2种检测变形杆菌PCR方法的比较研究

选取atpD基因作为靶序列设计了2对引物,分别采用不同的PCR扩增条件,对3株变形杆菌属标准菌株,67株样品分离株及13株非变形杆菌属菌株进行扩增实验,结果2种PCR方法对3株标准菌株和66株样品分离株分别扩增出348 bp和596 bp的特异性片断,实验结果呈阴性的1株分离株经全自动微生物鉴定仪鉴定为阴沟肠杆菌。13株非变形杆菌属菌株均未有特异性条带产生。2种PCR方法检测变形杆菌,均具有快速、准确、灵敏、特异的优点。建立的引物2的PCR方法在检测时间、特异性、灵敏度方面比引物1的PCR方法更具优势。     

PCR方法快速检测变形杆菌的研究

建立了一种快速、准确检测变形杆菌属的PCR方法.采用传统分离培养法、全自动微生物分析系统检测法和常规PCR方法对66株变形杆菌样本分离株进行鉴定.三种检测方法结果一致,但PCR方法检测时间只需5～6h,比传统分离方法节省40h左右.通过对13株非变形杆菌属菌株的特异性实验和标准菌株的灵敏度实验,进一步表明,PCR方法具有操作简便、准确可靠以及灵敏特异的特点,优于传统分离培养法和全自动微生物分析系统检测法.     

食品中副溶血性弧菌toxR和tdh基因双色荧光PCR检测方法的建立

目的 建立对副溶血性弧菌（Vibrio parahaemolyticus）特异性检测toxR（跨膜转录激活蛋白）基因和tdh（热稳定性直接溶血素）毒力基因的Taqman探针双色荧光PCR检测方法。方法 根据副溶血性弧菌toxR基因和tdh基因,分别设计引物和探针,建立Taqman探针双色荧光PCR扩增体系,进行特异性、灵敏度试验;对副溶血性弧菌分离菌株实施检测,了解其tdh基因和tdh基因分布情况。结果 结果表明,副溶血性弧菌标准菌株和3株从食物中毒患者中分离获得的分离株均出现toxR基因和tdh扩增曲线,而溶藻弧菌、单增李斯特菌等31株弧菌属其他菌株和肠杆菌科的菌株未见扩增曲线。从食品中分离的37株副溶血性弧菌分离株均未携带tdh毒力基因。副溶血性弧菌检测灵敏度可达到3.6×102 cfu/mL。结论 该方法可用于同时检测食品中副溶血性弧菌的特异性和毒力基因。     

空肠和结肠弯曲菌鞭毛蛋白基因fla A的检测

目的 运用一种快速、敏感、特异的检测空肠和结肠弯曲菌的方法.方法 以空肠和结肠弯曲菌所共有特异的鞭毛蛋白基因 fla A的一段高度保守序列为引物,用PCR法扩增fla A基因上的一段约1 700 bp的片断.用该引物对空肠和结肠弯曲菌的标准株、福建省的食品分离株进行PER扩增检测,并同时检测该PCR方法的敏感性.结果 扩增片断表现出极好的特异性,2株空肠和结肠弯曲菌标准菌株、8株分离自不同食品样品的空肠穹曲菌和结肠弯曲菌菌株均为阳性,且敏感性实验显示该PCR方法的反应体系最低检出菌量为6 CFU.结论 该方法快速、敏感、特异,可用于突发性食物中毒和暴发感染的调查.     

食品中单核细胞增生性李斯特氏菌PCR快速检测

通过扩增hly基因建立检测单核细胞增生性李斯特氏菌(Lm)的PCR方法.该方法具有较强的特异性,35株经传统方法鉴定的菌株PCR结果均为阳性,而其他三种同属异种菌,包括英诺克李斯特氏菌、绵羊李斯特氏菌和威尔斯李斯特氏菌及非李斯特氏菌均未扩增出特异性的片段.PCR方法对上Lm纯培养物的最低检测限为7.3CFU/μl,对模拟污染的生猪肉和蔬菜的检测低限为4CFU/g,牛奶为4CFU/ml.应用该方法对285份食品样品检测,17份样品Lm呈阳性,结果与常规的分离培养方法完全一致.该种方法具有敏感、特异、快速及准确的优点,可用于食品中Lm的快速检测.     

实时荧光LAMP技术快速检测变形杆菌

建立一种快速检测变形杆菌的方法。采用实时荧光LAMP(Real Amp)技术检测变形杆菌,分析Gen Bank中公布的变形杆菌atp D基因序列,针对其6个区域设计4条引物,利用实时荧光监测仪等温(62℃)扩增模板DNA,通过与电脑相连进行实时监测,对该方法检测变形杆菌的特异性、灵敏度、人工污染猪肉检出限等方面进行研究,并将该检测法的灵敏度与普通环介导等温扩增(LAMP)检测法进行比较。结果表明,Real Amp检测方法比普通LAMP检测方法更加方便快捷,20 min内即可判定结果;用于特异性试验的19株试验菌株中,6株变形杆菌呈阳性结果,其他13株非变形杆菌均呈阴性结果;检测纯菌的灵敏度为8.1CFU/m L,是普通LAMP检测方法的10倍;人工污染猪肉的检出限为81CFU/m L。本研究建立的Real Amp检测方法操作简单,耗时短,特异性强、灵敏度高,能够实现对变形杆菌的快速检测。     

食品中弓形菌16S rRNA特异性扩增检测方法的建立

针对弓形菌16SrRNA基因合成1对引物,通过对聚合酶链式反应(PCR)扩增条件的优化,建立了检测弓形菌的PCR方法。3株弓形菌标准菌株PCR产物测序结果与NCBI上公布的弓形菌16S rRNA基因序列进行比对,比对结果表明3株弓形菌测序结果与NCBI上公布的弓形菌16S rRNA基因序列同源性均在99%以上。3株弓形菌标准菌株均特异性地扩增出了长度为1202bp的片段,其他19株不同种类的菌株均无扩增产物出现。55份食品样品用Johnson-Murano肉汤增菌后用此法进行检测,其中6份样品为弓形菌阳性,阳性率为10.9%。上述实验结果表明,方法特异性强、操作简便,节省了检测时间,可用于食品中弓形菌的快速检测。     

LAMP实时浊度法快速检测食品中产志贺毒素大肠埃希氏菌

针对产志贺毒素大肠埃希氏菌(Shiga toxin-producing Escherichia coli,STEC)stx1和stx2基因的特异性序列分别设计4条特异性引物,采用环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术,利用实时浊度仪建立食品中STEC的LAMP实时浊度法快速检测方法。LAMP反应在实时浊度仪63℃恒温下1 h可完成。对方法的特异性、灵敏度、稳定性进行了评价,并在实际样品检测中进行应用。经优化该方法的检测灵敏度可达150拷贝/反应,4株STEC菌株LAMP扩增结果与其基因型一致,其他21株非STEC菌株均未出现非特异性扩增。395份食品样品检出STEC阳性68份(阳性率为17.2%),所检食品类别中畜产品阳性率最高(达31.3%),禽产品、水产品和可生食蔬菜均有少量阳性样品检出,阳性样品中以stx2基因阳性型为主。结果表明,LAMP实时浊度法具有快速、灵敏、特异性强、操作简便的优势,适用于食品中STEC的快速筛查。     

检测食品中大肠杆菌O157的多重荧光PCR方法的建立

针对大肠杆菌O157的志贺毒素编码基因stx1、stx2和O157血清型的标志基因wzy的保守序列,设计特异性的引物和探针,反复优化反应体系和条件,建立了一种新的多重荧光PCR检验方法.结果显示,该方法灵敏度高,stx1、stx2的扩增效率分别为96.4%和94.6%,实际样品检测时stx1、six2的最低检出限分别为4.2×102cfu/mL和4.2×103cfu/mL.该方法特异性强,扩增结果与各参考菌株基因型一致,能良好的区分出O157菌株和非O157犁大肠杆菌.该方法能用于食品样品的检测和流行病学分离株的快速鉴定.     

食品中大肠埃希氏菌O121荧光PCR检测方法的研究

针对大肠埃希氏菌(Escherichia coli,E.coli)O121的O抗原基因簇的vio A基因的特异性序列设计引物和探针,通过对荧光聚合酶链式反应(real-time PCR)扩增条件的优化,建立检测E.coli O121的血清型特异性荧光PCR方法。E.coli O121标准菌株呈现扩增曲线,其它22株非O121 E.coli和19株非E.coli细菌的菌株均无扩增,检测的灵敏度可达155拷贝/反应。339份食品样品用EC肉汤增菌后用本荧光PCR法进行检测,检出E.coli O121阳性9份,阳性率为2.7%,其中市场上采购获得的生猪肉和生牛肉阳性率达4.1%。上述试验结果表明,本方法特异性强、操作简便,食品中E.coli O121检测全过程可在1.5 d内完成,适用于食品中E.coli O121的快速检测。     

中山市生鲜畜禽肉中变形杆菌的污染分布及ERIC-PCR分型

调查中山市某菜市场生鲜畜禽肉中变形杆菌的污染情况,分析占优势的奇异变形杆菌的亲缘关系。采集268 份生鲜畜禽肉样品,使用聚合酶链式反应检测和生化实验的方法对污染的变形杆菌进行分离鉴定,并利用肠杆菌基因间保守重复共同系列-聚合酶链式反应（enterobacterial repetitive intergenic consensus-polymerase chain reaction,ERIC-PCR）技术对分离的奇异变形杆菌进行分子分型及同源性分析。结果表明：所有类型的样品受变形杆菌污染严重,程度由大到小依次为鸡肉、鸭肉和猪肉;PCR与生化鉴定结果一致,共检出123 株变形杆菌,变形杆菌阳性携带率为51.6%,其中117 株为奇异变形杆菌;ERIC-PCR指纹图谱条带均为3～9 条,呈现良好的多态性;101 株奇异变形杆菌集中分布在E、I、K簇,簇内相似性大于70%。ERIC-PCR技术适用于奇异变形杆菌的分型及同源性研究,对变形杆菌引起的食品污染和亲缘性溯源具有重要意义。     

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.     

一起检出多种致病菌的食源性疾病暴发调查与病因探讨

目的对一起食源性疾病暴发中检出多种致病菌的情况进行探讨分析,找出真正的致病菌,为研究类似的食源性疾病病因提供参考。方法利用标准化的食源性疾病暴发个案调查表收集信息,采用描述流行病学描述事件特征、分析流行病学探索危险食物,采集的样本按照GB 4789系列食品卫生微生物学检验标准进行可疑致病菌分离培养,分离出的副溶血性弧菌按照DNA指纹图谱分析方法进行基因分型。结果搜索到共同进餐者30例,失访5例,其中符合病例定义的17例,罹患率为56.67%(17/30);临床表现主要以腹泻(17/17)、腹痛(16/17)为主,腹痛以上腹部疼痛为主(10/16);发病潜伏期为11~25 h;回顾性队列研究未发现可疑食物。在病例肛拭子中检出9株副溶血性弧菌,8株奇异变形杆菌,1株沙门菌混合副溶血性弧菌,1株空肠弯曲菌;在从业人员肛拭子中检出1株奇异变形杆菌;在环境涂抹拭子中检出1株副溶血性弧菌,4株奇异变形杆菌;在食品样品中检出1株副溶血性弧菌,4株奇异变形杆菌。对从9例病例检测到的副溶血性弧菌进行脉冲场凝胶电泳分子分型,结果发现其遗传相似性达97%以上。结论综合现场流行病学调查和食品卫生学以及实验室检测结果分析,排除其他致病因子引起该起事件暴发的可能,认为这是一起由副溶血性弧菌引起的食源性疾病暴发事件。     

HISTAMINE PRODUCTION BY FOOD-BORNE BACTERIAL SPECIES12

A total of 112 bacterial strains representing 38 species were tested for their potential to elicit food poisoning outbreaks via histamine formation in foods. Proteus morganii and Enterobacter aerogenes displayed a quantitative superiority in terms of histamine production on a trypticase-soy broth-histidine (TSBH) medium and a tuna fish infusion broth (TFIB). When bacteria were incubated under standardized conditions in TSBH medium, histamine accumulated to levels exceeding 50 nmoles/ml of media with a total of 23 strains, including 13 of 15 P. morganii strains, 3 of 3 E. aerogenes strains, 3 of 12 Hafnia alvei strains, 1 of 4 Providencia alcalifaciens strains, 1 of 5 Enterobacter cloacae strains, 1 of 1 Proteus rettgeri strains, and 1 of 1 Citrobacter diversus strains. However, only 8 of the 15 P. morganii strains and the 3 E. aerogenes strains were capable of generating histamine in excess of 200 nmoles/ml in the TSBH medium. Of the 23 strains capable of appreciable histamine production in TSBH medium, P. morganii and E. aerogenes were, by far, the most prolific histamine producers in TFIB. Of the organisms tested, only P. morganii and E. aerogenes would appear to have the capability of forming sufficient histamine in scombroid fish products to elicit food poisoning outbreaks.     

DETECTION OF SALMONELLAE IN CHICKEN AND FISH SAMPLES USING DNA PROBE

The use of DNA probes for rapid identification of foodborne pathogens is an emerging field. We have standardized a method for the detection of Salmonella in food samples by using a specific insertion sequence (IS-200) of Salmonella chromosomal DNA as the probe. The procedure for colony hybridization, and washing conditions for the stringency were established. All the ten Salmonella strains, belonging to various serotypes tested were positive while 18 other bacterial cultures, mainly belonging to the family Enterobacteriaceae such as E. coli Shigella, Proteus etc., were negative in colony hybridization. The sensitivity of the detection was found to be 10⁵ cfu using ³²P-labeled probe. We surveyed 16 chicken and 6 fish samples from local market using IS-200 probe after preenrichment and selective enrichment. All the samples tested were found positive with DNA probe. The major strains of Salmonella isolated from these samples were S. gallinarum, S. typhimurium, S. enteritidis, S. choleraesuis and S. paratyphi A. These results were confirmed by standard Salmonella identification method.     

High genetic variability in non-aflatoxigenic A. flavus strains by using Quadruplex PCR-based assay

Aflatoxigenic Aspergillus flavus isolates always show, by using a multiplex PCR-system, four DNA fragments specific for aflR, nor-1, ver-1, and omt-A genes. Non-aflatoxigenic A. flavus strains give variable DNA banding pattern lacking one, two, three or four of these genes. Recently, it has been found and reported that some aflatoxin non-producing A. flavus strains show a complete set of genes. Because less is known about the incidence of structural genes aflR, nor-1, ver-1 and omt-A in aflatoxin non-producing strains of A. flavus, we decided to study the frequencies of the aflatoxin structural genes in non-aflatoxigenic A. flavus strains isolated from food and feed commodities. The results can be summarized as following: 36.5% of the examined non-aflatoxigenic A. flavus strains showed DNA fragments that correspond to the complete set of genes (quadruplet pattern) as found in aflatoxigenic A. flavus. Forty three strains (32%) showed three DNA banding patterns grouped in four profiles where nor-1, ver-1 and omt-A was the most frequent profile. Twenty five (18.7%) of non-aflatoxigenic A. flavus strains yielded two DNA banding pattern whereas sixteen (12%) of the strains showed one DNA banding pattern. In one strain, isolated from poultry feed, no DNA bands were found. The nor-1 gene was the most representative between the four aflatoxin structural assayed genes. Lower incidence was found for aflR gene. Our data show a high level of genetic variability among non-aflatoxigenic A. flavus isolates that require greater attention in order to design molecular experiment to distinguish true aflatoxigenic from non-aflatoxigenic A. flavus strains.     

白鱼腐败细菌的分离与鉴定

通过对不同温度条件下白鱼（Anabarilius）菌落数量的统计发现, 与37 ℃相比,25 ℃培养条件更有利于白鱼携带细菌的生长,也更容易导致白鱼的腐败。通过对分离自腐败的白鱼中21 株细菌的16S rDNA序列分析后,最终确定得到了11 株不同的菌株。同时结合生化鉴定结果,将这些菌种分属7 个不同的属：2 株摩根氏菌（Morganella）,3 株变形杆菌（Proteus）,2 株漫游球菌（Vagococcus）,1 株葡萄球菌（Staphylococcu）,1 株泰瑞芽孢杆菌（Terribacillus）,1 株赖氨酸芽孢杆菌（Lysinibacillus）,1 株透明颤菌（Vitreoscilla）。其中摩根氏菌和变形杆菌同属肠杆菌科,是腐败白鱼的优势菌,可能是白鱼的特定腐败菌 （specific spoilage organisms,SSO）。     

Proteus dysbioses in patients with ulcerative colitis

In patients with ulcerative colitis Proteus could be isolated from all the gastrointestinal portions studied: mouth cavity, stomach, small intestine, small intestinal mucous membrane, and mostly from faeces. In some patients with ulcerative colitis a decrease in membraneous hydrolysis of polysaccharides was noted. Severe Proteus dysbiosis was associated with a sharp decrease in absorption processes at the small intestinal mucous membrane. The Proteus strains obtained from the patients belonged mostly to 3 serogroups: OA, OB, OC. Studies in virulent properties of Proteus in experiments with white mice gave positive results in 96% cases. Enterotoxin could be found in no case. As the result of vaccinal therapy in most patients disappearance or decrease of signs of malabsorption, increase in weight, stool normalization, and better hematological indices was manifested.