食品中5种致病菌多重PCR快速检测技术的建立与应用

目的建立一种能同时检测食品中沙门氏菌、大肠杆菌O157∶H7、金黄色葡萄球菌、单核细胞增生李斯特菌和蜡样芽胞杆菌的多重PCR快速检测方法。方法分别针对沙门氏菌侵袭基因invA、大肠杆菌O157∶H7肠溶血素A基因HlyA、金黄色葡萄球菌耐热性核酸酶基因nuc、单核细胞增生李斯特菌李氏溶血素O基因hlyA和蜡样芽孢杆菌肠毒素FM基因entFM序列设计引物,进行PCR扩增及电泳检测。同时优化反应体系,测定特异性和灵敏性。结果初步建立的多重PCR方法可简便、快速、灵敏地实现对5种致病菌的同时检测,整个检测过程少于30h,且检测灵敏度均可达102CFU/ml。结论这种方法是对传统检测方法的有效改进,为食源性致病菌的快速高通量检测提供了理想手段,有良好的应用前景。     

食源性致病菌多重PCR检测技术建立及其应用

针对6种食源性致病菌特异性基因(金黄色葡萄球菌nuc基因、副溶血性弧菌tdh基因、单核细胞增生李斯特菌hly基因、阪崎克罗诺杆菌ompA基因、鼠伤寒沙门氏菌invA基因和大肠杆菌O157:H7 rfbE基因)建立了多重PCR检测技术,分析了其特异性和敏感性,并评估了其在人工染菌牛奶中的应用可行性.结果表明:该检测技术可...     

5种食源性致病菌PCR检测方法的建立

目的建立食品中的沙门氏菌、副溶血性弧菌、大肠埃希氏菌O157:H7、金黄色葡萄球菌和单核细胞增生李斯特氏菌的PCR快速检测方法。方法以缓冲蛋白胨水为增菌培养基,采用直接提取法提取DNA并测定其核酸浓度。对PCR的退火温度、模板浓度进行优化,把5种目标菌分为两组进行检测分析,并进行特异性和灵敏度实验。结果 5种目标菌培养后均表现出良好的生长趋势。PCR扩增最佳退火温度为59℃, 5种目标菌的引物特异性好,方法的检测灵敏度高。沙门氏菌、副溶血性弧菌、大肠埃希氏菌O157:H7、金黄色葡萄球菌和单核细胞增生李斯特氏菌最低检出核酸浓度分别为0.0202、0.158、0.187、2.30和1.05μg/mL。结论该方法简便、快速、灵敏度高,能满足食品安全检测要求。     

肉中4种致病菌的PCR快速检测方法的建立

目的:建立一种能同时检测肉中金黄色葡萄球菌、志贺氏菌、沙门氏菌和单核增生李斯特菌的多重PCR检测方法。方法:根据金黄色葡萄球菌的耐热核酸酶基因(nuc)、沙门氏菌的侵袭蛋白基因(invA)、志贺氏菌的侵袭性质粒抗原基因(ipaH)和单核细胞增生性李斯特菌的内化素基因(inlA)设计引物,通过优化好的反应体系进行多重聚合酶链反应(PCR)扩增目的基因。结果:特异性实验结果表明4种菌均能在相应位置扩增出特异性条带。对污染4种菌的猪肉进行检测,确定出金黄色葡萄球菌和沙门氏菌的检出限是102CFU/mL,志贺氏菌和单核增生李斯特菌的检出限是101CFU/mL。结论:本实验建立的多重PCR方法比传统细菌检测方法更特异、快速、灵敏,适用于肉中金黄色葡萄球菌、志贺氏菌、沙门菌和单核增生李斯特菌的快速检测。     

食品中3种致病菌多重PCR检测方法的建立及初步应用

建立用多重聚合酶链式反应(Multiplex polymerase chain reaction,mPCR)同时检测食品中沙门氏菌、金黄色葡萄球菌和单核细胞增生性李斯特菌的方法。以沙门氏菌的gyrB基因、单核细胞增生性李斯特菌的gyrB基因、金黄色葡萄球菌的coa基因作为目的基因,分别设计3对引物,通过优化反应体系,建立3种致病菌的多重PCR检测体系。采用单重PCR检测时,灵敏度可达0.423ng/mL(沙门氏菌)、2.5ng/mL(金黄色葡萄球菌)和0.36ng/mL(单核细胞增生性李斯特菌);而采用三重PCR检测时,灵敏度较单重PCR有所下降,分别为2.43ng/mL(沙门氏菌)、2.5ng/mL(金黄色葡萄球菌)、3.6ng/mL(单核细胞增生性李斯特菌)。初步建立能同时、快速、灵敏地检测食品中沙门氏菌、金黄色葡萄球菌和单核细胞增生性李斯特菌的三重PCR方法。     

五种食源性致病菌多重PCR的条件优化和检出限分析

对沙门菌、金黄色葡萄球菌、大肠杆菌O157∶H7、单核增生李斯特菌和福氏志贺菌5种食源性致病菌的多重PCR反应条件进行优化并分析方法的DNA检测限。方法 根据沙门菌的invA基因、金黄色葡萄球菌的16S rDNA基因、大肠杆菌O157∶H7的eaeA基因、单核增生李斯特菌的hlyA基因和福氏志贺菌的ipaH基因设计5对特异性引物进行多重PCR,对反应条件包括镁离子浓度、引物浓度和退火温度进行优化试验,并确定该检测方法的检出限。结果 最佳反应条件为金黄色葡萄球菌、沙门菌和福氏志贺菌引物浓度为0.25μmol/L,单核增生李斯特菌引物浓度为0.4μmol/L,大肠杆菌O157∶H7引物浓度为0.3μmol/L,Mg²⁺浓度为2.25mmol/L,退火温度为60℃;在此条件下金黄色葡萄球菌、肠炎沙门菌、大肠杆菌O157∶H7、单核增生李斯特菌、福氏志贺菌的多重PCR的DNA检出限分别为6.4、32、32、800和160pg。结论 通过对5种引物的PCR条件进行优化和检出限的分析,为食品中该5种致病菌的快速检测奠定基础,对实际应用具有指导意义。     

鲜切果蔬中4 种病原微生物多重PCR检测技术

研发可同时检测鲜切果蔬中的单核细胞性李斯特菌、鼠伤寒沙门菌、金黄色葡萄球菌和大肠杆菌O157:H7的多重聚合酶链式反应（polymerase chain reaction,PCR）检测方法。根据单核细胞性李斯特菌inlA基因、鼠伤寒沙门菌invA基因、大肠杆菌O157:H7 wzy基因、金黄色葡萄球菌nuc基因设计及筛选出4?对引物。对多重PCR体系及条件进行优化。该方法对单核细胞性李斯特菌、鼠伤寒沙门菌、金黄色葡萄球菌和大肠杆菌O157:H7的检出限分别为3.5×106、1.6×105、2.4×105、4.8×105?CFU/mL。将优化的多重PCR方法对不同接种量富集后验证,结果表明,经过9?h富集后,该方法检出限为1?CFU/mL。该方法在鲜切莴苣、鲜切黄瓜、鲜切木瓜、鲜切哈密瓜中应用同样可扩增出4?条目标菌。因此,利用所建立的多重PCR方法对鲜切果蔬中侵染的病原菌检出限可达到1?CFU/g。该方法相较于传统的培养检测方法具有节约大量的劳力、试剂、时间等优点,检测时间也由原来的5～7?d缩短至9～11?h,对于企业或分析检验中心大批量样品的监测具有指导意义。     

食品中3种致病菌多重PCR检测体系的建立及初步应用

目的:建立同步速测食品中沙门氏菌、金黄色葡萄球菌、单核细胞增生性李斯特菌的多重聚合酶链式反应(polymerase chain reaction,PCR)方法。方法:利用基因组比对法寻找3种致病菌的特异性序列——沙门氏菌的invA基因、金黄色葡萄球菌的nuc基因和单增李斯特菌的prs基因,运用Primer Premier 5.0分别设计3对片段大小不同的特异性引物;通过优化反应体系,建立3种致病菌的多重PCR检测体系。结果:建立的多重PCR方法灵敏度测试结果分别为7.6、3.8、5.1pg/μL,在此灵敏度下可以扩增出全部特异性引物条带,验证性实验结果出现相应的目的条带且未发生交叉影响。结论:初步建立能同步、简便、快速、灵敏地检测食品中沙门菌、金黄色葡萄球菌和单核细胞增生性李斯特菌的三重PCR方法。     

应用基因芯片技术检测肉及肉制品中5种致病菌

建立一种运用多重聚合酶链式反应(PCR)结合基因芯片技术检测大肠埃希氏菌、沙门氏菌、金黄色葡萄球菌、志贺氏菌和单核细胞增生李斯特菌5种食源性致病菌的快速、准确、灵敏的方法。分别选取编码大肠埃希氏菌的slt基因、沙门氏菌invA基因、金黄色葡萄球菌nuc基因、志贺氏菌ipaH基因和单核细胞增生李斯特菌inlA基因,并以细菌16S rDNA基因作为阳性对照,设计引物和探针,进行多重PCR扩增,产物与含特异性探针的芯片杂交。结果表明：该基因芯片可同时特异性地检测5种致病菌,多重PCR检测灵敏度为20pg,而DNA芯片检测灵敏度可达2pg;用所制备的基因芯片检测实际肉及肉制品样品,准确率高于传统培养法。所建立的基因芯片检测方法特异性好、灵敏度高,可为食源性致病菌的检测提供理想手段。     

牛奶中3种致病菌多重PCR的建立及初步应用

为了能同时在牛奶中快速检测金黄色葡萄球菌、大肠杆菌、沙门氏菌,本研究根据GenBank中的金黄色葡萄球菌femB、大肠杆菌phoA、沙门氏菌invA基因序列,分别设计3对特异性引物(目的片段分别为233 bp、444 bp、724 bp),通过对PCR反应条件的优化建立了多重PCR方法。敏感性、特异性试验结果显示,该多重PCR对金黄色葡萄球菌、大肠杆菌、沙门氏菌的最低核酸检测量分别为13.3 pg/mL、13.3 pg/mL、133.3 pg/mL,而对牛结核杆菌、多杀性巴氏杆菌、牛支原体、单核细胞增生李斯特氏菌、志贺氏菌的扩增结果均为阴性。对25分临床样品的检测结果表明,该多重PCR检测结果与单一PCR检测结果一致。结果表明,建立的多重PCR方法具有很好的特异性和敏感性,可用于金黄色葡萄球菌、大肠杆菌和沙门氏菌的快速鉴别诊断。     

多重PCR检测冷却肉中的3种致病菌

分别针对沙门氏菌(Salmonella choleraesuissubs.choleraesuis,S)编码DNA结合蛋白的基因hns、大肠O157:H7(Escherichia coliO157:H7,E)编码外膜蛋白紧密素的基因eaeA和单核细胞增生李斯特氏菌(Listeria monocytogenes,L)溶血素O基因Hly设计3对引物,建立同步检测肉制品中3中致病菌的多重PCR方法。通过对多重PCR特异性和灵敏度进行分析,对多重PCR反应条件进行优化,结果表明,此方法简便、快速,可使混菌检测灵敏度达到103cfu/mL。     

多重PCR检测4种常见食源性致病菌

目的 建立一种多重聚合酶链式反应法(multiplex polymerase chain reaction, MPCR)快速检测肉制品中金黄色葡萄球菌、沙门氏菌、志贺氏菌和单增李斯特氏菌的分析方法。方法 选取金黄色葡萄球菌nuc基因、沙门氏菌SipB基因、志贺氏菌ipaH基因、单增李斯特菌inlA基因作为目标基因, 设计4对PCR引物, 建立并优化多重PCR反应体系, 评价该体系的特异性和灵敏度, 并对人工污染的熟肉样品进行检测。结果 构建的多重PCR方法特异性强、灵敏度高, 人工污染熟肉匀浆中4种致病菌的检出限为103 CFU/mL。结论 构建的多重PCR检测方法能够快速、准确、高效地检测肉制品中金黄色葡萄球菌、沙门氏菌、志贺氏菌和单增李斯特氏菌, 为食源性疾病菌的快速检测提供参考依据。     

Rapid, sensitive, and simultaneous detection of three foodborne pathogens using magnetic nanobead-based immunoseparation and quantum dot-based multiplex immunoassay

Losses caused by foodborne diseases are enormous in terms of human life, illness, medical costs, and food product recalls. Rapid detection of multiple bacterial pathogens in foods is extremely important to ensure food safety. The objective of this research was to develop a multiplex immunoassay by integrating magnetic nanobeads (MNBs) for immunoseparation with quantum dots (QDs) as fluorescent labels for rapid, sensitive, and simultaneous detection of three major pathogenic bacteria, Salmonella Typhimurium, Escherichia coli O157:H7, and Listeria monocytogenes, in food products. In this research, both streptavidin-conjugated MNBs (30- and 150-nm diameter) and QDs (530-, 580-, and 620-nm emission wavelength) were separately coated with biotinylated anti-Salmonella, anti-E. coli, and anti-Listeria antibodies. The immuno-MNBs were mixed with a food sample to capture the three target bacteria. After being magnetically separated from the sample, the MNB-cell conjugates were mixed with the immuno-QDs to form the MNB-cell-QD complexes, and unattached QDs were removed. The fluorescence intensity of the MNB-cell-QD complexes was measured at wavelengths of 530, 580, and 620 nm to determine the populations of Salmonella Typhimurium, E. coli O157:H7, and L. monocytogenes, respectively. This multiplex immunoassay simultaneously detected Salmonella Typhimurium, E. coli O157:H7, and L. monocytogenes at levels as low as 20 to 50 CFU/ml in food samples in less than 2 h without enrichment. The change in fluorescence intensity was linearly correlated (R(2) > 0.96) with the logarithmic value of bacterial level in the range of 10 to 10(3) CFU/ml. More than 85% of the three target pathogens could be simultaneously separated from food samples. The multiplex immunoassay could be expanded to detect more target pathogens, depending on the availability of specific antibodies and QDs with different emission wavelengths.     

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.     

A novel multiplex PCR assay for rapid and simultaneous detection of five pathogenic bacteria: Escherichia coli O157:H7, Salmonella, Staphylococcus aureus, Listeria monocytogenes, and Vibrio parahaemolyticus

Rapid and sensitive detection techniques for foodborne pathogens are important to the food industry. However, traditional detection methods rely on bacterial culture in combination with biochemical tests, a process that typically takes 4 to 7 days to complete. Thus, this study was conducted to address the issue of time lag inherent in traditional methods by developing a novel PCR assay for each of five foodborne pathogenic bacteria. This new system consists of a simultaneous screening method using multiplex PCR in a single reaction tube for the rapid and sensitive detection of each of the five bacteria. Specific primers for multiplex PCR amplification of the Shiga-like toxin (verotoxin type II), femA (cytoplasmic protein), toxR (transmembrane DNA binding protein), iap (invasive associative protein), and invA (invasion protein A) genes were designed to allow simultaneous detection of Escherichia coli O157:H7, Staphylococcus aureus, Vibrio parahaemolyticus, Listeria monocytogenes, and Salmonella, respectively. To confirm the specificity of each primer pair for the respective target gene, three types of experiments were carried out using boiled cell lysates and their DNAs. In the multiplex PCR with mixed DNA samples, specific bands for corresponding genes were simultaneously detected from a single reaction. The detection of all five foodborne pathogenic bacteria could be completed in less than 24 h with this novel PCR method.     

A multiplex RTi-PCR reaction for simultaneous detection of Escherichia coli O157:H7, Salmonella spp. and Staphylococcus aureus on fresh, minimally processed vegetables

In this work, a new multiplex single-tube real-time PCR approach is presented for the detection of Escherichia coli O157:H7, Salmonella spp. and Staphylococcus aureus, three of the more frequent food-borne bacterial pathogens that are usually investigated in a variety of food matrices. The study includes the design and specificity testing, of a new primer and probe specific for Salmonella spp. Reaction conditions were adjusted for the simultaneous amplification and detection of specific fragments in the beta-glucuronidase (uidA, E. coli) and Thermonulease (nuc, Sta. aureus) genes, and in the replication origin sequence (oriC, Salmonella spp.). Melting-curve analysis using a SYBR Green I RTi-PCR approach showed characteristic T(m) values demonstrating the specific and efficient amplification of the three fragments. Subsequently, a TaqMan RTi-PCR approach was settled, using FAM, NED and VIC fluorescently labelled specific probes for an automated detection. It was equally sensitive than uniplex RTi-PCR reactions in Sta. aureus and E. coli O157:H7, using same amounts of purified DNA, and allowed detection of 10 genome equivalents in the presence of 10(2) or 10(4) genome equivalents of the other two pathogens. Finally, it was tested in artificially inoculated fresh, minimally processed vegetables, revealing a sensitivity of 10(3)CFUg(-1) each of these pathogens in direct detection, following DNA extraction with DNeasy Tissue Kit (Qiagen). The multiplex RTi-PCR developed scored the sensitivity recognised for PCR in food and it allows a high-throughput and automation, thus it is promising as a rapid and cost-effective test for the food industry.     

SIMULTANEOUS DETECTION OF LISTERIA MONOCYTOGENES, STAPHYLOCOCCUS AUREUS, SALMONELLA ENTERICA AND ESCHERICHIA COLI O157:H7 IN FOOD SAMPLES USING MULTIPLEX PCR METHOD

In this study, one multiplex polymerase chain reaction (MPCR) assay was developed for simultaneous detection of four foodborne pathogens, i.e., Listeria monocytogenes, Staphylococcus aureus, Salmonella enterica and Escherichia coli O157:H7. Five specific primer pairs were designed based on the nucleotide sequences of hemolysin gene (hly) of Listeria monocytogenes, thermostable nuclease gene (nuc) of Staphylococcus aureus, invasion gene (invA) of Salmonella enterica, shiga-like toxin gene (stx) and intimin gene (eae) of Escherichia coli O157:H7 in this assay. The specificity and sensitivity of the MPCR method were validated, and the limit of detection (LOD) of this method was about 10 copies. One cfu/mL each of these foodborne pathogens spiked in practical food samples, i.e., ground meat, beef, pork, fish, shrimp, cheese, canola leaf and cabbage, could be detected simultaneously after 24 h enrichment at a rate of 87.5%, indicating that the established MPCR detection method was effective and suitable for practical use.

PRACTICAL APPLICATIONS

This study presents a quick and effective identification method to simultaneous monitor four foodborne pathogens in food samples. The specificity and sensitivity of this method can be used to unambiguously identify these four foodborne pathogens in practical food samples based on the species-specific genes. Therefore, this detection method is applicable for surveillance measures of these four foodborne pathogens in the food production chain.     

高分辨率溶解曲线检测9种食源性致病菌方法的建立

当今食源性疾病已成为一个全球关注的食品卫生问题,常见的细菌性食物中毒的病原菌有:致病性大肠杆菌(特别是出血性大肠杆菌O157:H7)、沙门氏菌、志贺氏菌、致病性弧菌(包括霍乱弧菌和副溶血弧菌)、金黄色葡萄球菌、单增李斯特菌、空肠弯曲菌以及阪崎克罗诺杆菌等。单独检测一种致病菌已无法满足现今社会对同时高效检测多种致病菌的要求,为建立一种同时对上述9种食品中常见致病菌的检测方法,本研究分别以上述9种菌的特异性基因为靶基因,创新性地结合一对通用引物,建立能同时检测多种食源性致病菌的多重HRM-real time PCR检测体系。结果表明该多重HRM-real time PCR检测体系通过两管PCR反应可对上述9种食品中常见致病菌进行有效的检测与区分,且具有良好的特异性和灵敏度。