多重PCR检测金黄色葡萄球菌六型肠毒素基因的研究

目的:为了建立一种简单、快速检测金黄色葡萄球菌六型肠毒素基因的多重PCR方法。方法:根据相关文献和Genebank报道的编码金黄色葡萄球菌肠毒素A、B、C、D、E、H的基因序列,选择合成了6对特异性引物,建立多重PCR体系,并对反应条件进行了优化。结果:6对引物能同时特异地扩增出120、478、257、319、170、375bp的目的片段,表明6对引物具有良好的特异性。结论:成功地建立了一种同时检测金黄色葡萄球菌六型肠毒素基因的多重PCR方法,在金黄色葡萄球菌肠毒素快速筛查方面具有良好的应用前景。     

基于TaqMan探针荧光定量PCR技术快速检测乳中金黄色葡萄球菌肠毒素A

目的:建立检测金黄色葡萄球菌肠毒素A的Taq Man探针实时荧光定量PCR方法。方法:对金黄色葡萄球菌肠毒素A的基因序列分析、对比,设计特异性引物和Taq Man探针,建立对金黄色葡萄球菌肠毒素A的快速检测方法,并验证该方法的特异性、稳定性和灵敏性。结果:Taq Man探针荧光PCR检测金黄色葡萄球菌肠毒素A具有极强特异性,标准曲线相关系数为0.998,最低可检测出71个拷贝数的细菌DNA,检测乳中金黄色葡萄球菌肠毒素A菌灵敏度为1.3×102CFU/m L,不同浓度质粒重复性扩增试验Ct值的变异系数均3%,显示了良好重复性。结论:Taq Man探针荧光定量PCR方法可以在6h内快速、准确地检出金黄色葡萄球菌肠毒素A,为金黄色葡萄球菌快速检测和食物中毒快速诊断提供技术支撑,推动了荧光PCR技术在食品安全检测方面的实践应用。     

葡萄球菌肠毒素基因分型PCR检测技术的研究

通过生物信息学分析,设计了不同基因型肠毒素检测引物;应用含溶菌酶的碱裂解法提取葡萄球菌DNA及梯度PCR方法提高了肠毒素检出率和特异性.从国内28株葡萄球菌分离株中共检测出14种不同型的肠毒素基因(sea-see、tsst-1、seg-sel、sek-sel、sen-seo、seq-ser和seu),未检测到sei和sem基因.毒素基因携带率为16.67%,同时携带4种及以上毒素基因的菌株有11株,占39.29%,其中sek、seq和sea毒素基因在所研究菌株中分布最广,分别占到15.50%、14.30%和10.70%.结果表明不同葡萄球菌菌株间毒素型的关联度不同,sek与seq,seb和sed,sec、sei、sen和seu基因型呈现密切相关,各型肠毒素的分布还与菌株分离来源有着密切联系.所建立的PCR方法可用于金黄色葡萄球菌肠毒素基因分型和分布的研究.     

TaqMan探针荧光定量PCR检测金黄色葡萄球菌肠毒素D

建立了检测乳中产肠毒素D的金黄色葡萄球菌的荧光定量PCR方法,以SED基因为肠毒素D的检测靶序列,设计荧光定量PCR引物和Taq Man探针,将构建的重组质粒作为阳性对照,建立了对产肠毒素D金黄色葡萄球菌快速检测的Taq Man探针荧光定量PCR方法,并评价该方法的特异性、灵敏性和重复性。结果显示,Taq Man探针实时荧光定量PCR检测金黄色葡萄球菌肠毒素D的方法具有极强的特异性,标准曲线的相关系数为0.999,最低可检测到40copies/m L的阳性质粒,检测乳中产肠毒素D金黄色葡萄球菌的灵敏度为1.0×102CFU/m L。该检测方法具有较好的特异性和灵敏性,在产肠毒素D金黄色葡萄球菌快速筛查方面具有良好的应用前景。     

乳品中3种常见细菌多重PCR检测方法的建立

分别以大肠杆菌、金黄色葡萄球菌、沙门氏菌标准株为样本,提取其DNA,以大肠杆菌的pho A基因、金黄色葡萄球菌的nuc A基因和沙门氏菌的inv A基因作为靶基因,建立可以同时检测这3种菌株的多重PCR方法。与国标法对照,探讨多重PCR技术检测乳品样品中3种细菌的灵敏性和特异性。结果表明,建立的多重PCR检测方法与国标法检测的结果一致,人工模拟试验结果稳定,多重PCR具有快速、敏感和特异性强的特点。     

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.     

牛乳腺炎金黄色葡萄球菌肠毒素A基因的克隆及序列分析

根据Genbank上公布的金黄色葡萄球菌肠毒素A的全序列,利用生物学软件Primer 5.0和oligo 6.0设计了一对特异性引物来扩增靶序列片段,经克隆预测序,结果表明扩增片段长度为101 bp,锦州分离株与标准菌株的基因片段序列相似性为100%,与Genbank上公布的金黄色葡萄球菌菌株(EF520720.1)SEA基因相似性达到99.14%。高度相似性结果为进一步研究建立分子检测技术奠定了实验基础。     

鉴定金黄色葡萄球菌的多重PCR方法

建立了一种快速鉴定和鉴别金黄色葡萄球菌的多重PCR方法。利用金黄色葡萄球菌的特异基因、耐甲氧西林基因和4种肠毒素基因nuc、mecA和sea、sec、sed、see建立6重PCR反应体系,并对PCR体系,引物浓度进行优化。6对PCR引物均能特异地扩出相应的目的基因。对47株金黄色葡萄球菌的检测结果与应用单重PCR鉴定结果完全一致。实验所建立的多重PCR方法能在一个反应体系中鉴定和鉴别金黄色葡萄球菌,为食品中金黄色葡萄球菌的快速检测提供了一种有效的检测手段。     

食源性MRSA双色荧光PCR检测方法建立

目的建立快速检测食源性耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus,MRSA)Taqman探针双色荧光PCR方法。方法根据金黄色葡萄球菌种属鉴定nuc基因和MRSA决定因子mec A基因,设计合成引物探针,建立双色荧光PCR扩增体系。利用所建立的方法检测特异性及灵敏度。将金黄色葡萄球菌依次传代培养,检测不同代次的菌株验证方法的稳定性,并对实际样品分离株进行检测验证方法的可行性与实用性。结果该方法可准确并特异性检测出MRSA和甲氧西林敏感金黄色葡萄球菌(methicillin-susceptible Staphylococcus aureus,MSSA),检测MRSA的nuc基因和mec A基因的灵敏度可达2.7×103 CFU/m L,不同代次的菌株的检测结果一致。结论本实验所建立的双色荧光PCR检测方法具有良好的特异性、灵敏度及稳定性,可用于快速检测食源性MRSA。     

双重PCR-DHPLC技术快速检测水产品中金黄色葡萄球菌

根据编码金黄色葡萄球菌肠毒素A的sea基因、编码耐热核酸酶的nuc基因为目的基因,设计两对特异性引物,利用聚合酶链式反应结合变性高效液相色谱技术,建立水产品中金黄色葡萄球菌的双重PCR-DHPLC检测方法。以30株参考菌株进行特异性实验,除所试8株金葡菌出现目的片段和阳性吸收峰外,其余菌株均未检测到目的片段与阳性吸收峰,表明该方法具有良好的特异性。灵敏性实验结果表明,检测灵敏度达到菌液浓度50CFU/mL,比普通的凝胶电泳高一个数量级。利用建立的双重PCR-DHPLC检测方法对240份水产品进行检测,共检出22株金黄色葡萄球菌,检出率为9.2%,其中含有肠毒素基因有8株,占总样本的3.3%,与国标法检出阳性率比较无显著差异,证明该方法具有良好的实用性。实验证明,本研究建立的双重PCR-DHPLC方法不仅可以特异、灵敏、简便快速且高通量的实现对水产品中金葡菌的检测,而且也可通过对肠毒素基因的分析,方便地判断出该菌株致病性的强弱。     

Enterotoxin production and DNA fingerprinting in Staphylococcus aureus isolated from human and food samples. Relations between genetic types and enterotoxins

A total of 224 Staphylococcus aureus strains from human carriers (110 strains) and manually handled foods (114 strains) collected in the Principality of Asturias, Spain over 1995-1999 were analysed for the production of enterotoxins (SEs) A, B, C, and D by a reversed passive latex agglutination test and by amplification of ent genes (A, B, C, D, E, and J) using PCR. Sixty-two strains were enterotoxigenic and a good relation between detection of SEs and their ent genes was found. No strain carried entE and all strains producing SED carried entD and entJ genes. Among the enterotoxigenic strains the percentages registered were 29, 8, 35, 18, 2, 2, and 6 for SEA, SEB, SEC, SEDJ, SEAC, SEADJ and SECDJ, respectively. DNA fingerprinting of 77 strains (the SE prototypes, 62 enterotoxigenic and 10 non-enterotoxigenic [NE]) was carried out by randomly amplified polymorphic DNA using two selected primers independently. Combining results from both primers, 10 genetic types were defined, which showed a different degree of relationship (similarity coefficient: 0.9-0.36) and were clustered into three lineages. One lineage clustered five genetic types and a wide diversity of strains, mainly SEA, SEB, SEDJ, and NE. Another lineage clustered only SEC, SECDJ and NE strains. These two lineages showed a low genetic relationship and appeared as endemic in healthy humans living in the Principality of Asturias. The third lineage included only the prototype strains for SEA and SEE.     

PCR-焦磷酸测序检测单增李斯特氏菌研究

摘要 目的：建立结合PCR-焦磷酸测序检测单核细胞增生李斯特氏菌( Listeria monocytogenes, LMO)的方法。方法：根据LMO的hly基因设计扩增引物和测序引物,特异地扩增目的片段,再制备单链模版在测序引物引导下进行焦磷酸测序,测序结果与GenBank中的hly基因序列比对判断LMO。结果：扩增引物和测序引物表现出良好的特异性,16株LMO均扩增出大小249bp的DNA片段,焦磷酸测序结果与hly基因序列100%匹配,而非LMO对照菌株未扩增出DNA条带,焦磷酸测序结果阴性。结论：建立的方法特异性高,是快速从DNA序列水平上检测LMO的有效手段。     

A PCR assay based on a sequence-characterized amplified region marker for detection of emetic Bacillus cereus

A PCR assay for the detection of Bacillus cereus strains able to produce an emetic toxin (cereulide) was developed in this study based on a sequence-characterized amplified region (SCAR) derived from a random amplified polymorphic DNA (RAPD) fragment. One of the RAPD fragments generated was selected, cloned, and sequenced. A set of PCR primers was newly designed from the SCAR obtained (the sequence of the cloned RAPD fragment) and used in this assay. To determine the specificity of the assay, 30 different B. cereus strains, 8 other Bacillus strains (of six species), and 16 other non-Bacillus strains (from 16 genera) were tested. Results were positive for every emetic B. cereus strain and for only one nonemetic B. cereus strain. For all other bacterial strains, results were negative. Bacterial DNA for PCR was prepared by a simple procedure using Chelex 100 resin from the bacterial colony on the agar plate or from culture after growth in brain heart infusion medium. This PCR assay enabled us to detect the bacteria of emetic B. cereus grown on agar plates but not the bacteria of nonemetic B. cereus. To test this PCR assay for the monitoring of the emetic bacteria, 10 to 70 CFU of B. cereus DSM 4312 (emetic) per g of food was inoculated into several foods as an indicator, followed by a 7-h enrichment culture step. Because this PCR assay based on the SCAR derived from the RAPD fragment was able to detect bacterial cells, this assay should be useful for rapid and specific detection of emetic B. cereus.     

PCR-焦磷酸测序检测单核细胞增生李斯特氏菌研究

目的 建立结合PCR-焦磷酸测序检测单核细胞增生李斯特氏菌的方法.方法 根据单核细胞增生李斯特氏菌的hly基因设计扩增引物和测序引物,特异地扩增目的片段,再制备单链模板,在测序引物引导下进行焦磷酸测序,通过测序结果与GenBank中的hly基因序列的比对进行鉴定.结果 扩增引物和测序引物表现出良好的特异性,16株单核细胞增生李斯特氏菌菌株均扩增出大小249 bp的DNA片段,焦磷酸测序结果与hly基因序列100％匹配,而阴性对照菌株均未扩增出DNA条带,焦磷酸测序结果为阴性.结论 建立的方法特异性高,是快速从DNA序列水平上检测单核细胞增生李斯特氏菌的有效手段.     

Gold nanoparticle-based lateral flow assay for detection of staphylococcal enterotoxin B

The lateral flow assay (LFA), a rapid, sensitive, and reproducible technique, was successfully applied to detect staphylococcal enterotoxin B (SEB). The assay was based on a double-antibody sandwich format on a porous nitrocellulose membrane. When SEB-containing samples were applied to the LFA-device, the toxin initially reacted with polyclonal antibody (Pab)-coated colloidal gold particles and then reacted with the fixed Pab on the membrane. These reactions resulted in a red line at the detection zone, with intensity proportional to the SEB concentration (under 100 ng/ml). With this method, 1 ng/ml of SEB can be detected in less than 5 min and was highly reproducible. Signal can be amplified to 10 pg/ml by silver enhancement. This assay also showed no cross-reaction with other SEs, such as SEA, SEC, SED and SEE. The assay was significantly faster than the ELISA or real-time PCR assay and should facilitate early and rapid SEB detection in clinical and food samples.     

胶体金免疫层析技术快速定量检测金黄色葡萄球菌肠毒素B

目的建立胶体金免疫层析技术快速定量检测金黄色葡萄球菌肠毒素B的方法。方法利用胶体金标记和双抗体夹心免疫层析技术,建立金黄色葡萄球菌肠毒素B的快速检测方法,评价其特异性和敏感性,并拟合检测曲线进行定量检测;在牛奶样品中添加金黄色葡萄球菌肠毒素B作为模拟污染样品进行检测。结果该法可在5～10min内完成定性和半定量检测,检出限为8ng/ml,线性范围8～1000ng/ml。结论建立的检测金黄色葡萄球菌肠毒素B的胶体金免疫层析方法,能快速、灵敏、特异、准确地检测样品中的金黄色葡萄球菌肠毒素B,并可实现定量,适用于现场快速检测。     

Design of PCR primers and gene probes for the general detection of bacterial populations capable of degrading aromatic compounds via catechol cleavage pathways

For the general detection of bacterial populations capable of degrading aromatic compounds, two PCR primer sets were designed which can, respectively, amplify specific fragments from a wide variety of catechol 1,2-dioxygenase (C12O) and catechol 2,3-dioxygenase (C23O) genes. The C12O-targeting primer set (C12O primers) was designed based on the homologous regions of 11 C12O genes listed in the GenBank, while the C23O-targeting one (C23O primers) was designed based on those of 17 known C23O genes. Oligonucleotide probes (C12Op and C23Op) were also designed from the internal homologous regions to identify the amplified fragments. The specificity of the primer sets and probes was confirmed using authentic bacterial strains known to carry the C12O and/or C23O genes used for the primer and probe design. Various authentic bacterial strains carrying neither C12O nor C23O genes were used as negative controls. PCR with the C12O primers amplified DNA fragments of the expected sizes from 5 of the 6 known C12O-carrying bacterial strains tested, and positive signals were obtained from 4 of the 5 amplified fragments on Southern hybridization with the C12Op. The C23O primers amplified DNA fragments of the expected size from all the 11 tested C23O-carrying bacterial strains used for their design, while the C23Op detected positive signals in the amplified fragments from 9 strains. On the other hand, no DNA fragments were amplified from the negative controls. To evaluate the applicability of the designed primers and probes for the general detection of aromatic compound-degrading bacteria, they were applied to wild-type phenol- and/or benzoate-degrading bacteria newly isolated from a variety of environments. The C12O and/or C23O primers amplified DNA fragments of the expected sizes from 69 of the 106 wild-type strains tested, while the C12Op and/or C23Op detected positive signals in the amplified fragments from 63 strains. These results suggest that our primer and probe systems can detect a considerable proportion of bacteria which can degrade aromatic compounds via catechol cleavage pathways.     

Development of two multiplex polymerase chain reactions for the detection of enterotoxigenic strains of Staphylococcus aureus isolated from foods

Two multiplex polymerase chain reactions were developed for the detection of enterotoxigenic strains of Staphylococcus aureus: one multiplex reaction for the simultaneous detection of enterotoxigenic strains type A (entA), type B (entB), and type E (entE) and another for the simultaneous detection of enterotoxigenic strains type C (entC) and type D (entD). Both reactions were standardized with the use of the reference enterotoxigenic strains of S. aureus: FRI 722, producer of staphylococcal enterotoxin (SE) type A (SEA); FRI 1007, producer of SEB; FRI 137, producer of SEC1; FRI 472, producer of SED; and FRI 326, producer of SEE. Optimized methods were used to determine the presence of enterotoxigenic types for 51 S. aureus strains isolated from meat (sausage, ham, and chorizo) and dairy (powdered milk and cheese) products by the Baird-Parker technique. The enterotoxigenic capacities of the strains were determined by the indirect enzyme-linked immunosorbent assay (ELISA) with the use of reference staphylococcal toxins and antitoxins. Fifty of the 51 strains isolated were enterotoxigenic and produced one to four enterotoxin types, with the most frequently produced types being SEA and SED. Levels of correlation between the presence of genes that code for the production of SE (as determined by polymerase chain reaction) and the expression of these genes (as determined by the indirect ELISA) were 100% for SEA and SEE, 86% for SEC, 89% for SED, and 47% for SEB.