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。     

基于内参的副溶血性弧菌实时荧光定量PCR快速检测方法的建立

目的建立基于内参的副溶血性弧菌实时荧光定量PCR方法,快速检测样品中的副溶血性弧菌。方法根据Gen Bank已公布的副溶血性弧菌基因组序列,筛选特异性靶基因,设计特异性引物探针,优化反应体系,并在体系中加入内参(IAC),通过标记不同荧光基团的Taq Man探针来监测IAC,进而实时监控整个PCR反应。按照5~50 cfu/25 g的细菌量人工污染样品,以评价所建立反应的体系。结果以副溶血性弧菌基因组DNA为模板,最低检测限为1 pg/μl;以10倍梯度稀释的菌液经水煮法提取的DNA为模板,最低检测限为4×102cfu/ml;以含有gyr B的质粒为模板,最低检测极限可以达到100 copies/μl;建立gyr B和gyr B-IAC标准曲线,Ct值与模板拷贝数均呈良好线性关系(r2=0.999);人工污染初始菌量为7 cfu/25 g时,样品中副溶血性弧菌增菌6 h即可检出。结论本研究所建立的gyr B-IAC实时荧光定量PCR方法,既能有效检测食品中副溶血性弧菌,又能实时监测PCR反应过程,有效防止"假阴性"的发生,结果可靠,有利于实现海产品中副溶血性弧菌实时荧光定量PCR检测方法的标准化。     

单增李斯特菌毒力因子多重荧光PCR法 建立与应用

目的建立同时检测单增李斯特菌(Listeria monocytogenes)及其3种毒力因子的多重荧光PCR快速检测方法,并应用于日常食品的检测。方法根据单增李斯特菌溶血素基因hly A、内化素基因inl A和表面蛋白act A基因的保守序列,分别设计合成特异性引物和探针,优化多重荧光PCR反应体系。对该方法的特异性、敏感性和重复性进行评估。结果该法特异性强、敏感性高,对单增李斯特菌纯培养物的最低检出限410cfu/m L;重复性好,变异系数均小于2%。对84份食品检测结果与传统国标法相符,共检出单增李斯特菌4份,检出率为4.76%。多重荧光PCR检测方法耗时1 h,比传统方法节约2~5 d。4株单增李斯特菌分离株中2株同时含有inl A、act A、hly A 3种毒力基因,另2株为毒力基因act A缺失株,提示目前流行株并非同一来源。结论本研究建立的多重实时荧光PCR方法能同时对单增李斯特菌及其3种毒力因子进行快速检测,且灵敏度高、特异性好,为食源性疾病的病原学检测提供了快速可靠的方法。     

多重PCR联合同源加尾技术检测四种食源性致病菌

[目的]建立快速、准确检测食品中沙门氏菌、大肠埃希氏菌0157、副溶血性弧菌、单增李斯特菌的多重荧光PCR方法。[方法]分别以沙门氏菌、大肠埃希氏菌0157、副溶血性弧菌和单增李斯特菌的fimY、rfbE、ToxR和hly作为靶基因,将上下游引物采用同源加尾的方式,构建四重荧光PCR体系,分析该体系的特异性、灵敏度,并进行模拟污染实验,将该方法与国家标准检测方法进行方法比对实验。[结果]该体系成功地检测出了四种目标致病菌。初始菌浓度分别为2、3、8、1CFU／m1的沙门氏菌、大肠埃希氏菌0157、副溶血性弧菌和单增李斯特菌增菌液,在增菌18h后均被检出。[结论]采用同源加尾的方法成功构建了同时检测四种致病菌的多重荧光PCR方法,该方法特异性强、灵敏度高。     

双重荧光定量PCR检测沙门菌和单核细胞增生李斯特菌方法的建立

目的建立双重荧光定量PCR方法,快速检测沙门菌和单核细胞增生李斯特菌。方法通过设计特异性引物和探针,扩增沙门菌的fimY基因和单核细胞增生李斯特菌的hly基因,采用倍比梯度稀释法检测该体系的灵敏度,以另外7株肠道致病菌评价检测体系的特异性;建立了沙门菌和单核细胞增生李斯特菌感染小鼠的检测模型以验证方法的适用性。结果建立了同时检测沙门菌和单核细胞增生李斯特菌的双重荧光定量PCR方法,从DNA提取到检测完毕仅需2.5 h。检测两种病原菌的灵敏度分别为11和12.8 copies/μl,特异性为100%,符合率为93.3%。结论该法缩短了检测时间,并有良好的灵敏性和特异性,在疾病防控及食品卫生行业中很有应用前景。     

水产品三种致病菌多重PCR检测方法的建立

根据副溶血弧菌(Vibrio parahaemolyticus)的耐热直接溶血素基因(tdh)、金黄色葡萄球菌(Staphylococcus aureus)的耐热核酸酶基因(nuc)和单增李斯特菌(Listeria monocytogenes)的侵入关联蛋白基因(iap)分别设计引物,进行PCR扩增及反应条件的优化,建立了对3种菌的多重PCR检测方法.结果表明:3对引物能特异性地扩增出202 bp、484 bp和714 bp的目的片段.优化后的多重PCR反应体系为:10×PCR buffer(Mg2+)2.5 μL、200 μmol/L dNTP、2 mmol/L Mg2+、模板2 μL、2.5 U Taq酶,引物浓度分别为:副溶血弧菌200 nmol/L、金黄色葡萄球菌40 nmol/L、单增李斯特菌320 nmol/L,总反应液25 μL.对贝类模拟样品的检测结果显示,应用多重PCR技术对3种菌的检测限分别为副溶血弧菌2.3×102 CFU/mL、金黄色葡萄球菌2.5×101 CFU/mL、单增李斯特菌1.5×103 CFU/mL.作者通过多重PCR技术实现了对副溶血弧菌、金黄色葡萄球菌和单增李斯特菌的同时检测,具有快速、灵敏度高、特异性强等优点.     

食品中金黄色葡萄球菌、单增李斯特氏菌和副溶血性弧菌的MPCR法检测

为快速检测食品中的金黄色葡萄球菌(SA)、单增李斯特氏菌(LM)和副溶血性弧菌(VP),根据各菌的相关基因设计引物,分别扩增金黄色葡萄球菌的耐热核酸酶基因-nuc、单增李斯特氏菌溶血素O上的hlyA基因和副溶血性弧菌的热稳定直接溶血素基因-tdh,建立一种MPCR快速检测食品中致病菌的方法,结果表明,三条特异性扩增片段分别为279bp、 243bp和202bp,经DNA测序证明其序列与模板被扩增片段一致.该方法具有良好的灵敏性和特异性.     

冻虾类海产品中3种致病菌的多重PCR快速检测技术的建立

建立快速检测冻虾类海产品中沙门氏菌(Salmonella typli)、副溶血性弧菌(Vibrio parahae-molyticus)和单核细胞增生李斯特菌(Listeria monocytogenes)的多重PCR方法。根据3种致病菌的靶基因,分别设计了3对引物,进行PCR扩增及反应条件的优化,建立了三重PCR检测虾类海产品中沙门氏菌、副溶血性弧菌和单核细胞增生李斯特菌的方法,实现了对这3种致病菌的同时检测。该方法操作简单,检测周期短,具有灵敏度高、特异性强等优点,能够快速地实现对海产品中多种致病菌的诊断和监控。     

水产品中副溶血性弧菌特异性二重PCR检测方法的研究

建立快速检测水产品中副溶血性弧菌(Vibrio parahae-molyticus)的二重PCR方法.以副溶血性弧菌特异性基因tlh和toxR为靶基因,选择2对引物,对5株副溶血性孤菌和40株非副溶血性弧菌进行特异性检测;梯度稀释副溶血性弧菌基因组DNA,以不同稀释度DNA作PCR扩增;在鱼肉样品中以不同菌量人工污染,不同增菌时间培养,提取DNA进行PCR扩增;应用该方法对实际样品进行检测.以tlh和toxR为靶基因的两对引物对副溶血性弧菌的检出有很好的特异性.PCR检测的灵敏度在DNA水平上达到28.76 pg;人工污染样品,当起始污染量为1 CFU/mL时,37℃增菌培养10 h即可检出.本试验一共检测了21份水产品样品,有14份检出了副溶血性弧菌.     

3种常见食源性致病菌多重重组酶聚合酶扩增检测方法研究

目的建立了食品中沙门菌、单核细胞增生李斯特菌和蜡样芽胞杆菌多重重组酶聚合酶扩增（recombinase polymerase amplification,RPA）快速检测方法。方法选择沙门菌invA基因、单增李斯特菌hlyA基因和蜡样芽孢杆菌16S RNA序列为目标基因进行扩增,建立并优化多重RPA扩增体系和扩增条件;评价反应体系的特异性和灵敏度,并对人工污染食品样品和实际样品进行检测。结果多重RPA反应体系能够在20 min完成三种目标基因的扩增,特异性强;对沙门菌、单增李斯特菌和蜡样芽孢杆菌的灵敏度分别为2.70×105、1.30×105、1.44×104 CFU/mL;能够用于人工污染样品和实际样品的检测。结论本研究建立的多重RPA等温扩增方法特异性强,操作快速、简单,为食源性致病菌的快速检测提供新方向     

Combined PCR and slot blot assay for detection of Salmonella and Listeria monocytogenes

Detection of Salmonella typhimurium and Listeria monocytogenes by the polymerase chain reaction (PCR) assay coupled with slot blot detection was investigated in this study. After being extracted from diluted bacterial culture with the extraction buffer, bacterial DNA was subjected to PCR. The slot blot assay was optimized and used to detect PCR products. The lowest detection level of this method was 10(3) cfu/ml in the original culture media for both pathogens, or 5 bacterial cells in the PCR reaction. Combined with immunomagnetic separation (IMS) to separate and concentrate bacteria from samples, the detection limit could be 40 cfu/ml of bacteria from milk samples. The whole detection procedure was completed within 7 h. After multiplex PCR (amplification of DNA from two different bacteria in the same PCR tube) and slot blot, a detection level of 10(3) cfu/ml was achieved in the simultaneous detection for both pathogens, which was similar to that of individual detection for each pathogen. The combination of PCR and slot-blot seems to be highly sensitive and time-efficient, and is therefore promising for routine use in the detection of Salmonella and L. monocytogenes in food samples such as milk.     

丹东口岸2003-2005年进口海产品中3种致病菌的检验

目的掌握丹东口岸进口海产品中单核细胞增生李斯特菌、沙门菌和副溶血性弧菌污染情况。方法对丹东口岸2003-2005年进口的海产品3种致病菌的检验结果进行了分析。结果1965批进口海产品中检出不合格产品44批(2.24%)。其中32批(1.63%)检出单核细胞增生李斯特菌、6批(0.31%)检出沙门菌和6批(0.31%)检出副溶血性弧菌。不合格产品主要为冻章鱼、冻海螺、冻紫石房蛤、活河螺、冻河螺肉等。结论从丹东口岸进口的海产品3种致病菌的污染比较严重,特别是被单核细胞增生李斯特菌的污染最严重,海产品中冻章鱼和冻海螺被这3种致病菌的污染比较普遍。     

Multiplex PCR for simultaneous detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 in meat samples

A multiplex PCR method was developed for simultaneous detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 in meat samples. DNA detection sensitivity for this method was 10(3) CFU/ml for each pathogen. When this protocol was used for the detection of each of the above pathogenic bacteria in spiked pork samples, 1 cell per 25 g of inoculated sample could be detected within 30 h. In the samples of naturally contaminated meat, Salmonella spp., L. monocytogenes, and E. coli O157:H7 were detected over the same time period. Excellent agreement was obtained for the results of multiplex PCR and the conventional culture method, which suggests that the multiplex PCR is a reliable and useful method for rapid screening of meat products for Salmonella spp., L. monocytogenes, and E. coli O157:H7 contamination.     

传统发酵豆制品中3种致病菌的三重荧光PCR快速检测方法的建立

为了建立传统发酵豆制品中单增李斯特菌、蜡样芽孢杆菌和金黄色葡萄球菌的三重荧光PCR快速检测方法。以单增李斯特菌hly A基因、蜡样芽孢杆菌Cereolysin AB基因和金黄色葡萄球菌nuc基因为靶基因设计引物与TaqMan探针,通过优化PCR反应体系,建立了可同时检测单增李斯特菌、蜡样芽孢杆菌和金黄色葡萄球菌的三重荧光定量PCR体系,并进行了特异性和敏感性试验。结果显示,该方法灵敏度高,特异性强,重复性好。对26株非目标菌进行检测,结果均为阴性,而定量检测批内和批间的变异系数均小于2%。单增李斯特菌、蜡样芽孢杆菌和金黄色葡萄球菌敏感性试验结果表明,这三种细菌的最低检测浓度分别为3×103cfu/mL、2×104cfu/mL、2×104cfu/mL。应用该方法可在8h内完成对样品中单增李斯特菌、蜡样芽孢杆菌和金黄色葡萄球菌的同步检测。     

食源性致病菌快速检测试剂盒的研制

目的:研制一种快速检测沙门氏菌、金黄色葡萄球菌和单核细胞增生李斯特菌的三重PCR试剂盒,并在山东泰安地区禽肉市场采样检测。方法:以沙门氏菌的invA基因、单增李斯特菌溶血素hlyA基因和金黄色葡萄球菌耐热核酸酶Nuc基因序列作为目的基因片段金黄色葡萄球菌、沙门菌和单核细胞增生李斯特菌的特异性抗原基因序列,分别设计1对引物,优化各种工作条件,建立一种多重PCR诊断试剂盒。结果:该试剂盒特异性好,敏感性高,沙门氏菌检出极限为4.7cfu/mL,单核细胞增生李斯特菌检出极限17cfu/mL,金黄色葡萄球菌检出极限4.5cfu/mL,可在5h之内得到检测结果。在山东泰安地区取样58份,其中25份样品中检出沙门氏菌阳性,带染率43.1%;6份样品中检出单增李斯特菌阳性,带染率为10.3%;3份样品中检出金黄色葡萄球菌阳性,带染率为5.2%。结论:建立的试剂盒能同时检测上述3种病原菌,可用于食品及其原料的生物安全监测,也可用于兽医临床诊断。     

Enrichment and DNA extraction protocols for the simultaneous detection of Salmonella and Listeria monocytogenes in raw sausage meat with multiplex real-time PCR

A novel method of DNA extraction and purification was developed and was used in conjunction with a multiplex real-time PCR assay for the simultaneous detection of Salmonella and Listeria monocytogenes in a raw meat sample. The PCR used primers targeting the invA gene of Salmonella and the hlyA gene of L. monocytogenes, and PCR products were detected with a LightCycler on the basis of fluorescence from SYBR Green and melting temperature. The assay allowed the detection of 3 Listeria cells and 4 Salmonella cells per g of the original sausage within 10 h, including an enrichment period of 6 to 8 h.     

The incidence of Salmonella and Listeria in raw milk from farm bulk tanks in England and Wales

Raw milks from farm bulk tanks in England and Wales were sampled over a 15 month period in 1992–93 and analysed for Salmonella spp, Listeria spp and Listeria monocytogenes. Of 1673 samples tested for Salmonella spp, six (0.36%) yielded a positive result. A total of 2009 samples were analysed for Listeria spp. Of these, 310 (15.43%) were found positive for Listeria spp, 102 (5.08%) of the positive samples yielded L monocytogenes, which represented 33% of the Listeria isolations. There was a significant rise in the isolation rate for listerias between October and March broadly in line with the period during which the cows were housed indoors. The highest isolation rate was in January, 25.89% in 1992 and 28.4% in 1993, and the lowest isolation rate (3.1%) in August. Although the incidence of L monocytogenes broadly paralleled the trend for total Listeria, the highest percentage of L monocytogenes to total Listeria was found in the months of April to September when the total Listeria isolations were at a minimum. Thirty two (1.59%) samples were found to be positive for Listeria spp prior to enrichment procedure, with 17 (0.85%) positive for L monocytogenes. The highest counts reported for L monocytogenes were 62 and 30 colony forming units (cfu)/ml with over 60% at a level of less than 10 cfu/ml. Of the direct isolations, some 78% were obtained between the months of March and July.     

The development of a combined surface adhesion and polymerase chain reaction technique in the rapid detection of Listeria monocytogenes in meat and poultry.

A procedure combining enrichment surface adhesion and polymerase chain detection (SA-PCR) was developed and applied in the detection of Listeria monocytogenes in meat products. Minced beef samples were inoculated with L. monocytogenes (log(10)3 cfu g(-1)) and incubated for 10 h at 30 degrees C in buffered peptone water. L. monocytogenes was recovered from the culture by attachment to a polycarbonate membrane immersed for 15 min in the enriched meat culture. The membrane and attached bacteria were removed from the culture and the membrane dissolved in phenol:chloroform. The DNA was extracted from the bacteria and a PCR assay was carried out using primers directed against the listerolysin O gene of L. monocytogenes. The combined (SA-PCR) technique had a detection limit of log10 4.0 cfu ml(-1) in enriched meat cultures.The rapid technique was applied to a small number of retail samples (n = 100) and was found to compare favourably to the standard cultural method. A total of 12 samples were confirmed positive for L. monocytogenes using the standard cultural method and the SA-PCR assay. No false positives or negatives were recorded by either method.