~2=0.994),PMA-qPCR定量结果与平板计数结果一致性较好。当sea阳性菌株污染量大于10~3CFU/g时,应用PMA-qPCR方法可有效评估食品污染肠毒素A的风险。综上所述,本研究建立的三重PMA-qPCR检测方法操作简便,可快速定量检测速冻面米制品中金黄色葡萄球菌,评估食品污染肠毒素A的风险。     

食品基质中不同金黄色葡萄球菌肠毒素基因型表达差异研究

目的 比较研究食品基质中不同金黄色葡萄球菌肠毒素(Staphylococcal enterotoxins ,SEs)基因型的蛋白表达差异,为预防金黄色葡萄球菌食物中毒提供参考依据。方法 采用特异聚合酶链反应方法(polymerase chain reaction,PCR)对食品中分离的金黄色葡萄球菌菌株进行肠毒素基因型检测;选择sea、seb、sec、sed等基因型阳性菌株,分别接种于胰酪大豆胨液体培养基(trypticase soy broth,TSB)、牛奶、鸡肉中,按照国家标准GB 4789.10—2016酶联免疫吸附试验法(enzyme linked immunosorbent assay,ELISA)定量检测TSB培养基、牛奶和鲜鸡肉中不同金黄色葡萄球菌肠毒素基因型的蛋白表达量。结果 30株金黄色葡萄球菌中检测到14株肠毒素基因型阳性菌株,所占比例为46.67%,其中sea基因携带率最高(16.67%),而seb、sec、sed、seh则各占6.67%。SEA 在TSB、牛奶、鸡肉3种基质中的平均表达量为7.37 ng/mL,高于SEB、SEC、SED;不同基质环境对肠毒素的表达具有一定影响,如SEA、SEB、SEC、SED在TSB中的表达水平最高,平均表达量为9.04 ng/mL,牛奶次之,鸡肉最低。结论 肠毒素基因型的表达与菌株自身的调控及环境作用密切相关,本研究对肠毒素的产生机制进行初步了解,有助于进一步降低食物中毒风险。     

金黄色葡萄球菌肠毒素及移动基因元件研究进展

金黄色葡萄球菌肠毒素是一种热源性的超抗原。食用被肠毒素污染的食物能够引起食物中毒,导致恶心、呕吐、腹痛有时伴随腹泻。本文综述了肠毒素的分类命名、理化性质、以及编码不同肠毒素的基因在移动基因元件中的存在情况。这些移动基因元件在金黄色葡萄球菌毒力传播和进化过程中起着至关重要的作用,了解它们对于金黄色葡萄球菌流行病学溯源以及理解毒力机制具有一定的指导意义。     

Detection of genes for enterotoxins (ent) and toxic shock syndrome toxin-1 (tst) in mammary isolates of Staphylococcus aureus by polymerase-chain-reaction

During a two-year-period (1997–1998) 94 field strains of Staphylococcus aureus isolated from cases of bovine mastitis were investigated for the presence of genes for staphylococcal enterotoxin (ent) and toxic shock syndrome toxin-1 (TSST-1; tst). Polymerase-chain reaction (PCR) was performed using six pairs of relevant oligonucleotide primers. Thirty-four isolates were positive for one (18 strains) or two (16 strains) toxin genes. Three field strains were positive for enterotoxin A gene (sea), two for enterotoxin B gene (seb), 22 for enterotoxin C gene (sec), four for enterotoxin D gene (sed) and 19 for TSST1 gene (tst). The enterotoxin E gene (see) sequence was not found. The combination of sec and tst showed the highest incidence. A high correlation between PCR results and toxin protein detection by a commercial enzyme linked immunosorbent assay (ELISA) system and a reversed passive latex-agglutination (RPLA) test was observed. In these immunoassays, only one strain of S. aureus produced equivocal results for production of enterotoxin A, giving an overall concordance between genotypic and phenotypic identification of 98.9%.     

大豆天冬氨酸途径关键酶基因GmASADH的克隆与分析

本研究基于电子克隆获得的大豆GmASADH基因eDNA序列,根据其编码区设计特异引物,以大豆品种小鹦哥豆总RNA为模板,通过RT—PCR获得了约1130bp的eDNA片段,T／A克隆后进行序列测定。测序结果显示,GmASADH基因家族有两个成员,命名为GmASADHI（FJ581425）和GmASADI-12（HM015631）。GmASADHl编码区长度为1134bp,编码378个氨基酸,由7个外显子和6个内含子构成,定位于Gm08染色体。GmASADH2编码区长度为1131bp,编码377个氨基酸,由7个外显子和6个内含子构成,定位于Gm05染色体。两个基因在氨基酸水平上的相似性达到了96．02％,在二级及三级结构上均有不同程度的差异。ASADH蛋白含有NADB—Rossmannsuper-family和Semialdhyde—dhCsuperfamily结构域。     

山药颗粒结合型淀粉合成酶基因DaGBSS的克隆与分析

颗粒结合型淀粉合成酶（granule-bound starch synthase, GBSS）是一种能够决定直链淀粉生物合成的关键性酶。通过对山药转录组分析以及分子克隆获得一个具有完整ORF的DaGBSS基因,cDNA片段长度为1845 bp,编码614个氨基酸。生物信息学分析结果显示DaGBSS蛋白为一酸性稳定蛋白,含有淀粉合酶催化和糖基转移酶两个功能结构域。多序列比对结果显示：糯米山药DaGBSS蛋白与所选的物种具有较高的同源性,且含有典型的功能结构域;生物进化分析结果显示DaGBSS蛋白与圆形薯蓣亚种进化亲缘关系最近。对采收15天山药块茎进行处理,qRT-PCR以及指标分析结果显示DaGBSS基因在山药块茎的表达存在明显的时空差异性,且在距离地表位置最远的部位DaGBSS基因的表达量、GBSS和直链淀粉的含量均为最高。该研究通过对山药块茎淀粉合成的早期阶段进行生理以及关键调控因子的克隆分析,为探究山药淀粉合成的调控提供理论依据,同时对糯米山药的分子育种提供了重要的基因资源。     

Staphylococcal enterotoxin D production by Staphylococcus aureus FRI 100

Staphylococcus aureus FRI 100 is commonly used as a control strain for staphylococcal enterotoxin A (SEA) assays. When FRI 100 was used in PCR-based enterotoxin detection methods, the strain gave a positive result for both SEA and staphylococcal enterotoxin D (SED). Production of SED was confirmed by testing concentrated and unconcentrated culture supernatants with the TECRA staphylococcal enterotoxin visual immunoassay. SED was detected after 24 h of growth in Trypticase soy broth. Primers were created to amplify the entire sed gene by PCR for subsequent sequencing. The sequenced gene showed high similarity to a previously sequenced sed gene. The SED-like gene in FRI 100 exhibited four point mutations and two deletions. Changes in the FRI 100 open reading frame altered the primary structure of the SED-like protein, allowing for coding of only the first 150 amino acids followed by a stop codon. Because the SED active site is at the proximal end, where there was no change in DNA sequence, we conclude FRI 100 produces a variant form of SED. It is necessary to note that, when using FRI 100 as an SEA control strain, it does produce a variant of the SED protein, which exhibits immunological activity, and the sed-like gene is detected by commonly used PCR primers. This phenomenon may be an important general consideration when using PCR to characterize strains of toxin-producing S. aureus. S. aureus enterotoxin-positive PCR results should be confirmed by immunological techniques.     

Evaluation of enzyme-linked immunosorbent assay (ELISA) for detection of staphylococcal enterotoxin in foods

Two ELISA kits were employed to detect staphylococcal enterotoxin A, B, C and D in foods to which enterotoxin had been added or which had been artificially contaminated with enterotoxin-producing strains of Staphylococcus aureus. The sensitivity was satisfactory, and enterotoxins were detected by both ELISA kits in all positive samples. Weak positive reactions with one ELISA kit caused some difficulties in evaluation of samples. Enterotoxins A, B and C detected in small amounts (2-22 ng/ml) in minced fried beef were not detectable after heating at 80 degrees C, whereas enterotoxin D detected in considerably greater amounts (783 ng/ml) was reduced to 0.4% of this amount.     

Growth and toxin profiles of Bacillus cereus isolated from different food sources

Eleven strains of Bacillus cereus isolated from milk and meat products have been used to study growth and sporulation profiles in detail. Polymerase chain reaction (PCR) using primers detecting cold shock protein A gene signatures (cspA), showed that none of the strains were the newly suggested species in the B. cereus group, B. weihenstephanensis, comprising psychrotolerant cereus strains, although one of the strains grew at 4 degrees C, two at 6 degrees C and seven grew at 7 degrees C. One of the two strains that grew at 6 degrees C had a maximum growth temperature of 42 degrees C, while the remaining 10 strains all grew at temperature of 43 degrees C or higher. Only three strains grew at 48 degrees C. At 42 degrees C, the generation time varied between 11 and 34 min. Spore germination was much faster for the two strains that grew at 6 degrees C than for the other nine strains in milk at 7 degrees C and 10 degrees C. All strains were cytotoxic and contained the non-haemolytic enterotoxin gene (nhe), 10 strains contained the enterotoxin T gene (bceT), and only six had the gene (hbl) encoding haemolytic enterotoxin. Two strains showed some microheterogeneity in the nhe operon. but contained all three genes. We can conclude that true B. cereus strains can have growth profiles as expected for B. weihenstephanensis, and that nhe and bceT were not correlated with growth profiles. However, the two psychrotolerant strains with minimal growth temperature of 4 degrees C and 6 degrees C did not contain hbl, as judged from our PCR results.     

Rapid detection of enterotoxigenic clostridium perfringens by real-time fluorescence resonance energy transfer PCR

Clostridium perfringens is one of the etiologic agents of gas gangrene that can occur when a wound is contaminated with soil. Type A C. perfringens can cause foodborne and nonfoodborne gastrointestinal illnesses due to an enterotoxin (CPE) produced by some strains during sporulation. We developed a quantitative real-time PCR assay based on fluorescence resonance energy transfer hybridization chemistry that targets the C. perfringens-specific phospholipase C (plc) gene and the enterotoxigenic gene (cpe) with the LightCycler and the Ruggedized Advanced Pathogen Identification Device (R.A.P.I.D.). The assay can detect as few as 20 copies of target sequences per PCR. The total assay time, from extraction to PCR analysis, is 90 min. This assay is rapid, sensitive, and specific and will allow direct detection of C. perfringens in water, food, and stool samples. It should prove helpful in investigating foodborne illnesses due to C. perfringens and can be used as a tool to ensure the safety of food and water supplies.     

猪肉源大肠杆菌分离鉴定及其生物被膜形成

为研究猪肉来源大肠杆菌分离菌株的生物被膜形成及相关基因表达变化，首先从生猪屠宰线、猪胴体表面及生鲜猪肉中采用选择平板和特异性聚合酶链式反应（polymerase chain reaction，PCR）分离鉴定大肠杆菌，采用微孔板结晶紫染色法评价分离菌株15 ℃培养72 h时生物被膜形成能力，进而选取代表菌株研究生物被膜形成过程中被膜量、胞外聚合物（extracellular polymeric substances，EPS）组成，以及基于反转录荧光定量PCR的成膜基因表达的变化。结果表明：共分离到猪肉源大肠杆菌31 株，包括生猪屠宰线11 株、猪胴体表面4 株、生鲜猪肉16 株；微孔板结晶紫染色结果显示，被膜形成能力存在明显菌株差异，64.52%菌株成膜能力较弱，选取其中1 株（D4-18）进行成膜过程研究；微孔板中菌株D4-18 15 ℃培养168 h过程中被膜量持续增加，培养72 h和168 h时，菌株D4-18分泌EPS，培养72 h时，papC、fimH、csgA基因表达量分别为0.095、0.933、0.435 copies/cm2，随着培养时间延长，松散型EPS中蛋白质及多糖含量、紧密型EPS中蛋白质含量极显著增加（P＜0.01），培养168 h时，papC和fimH基因表达量增加，csgA基因表达量无显著变化，表明上述基因在大肠杆菌生物被膜形成过程中发挥了不同程度的调控作用。     

产气荚膜梭菌肠毒素基因地高辛探针制备与检测方法建立的初步研究

产气荚膜梭菌（ｃｌｏｓｔｒｉｄｉｕｍ ｐｅｒｆｒｉｎｇｅｎｓ,ＣＰ）是人类和家畜重要的厌氧性病原菌,产生肠毒素的ＣＰ是人类食物中毒病原菌。本研究用地高辛标记肠毒素基因制备探针,并建立了原位杂交检测方法,直接检测肠毒素基因（ｃｐｅ）。这是继ＰＣＲ检测方法后又一项特异性基因诊断技术。由于利用了光学显微镜观察结果,形态学上比较直观,增加了实验的准确性。与ＰＣＲ、乳胶凝集相比,原位杂交检测方法具有较高的敏