食品中沙门菌PCR检测方法的建立

为建立食品中快速检测沙门菌的PCR方法。选取沙门菌属侵袭性抗原保守基因invA基因上的靶序列设计一对引物,选择最适Mg 浓度和退火温度,建立最适PCR反应体系,用2%琼脂糖,5μl反应产物(包括EB),100V,40min进行电泳,显像。用该引物对已经传统方法鉴定的22种77株沙门菌和24种24株非沙门菌进行特异性检测,并对人工污染的食品进行检测条件的研究。Mg 浓度和退火温度对该反应体系的影响较小,稳定性较好;经传统方法鉴定的22种77株沙门菌和24种24株非沙门菌验证了该检验方法具有很好的特异性;该检测方法可以在19h内检出含有沙门菌102CFUg的食品(火腿肠、鸡蛋、散装肉馅)。与传统方法比较,该方法快速、敏感、特异,能在较短的时间内对大量样品同时进行检测,适用于食品中沙门菌的快速、敏感、特异检测。     

G-四联体与聚合酶链式反应联用可视化检测沙门氏菌

建立对沙门氏菌的G-四联体与聚合酶链式反应（polymerase chain reaction,PCR）联用可视化检测方法。以沙门氏菌属特异性基因invH为检测目标,设计5’端含有G-四联体互补序列的上下游引物,通过PCR的特异性识别并扩增目标序列,获得大量含G-四联体的双链DNA,变性后与氯高铁血红素结合生成具有过氧化物酶活性的模拟酶（DNAzyme）,并催化H2O2与2,2’-联氮-双(3-乙基-苯并噻唑琳-6-磺酸)二胺盐由无色变为绿色,实现G-四联体与PCR联用对沙门氏菌的可视化检测。在优化的检测体系下,沙门氏菌基因组的质量浓度对数与421 nm处的吸光度具有良好的线性关系,其回归方程为y=0.129 9x＋0.217 9,R2=0.994 5,线性范围0.07～771.6 ng/μL,灵敏度为0.07 ng/μL。特异性分析表明：此方法适用于沙门氏菌属的检测,可成功应用于人工污染沙门氏菌牛奶的检测,检出限为1.2×102 CFU/mL。通过检测30 种市售样品,发现其结果与国标检测方法结果一致。本研究为食源性致病菌的检测提供了新的策略。     

PCR法检测肉鸭屠宰加工生产链中沙门氏菌的污染

为建立肉鸭加工中快速、准确的沙门氏菌PCR 检测方法,并应用于肉鸭屠宰生产加工链沙门氏菌的实时监测。采用Primer Premier 5.0 软件针对沙门氏菌特有的fimY 基因设计合成一对引物5′- GCATTCCGCTCATTAGAT-3′和5′-TGGAGGCTGATAACAAGG-3′,并对沙门氏菌DNA 扩增PCR 体系的退火温度、引物浓度、Mg2+ 浓度和聚合酶浓度进行优化。结果表明：成功扩增出沙门氏菌标准株和分离株的2 7 5b p 目的片段,灵敏度达到了1.2pg;应用建立的PCR 方法对国内某典型肉鸭屠宰厂的肉鸭屠宰链环节进行沙门氏菌污染的检测,发现在屠宰环境、拔毛浸烫水、浸蜡冷却水、清洗池水、宰前毛、食道、粪便和沥水体腔内共有25 个样品中扩增出了275bp大小的特异性片段,而空白对照无扩增条带。     

PCR Detection and Microbiological Isolation of Salmonella spp. from Fresh Beef and Cantaloupes

ABSTRACT:  Species belonging to the genus Salmonella are an important cause of enteric fevers, gastroenteritis, and septicemia, and the pathogens are commonly transmitted through contaminated food. In this study, polymerase chain reaction (PCR) amplification of a 287-bp region of the invA gene was compared to a microbiological technique to determine the presence of Salmonella in retail beef and in cantaloupe rinse samples. Both methods showed the same level of sensitivity, detecting 1 CFU/25 g of meat after enrichment for 24 h at 42 °C. The presence of Salmonella was determined in 50 commercial top sirloin beef samples that were not artificially inoculated. Three samples were positive by the microbiological method, and these samples and an additional sample were positive by the PCR. Both methods were also used to test surface rinses of cantaloupes collected from 4 farms in Nayarit, Mexico. Salmonella was detected by the microbiological method in 9 of 20 samples (45%), whereas the pathogen was detected by the PCR in 11 samples (55%). This study demonstrates the utility of the PCR targeting the invA gene to determine the presence of Salmonella spp. in beef and cantaloupe samples.     

PCR和实时PCR技术快速检测牛奶样品中的沙门氏菌

根据沙门氏菌组氨酸转运操纵子基因的保守序列设计特异性引物,建立快速检测牛奶中沙门氏菌的方法.比较不同的DNA模板制备方法,将快速常规FCR与实时PCR结合,对阳性培养液及牛奶中的沙门氏菌进行检测.结果表明,采用设计的引物能有效地扩增出沙门氏茵特有的大小为495 bp的基因片段,与GeneBank 上的序列大小一致.常规PCR菌液敏感度可达到8 cfu/mL,检出时间少于20 h.实时PCR的DNA模板最低可达1.59x10-5μg/μL,检测时间仅需3 h.新建的PCR检测方法准确、敏感、特异、快速.     

鼠伤寒沙门氏菌ompc基因PCR的检测方法

建立一种鼠伤寒沙门氏菌的聚合酶链式反应检测方法。根据GenBank中已发表的鼠伤寒沙门氏菌的ompc的基因序列,设计和合成了一对特异性引物,并优化了反应条件,检测了该方法的敏感性和特异性。结果成功扩增出了鼠伤寒沙门氏菌470 bp的ompc特异性基因片段,而对致病性大肠杆菌、金黄色葡萄球菌、绿脓杆菌和志贺氏菌4 种食品中常见的病原菌均未扩增出相应片段。敏感性实验结果表明本方法可检测到最低1 pg/μL的鼠伤寒沙门氏菌基因组DNA。     

基于TaqMan探针双重荧光PCR检测食品中肠道沙门氏菌和肠炎沙门氏菌

建立基于TaqMan探针双重重实时荧光PCR检测肠道沙门氏菌(Salmonella enterica,SP)和肠炎沙门氏菌(Salmonella Enteritidis,SE)的方法。根据SP的aceA基因(Gen Bank:U43344.1)、肠炎沙门氏菌特异序列SEP(GenBank:AF370707.1),分别设计引物和探针,在ace A探针的5′端标记FAM和SEP探针的5′端标记VIC,建立基于TaqMan探针双重荧光PCR检测方法。试验结果,58株29种不同血清型肠道沙门氏菌均扩增出ace A基因扩增曲线,SEP特异性地扩增出15株SE,而28种不同血清型沙门氏菌和17株变形杆菌等阴性对照株扩增结果均为阴性。ace A和SEP的双重荧光PCR扩增效率分别为100%和104%,R2分别为0.999和0.998,最低检测浓度分别达到280 cfu/m L和260 cfu/m L。建立的方法特异性好、灵敏度高,整个试验可在31 h完成,是快速检测SP和SE的有效方法,可用于食品中SP和SE的特异性检测。     

环介导等温扩增技术快速检测沙门菌

环介导等温扩增(loop-mediated isothermal amplification,LAMP)是一种在等温条件下高特异、高效、快速地扩增靶序列的DNA扩增新技术.以沙门菌(Salmonella spp.)为研究对象,根据其特异性的invA基因,设计了一套特异性引物对该基因进行了LAMP,同时优化了其反应条件,建立了沙门菌的LAMP快速检测技术.结果表明,LAMP的最佳反应条件为外引物浓度5 pmol/L、内引物浓度40pmol/L,Mg2 浓度6mmol/L,dNTP浓度0.8mmol/L,甜菜碱浓度0.8mmol/L,Bst DNA聚合酶8u,反应温度63℃,反应时间1 h.在此条件下,LAMP检测沙门菌DNA的敏感度达10fg/反应,且与其他常见的细菌无交叉反应.其对牛奶样品的检出量为102cfu/mL,适合于食品中污染沙门菌的快速检测.     

Multiplex PCR for the simultaneous detection of Salmonella spp. and Salmonella Enteritidis in food

Multiplex PCR assay (mPCR) for the detection of Salmonella spp. and S. Enteritidis was developed in this study using artificially contaminated chicken carcasses. The assay showed 100% specificity to detect approximately 1 CFU of Salmonella in 10 g of chicken skin after non‐selective enrichment. The mPCR was evaluated in Minas cheese, fresh pork sausage and chicken carcasses commercially available. Salmonella spp. was detected in nine of sixty‐six chicken carcasses, five of fifty‐two cheese samples, and five of fifty‐two sausage samples. The serovar Enteritidis was detected in two samples of contaminated sausage. The mPCR results were confirmed by conventional culture and biochemical identification of the isolates. Serotyping confirmed the presence of S. Enteritidis in sausage samples and showed contamination by serovars Schwarzengrund and Montevideo in chicken carcasses.     

Innovative Methods for Removal of PCR Inhibitors for Quantitative Detection of Plesiomonas shigelloides in Oysters by Real-Time PCR

A quantitative assay for Plesiomonas shigelloides in pure culture and oysters based on the real-time polymerase chain reaction (Rti-PCR) was developed. The methodology involved the treatment of oyster tissue homogenates with formaldehyde, differential centrifugation, and treatment of samples with activated charcoal coated with Pseudomonas fluorescens. With seeded oyster tissue homogenates, without formaldehyde or coated charcoal treatments, the lowest level of detection for P. shigelloides was 1?×?10⁷ genomic targets per gram of tissue, equivalent to 2.5?×?10⁵ genomic targets per Rti-PCR. The addition of 4% formaldehyde to tissue homogenates reduced the minimum level of detection of P. shigelloides to 1?×?10⁵ genomic targets per gram of tissue, equivalent to 2.5?×?10³ genomic targets per real-time PCR. The treatment of tissue homogenates with only activated charcoal coated with P. fluorescens reduced the minimum level of detection of P. shigelloides to 1?×?10⁴ genomic targets per gram, equivalent to 2.5?×?10² genomic targets per real-time PCR, without DNA purification or enrichment. The combination of adding 4.0% formaldehyde to oyster tissue homogenates and treating with coated charcoal reduced the level of detection of P. shigelloides to 1?×?10³ genomic targets per gram, equivalent to 25 genomic targets per real-time PCR. The linear range of detection was from 1?×?10³ to 1?×?10⁶ genomic targets per gram without enrichment.     

Innovative Methods for Removal of PCR Inhibitors for Quantitative Detection of Plesiomonas shigelloides in Oysters by Real-Time PCR

A quantitative assay for Plesiomonas shigelloides in pure culture and oysters based on the real-time polymerase chain reaction (Rti-PCR) was developed. The methodology involved the treatment of oyster tissue homogenates with formaldehyde, differential centrifugation, and treatment of samples with activated charcoal coated with Pseudomonas fluorescens. With seeded oyster tissue homogenates, without formaldehyde or coated charcoal treatments, the lowest level of detection for P. shigelloides was 1 × 10⁷ genomic targets per gram of tissue, equivalent to 2.5 × 10⁵ genomic targets per Rti-PCR. The addition of 4% formaldehyde to tissue homogenates reduced the minimum level of detection of P. shigelloides to 1 × 10⁵ genomic targets per gram of tissue, equivalent to 2.5 × 10³ genomic targets per real-time PCR. The treatment of tissue homogenates with only activated charcoal coated with P. fluorescens reduced the minimum level of detection of P. shigelloides to 1 × 10⁴ genomic targets per gram, equivalent to 2.5 × 10² genomic targets per real-time PCR, without DNA purification or enrichment. The combination of adding 4.0% formaldehyde to oyster tissue homogenates and treating with coated charcoal reduced the level of detection of P. shigelloides to 1 × 10³ genomic targets per gram, equivalent to 25 genomic targets per real-time PCR. The linear range of detection was from 1 × 10³ to 1 × 10⁶ genomic targets per gram without enrichment.     

沙门氏菌多重PCR检测方法的建立

采用CTAB/NaCl法提取沙门氏菌及对照菌株的基因组DNA。对引物浓度、TaqDNA聚合酶用量进行优化,建立了沙门氏菌属特异基因-hut基因(495bp)、hilA基因(490bp)、invA基因(284bp)和hns基因(152bp)间的多重PCR检测方法,并进行了灵敏度测试。结果表明,建立的多重PCR检测结果与预期一致,二重PCR的检测灵敏度与单一PCR的一致,三重PCR检测灵敏度有所下降。     

速冻食品中沙门氏菌和金黄色葡萄球菌多重PCR检测方法的建立与应用

为实现速冻食品中沙门氏菌(Salmonella spp.)和金黄色葡萄球菌(Staphylococcus aureus)的多重聚合酶链式反应(PCR)检测,首先优化多重PCR扩增的反应条件,比较基因组DNA提取方法,结果表明：退火温度采用60℃、各引物浓度200nmol/L及扩增循环35次,本多重PCR检测技术可以有效地将沙门氏菌和金黄色葡萄球菌同时检出,检测特异性为100%。3种DNA提取方法中试剂盒法纯度最高,检出限分别是31、26DNA copies/reaction。经过在人工污染致病菌的速冻水饺中应用试验后,该多重PCR方法经过4h的增菌培养即可从速冻水饺中同时检测出起始菌落数低至100CFU/g的沙门氏菌和金黄色葡萄球菌。     

基于核酸适配体杂交链式反应比色法检测鼠伤寒沙门氏菌

该研究探讨了基于核酸适配体特异性识别机制和杂交链式反应（hybridization chain reaction,HCR）扩增策略,以金纳米粒子（gold nanoparticles,AuNPs）颜色变化为比色信号,设计了一种无标记、无酶、灵敏的鼠伤寒沙门氏菌（Salmonella typhimurium,S. typhimurium）比色检测法。根据鼠伤寒沙门氏菌核酸适配体设计引发链和两个发夹探针,核酸适配体捕获鼠伤寒沙门氏菌,触发引发链打开发夹探针,发生杂交链式反应,在实现目标菌信号放大同时,利用反应前发夹探针粘性末端以及反应后形成的杂交长链对金纳米粒子结合差异性,产生比色信号,实现鼠伤寒沙门氏菌的快速检测。通过对杂交链式反应时间、发夹探针与金纳米粒子结合时间以及发夹探针浓度等实验参数进行优化,提高实验灵敏度。在最优实验条件下,鼠伤寒沙门氏菌浓度对数值与紫外吸光比值（A630/525）在103~107 CFU/mL范围内呈现良好的线性关系,检测限为6.3×101 CFU/mL,在牛奶样品中加标回收率为90.05%~109.97%。本比色法操作方便,无需要化学修饰以及复杂仪器且实验结果可视,为鼠伤寒沙门氏菌监测提供一种新的方法。     

基于免疫磁分离-双重荧光定量PCR的鲜猪肉中沙门氏菌和志贺氏菌的检测

目的使用免疫磁分离-双重荧光定量PCR方法实现对鲜猪肉中金黄色葡萄球菌和志贺氏菌的同时、快速检测。方法在37℃的条件下,利用特异性免疫磁球从250 m L循环体系中快速、有效地捕获目标菌。再通过特异性的引物与探针,对沙门氏菌和志贺氏菌进行双重荧光定量PCR检测。结果本研究方法针对鲜猪肉中沙门氏菌和志贺氏菌的检测限分别达到3.4 cfu/g和9.4 cfu/g。方法总体灵敏度、特异性和准确度均达到100%。此外,通过对30组实际样品的检测,该方法与传统标准方法的结果保持一致。结论本研究建立的免疫磁分离-双重荧光定量PCR方法比国标方法更快速和高效,适用于鲜猪肉中沙门氏菌和志贺氏菌的快速检测。     

牛奶样品中志贺氏菌的快速PCR检测技术研究

建立牛奶中快速检测志贺氏菌的有效方法。根据GenBank公布的志贺氏菌ipaH基因的保守序列设计特异性引物,筛选合适的DNA模板制备方法,采用快速常规PCR和定量实时PCR结合,对培养液中及牛奶阳性样品中的志贺氏菌进行检测,检测敏感度可达到2CFU/ml,检出时间小于20h。新建的PCR方法具有特异性好,灵敏度高等特点,适用于快速、准确检测牛奶中志贺氏菌的需要。     

沙门氏菌重组酶聚合酶检测方法的建立及应用

摘 要：建立一种重组酶聚合酶扩增技术（recombinase polymerase amplification,RPA）检测沙门氏菌（Salmonella）的方法。本研究依据沙门氏菌侵袭蛋白A基因（invA）的保守序列设计特异性引物,通过对反应时间的优化,建立的RPA方法在38?℃水浴锅中恒温反应20?min,即可实现对目的片段的有效扩增;除沙门氏菌外,其他26?种食源性致病菌均无扩增,具有良好的特异性;以沙门氏菌基因组DNA作为模板,该方法的检测灵敏度为1.1×10－3?ng/μL,与本研究应用的实时荧光聚合酶链式反应（real-time polymerase chain reaction,PCR）方法一致;人工污染实验表明,当羊肉、鸡肉和西兰花样品污染量为4?CFU/25?g,增菌8?h,即可通过RPA方法检出沙门氏菌。在人工污染实验中,RPA和PCR检测结果一致。本研究建立的沙门氏菌的RPA检测方法特异性强、操作简单、为食源性致病菌的鉴定提供了一种新的方向。     

实时荧光PCR法检测食品中亚利桑那沙门氏菌

沙门氏菌（Salmonella）是食品检测过程中最常见的致病菌之一,亚利桑那沙门氏菌（Salmonella arizonae）又是沙门氏菌中比 较难鉴定的亚种。该实验通过实时荧光聚合酶链式反应（PCR）方法快速准确的检测亚利桑那沙门氏菌和其他沙门氏菌。根据GenBank 公布的亚利桑那沙门氏菌和其他沙门氏菌gud D基因序列, 分别设计引物和Taqman探针。 使用10株不同血清型的沙门氏菌标准菌 株、88株沙门氏菌分离株和29株食品中常见食源性致病菌进行实时荧光PCR特异性实验。结果显示,该实验所设计的引物探针特异 性非常好。 实时荧光PCR灵敏性试验结果表明,检测灵敏度可达到1～10 CFU/mL的添加浓度。 经模拟污染样品验证,所建立的实时 荧光PCR方法与传统方法的检测结果相一致,具有检测周期短、操作简便的优势。     

食品中沙门氏菌恒温隔绝式PCR检测方法的建立

为弥补传统培养方法耗时长和现场检测步骤繁琐等缺陷,该研究建立了一种针对食品中沙门氏菌的恒温隔绝式PCR快速检测方法.根据沙门氏菌的invA基因设计特异性引物和探针,通过水浴法快速提取细菌DNA,优化引物、探针以及模板用量,建立了一种基于恒温隔绝式PCR快速检测沙门氏菌的方法,并对方法的特异性和灵敏度及稳定性进行评价,最...