食品中单核细胞增生李斯特菌PCR快速检测方法

[目的]建立食品中单核细胞增生李斯特菌的快速、敏感、特异的PCR检测方法。[方法]选择Hly基因作为靶序列设计一对引物，用该引物对单增李斯特菌和10株非单增李斯特菌进行PCR扩增，并且用此方法对30份食品进行检测。[结果]扩增片断表现出极好的单增李斯特菌特异性，最低检出限为190cfu/ml。[结论]本实验建立的PCR检测方法为一种简便、快速、敏感、特异的单增李斯特菌检测方法。     

牡蛎中单核细胞增生李斯特菌PCR快速检测方法的建立

目的 建立一种无需前增菌快速检测牡蛎中单核细胞增生李斯特菌的PCR方法。方法 利用β-环糊精和膨润土包被活性炭处理牡蛎样品，去除聚合酶链反应(polymerase chain reaction, PCR)反应抑制因子，以单核细胞增生李斯特菌特异基因inlB为靶基因，建立无需样品前增菌的PCR快速检测方法。同时验证该方法的特异性、灵敏度及实用性参数，并评估该方法所用试剂在冷冻保存后的稳定性与实用性。结果 建立的PCR方法对130株目标菌和63株非目标菌的检测特异性为100%,灵敏度达10 CFU/25 g。该方法无需前增菌，4 h即可完成检测。PCR方法和国标方法对实际样品中单核细胞增生李斯特菌的检出率(均为13%)和活菌检测率(均为100%)一致，即符合率100%。此外，该方法所用试剂在-20℃冷冻条件下可稳定保存至少1年。结论 建立的PCR方法特异性强、灵敏度高，可快速、准确检测出牡蛎中的单核细胞增生李斯特菌。     

RT-PCR方法检测单核细胞增生李斯特活菌研究

目的:建立单核细胞增生李斯特活菌快速检测技术,解决PCR带来的假阳性问题。方法:采用以mRNA为基础的RT-PCR方法对单核细胞增生李斯特氏菌的hlyA基因进行检测,设计引物,扩增,并作了特异性和灵敏度比较。结果:该引物能较好地扩增单核细胞增生李斯特菌的特异性片断,而对其它的李斯特菌以及常见食源性致病菌无特异性扩增条带;检测的灵敏度纯菌可达到1×104cfu/m l,人工污染样品猪肉、虾肉和牛奶等培养12~16 h,可达4.5 cfu/m l(g)。结论:可以应用该方法对样品中单核细胞增生李斯特活菌进行检测,能较好地解决PCR检测所带来的假阳性问题。     

单核细胞增生李斯特菌PCR鉴定技术的研究

目的建立单核细胞增生李斯特菌PCR鉴定技术.方法选择Hly Inl基因设计扩展片段分别为300～400bp、600～700bp的二对特异性引物,进行PCR扩增.结果单增李斯特菌标准菌株及本实验室分离保存的单增李斯特菌均扩增出两条明显条带,其它食品中常见致病菌及非单核细胞增生李斯特菌均不见扩增条带.52株李斯特菌生化符合菌中,有30株菌同时扩增出两条明显条带,与Mini-VIDSA测定法比较,两者符合率达92.31%.结论本实验建立的PCR鉴定技术为一种简便、快速、敏感、特异的单增李斯特菌鉴定方法.     

荧光实时定量PCR检测单核李斯特菌方法学建立及应用

目的 建立检测单核细胞增多性李斯特菌的荧光定量PCR方法，并利用此方法检测人工污染的牛奶中单增李斯特菌菌量。方法 选择单增李斯特菌特异性的基因Listeriolysin O基因为靶目标设计引物，采用煮沸法抽提纯单增李斯特菌株（1／2a）DNA，建立SYBRGREEN实时定量PCR检测体系：结果 定量PCR对李斯特菌可产生特异扩增信号，对其他菌（大肠杆菌）无响应。标准曲线的相关系数为0．9521。最小检菌量约为100个菌落形成单位／反应体系。在人为污染牛奶检测中，与传统细菌计数方法结果相比，相关系数为0．839。结论 实时定量PCR是一种快速、灵敏的定量检测单增李斯特菌的方法，可用于牛奶样品的检测。     

荧光定量PCR监测单核细胞增多性李斯特菌研究

目的 探索一种快速、敏感、特异的单核细胞增多性李斯特菌检验方法。方法 以单核细胞增多性李斯特菌hlyA基因作为靶序列设计一对引物，并以标准单核细胞增多性李斯特菌菌株中提取的DNA作为模板，用常规PCR和荧光定量PCR对单核细胞增多性李斯特菌进行监测与鉴定。结果 含有一段特征基因的600bp片段，具有良好的单核细胞增多性李斯特菌检测特异性。结论 荧光定量PCR比普通PCR具有特异性强、灵敏度高、重现性好。可以定量、快捷的应用于监测检验单核细胞增多性李斯特菌。     

三种食源性致病菌多重PCR检测技术建立及应用研究

目的建立多重PCR方法同时检测水产品中沙门菌、副溶血性弧菌和单核细胞增生性李斯特菌。方法分别以沙门菌invA基因、副溶血性弧菌toxR基因和单核细胞增生性李斯特菌iap基因为靶基因,建立并优化了同时检测水产品中这3种致病菌的多重PCR体系,扩增产物分别为495bp(invA)、368bp(toxR)和660bp(iap)。结果建立的多重PCR方法可以简便、快速、灵敏地实现水产品中沙门菌、副溶血性弧菌和单核细胞增生性李斯特菌的同时检测,人工污染水产品检测限为10cfu/ml。结论为无公害水产品食源性致病菌的快速检测提供了理想手段,有良好的应用前景。     

单增李斯特菌不同PCR快速检测方法比较

目的 比较3种PCR快速检测方法在检测单核细胞增生李斯特菌时的特异性和灵敏度方面的差异.方法 针对单核细胞增生李斯特菌的毒力基因iap和prfA基因,分别设计引物,进行单个PCR、多重PCR、套式PCR等3种方法检测.结果 3种PCR方法均具有较强的特异性;套式PCR的灵敏度为102 cfu/ml,高于单个PCR(103cfu/ml)和多重PCR(104cfu/ml).结论 多重PCR在特异性检测方面具有优势,而在灵敏度方面,套式PCR要明显优于单个及多重PCR方法.     

应用RCP方法检定单核细胞增多性李斯特菌

目的 建立单核细胞增多性李斯特菌（单增李斯特菌）快速、敏感、特异的ＰＣＲ诊断方法。方法 选取ｈｌｙＡ基因作为靶序列设计一对引物，用该引物对５４株标准李斯特菌和２１株无关菌进行ＰＣＲ扩增，得到进一步验证，采用ＰＣＲ和常鉴定方法同时对从国内食品分离的３３株李斯特菌进行鉴定。结果 可扩增含有保守Ｈｉｎｄ Ⅲ切点的７４３ｂｐ片段。表现出极好的单增李斯特菌种特异性。纯培养的检测极限为５５个菌。发现其中１８株     

应用液相芯片技术快速检测常见食源性致病菌的研究

目的建立一种基于多重PCR技术联合液相芯片技术快速、准确同时检测沙门菌、副溶血性弧菌、霍乱弧菌、单核细胞增生李斯特菌、金黄色葡萄球菌的方法。方法针对5种食源性致病菌的目标基因设计特异性的引物探针,建立5种食源性致病菌的多重PCR检测方法,将PCR产品与偶联核酸探针的微球混合物进行杂交,利用液相芯片检测仪来检测杂交结果,并对检测方法的重复性、灵敏度、特异性进行评价,以及使用实际样本对该方法进行初步验证。结果该方法检测5种食源性致病菌,重复性良好,变异系数均2%;沙门菌inv A与霍乱弧菌hly A的检测灵敏度为50 cfu/ml,单核细胞增生李斯特菌Hly、金黄色葡萄球菌Sa442、副溶血性弧菌toxR的检测灵敏度为100 cfu/ml;检测的特异度为100%。结论本研究建立的液相芯片检测方法能快速、准确、特异地同时检测沙门菌、副溶血性弧菌、霍乱弧菌、单核细胞增生李斯特菌、金黄色葡萄球菌5种常见的食源性致病菌。     

Development and evaluation of a real-time polymerase chain reaction assay targeting iap for the detection of Listeria monocytogenes in select food matrices

Listeria monocytogenes is an intracellular foodborne pathogen that has been associated with severe human illnesses. Various rapid detection methods have been developed for the specific detection of this pathogen. In the present study, a real-time quantitative polymerase chain reaction (PCR) assay targeting iap, a gene encoding extracellular protein p60, was developed for L. monocytogenes. The PCR efficiency is above 85% and the limit of detection (LOD) is 30 copies of genome per reaction for all strains tested. The assay exhibited 100% inclusivity and exclusivity rates. The detection of L. monocytogenes in five food matrices, whole milk, soft cheese, turkey deli meat, smoked salmon, and alfalfa sprouts, was evaluated with and without enrichment. Without enrichment, the LOD for all food matrices were 4×10(3)?CFU/mL food enrichment mix for whole milk and 4×10(4)?CFU/mL for all other foods. With 24?h incubation in Buffered Listeria Enrichment Broth, the LOD was 3?CFU/25?g food for whole milk, turkey deli meat, and smoked salmon and 9?CFU/25?g food for soft cheese and alfalfa sprouts. With 48?h incubation, the LOD was 3?CFU/25?g food for all matrices. This quantitative PCR appears to be a promising alternative for rapid detection of L. monocytogenes in select foods.     

单增李斯特菌PCR-ELISA快速检测技术研究

目的建立快速检测单增李斯特菌的PCR-ELISA方法,将其应用于人工污染的食物样品检测。方法根据GenBank数据库资料,应用分子生物学软件DNAMAN6.0和Primer Premier5.0,以单增李斯特菌的致病基因hlyA为基础,选用PCR引物,应用地高辛标记试剂盒,获得单增李斯特菌特异的地高辛标记片段。根据PCR目的片段序列,设计特异性捕获探针,建立了单增李斯特菌PCR-ELISA快速检测方法。应用该方法对不同血清型的单增李斯特菌食品分离株进行了检测,并将PCR方法与PCR-ELISA方法对人工污染单增李斯特菌的牛奶样品的检测敏感性进行了比较。结果应用单增李斯特菌特异的PCR-ELISA方法完成检测约需6h。该方法对单增李斯特菌分离株检测的结果,与国家食源性疾病检测网的鉴定结果100%符合。经过12h的增菌培养,PCR-ELISA方法最低可从25ml样品中检出1CFU,检测敏感性为传统PCR方法的10～100倍。结论建立了单增李斯特菌PCR-ELISA快速检测方法。该方法敏感性高、特异性强、可靠性好,对于提高食源性疾病预警、预测能力,增强检测的时效性和准确性,具有推广应用价值。     

改良分子信标-实时PCR快速检测产单核李斯特菌

目的：建立改良分子信标-实时PCR检测产单核李斯特菌（LMO）的快速方法，应用于食品中LMO的污染状况调查及食物中毒快速诊断。方法：根据GenBank公布的LMO hlyA基因的保守序列，设计引物和改良分子信标探针，建立改良分子信标-实时PCR检测体系，应用于食品中LMO检测。结果：改良分子信标-实时PCR反应体系DNA灵敏度为110fg，菌液灵敏度为99cfu／ml或4cfu／PCR反应体系，无交叉反应。以此反应体系检测28株LMO，均出现特异的荧光信号。上述方法可将检测时问由原来的至少4d缩短至1d。对228份食品进行LMO检测，8份增菌液LMO实时荧光PCR阳性，其中6份1310细菌培养阳性。结论：改良分子信标-实时PCR反应体系快速、灵敏度高，特异性强．能提高LMO的检出率和准确性，可应用于LMO食品污染状况调查及食物中毒的快速诊断。     

Evaluation of effects of primary and secondary enrichment for the detection of Listeria monocytogenes by real-time PCR in retail ground chicken meat

A SYBR Green I based real-time PCR assay with inlA-specific oligonucleotide primers was developed for easy and rapid detection of Listeria monocytogenes in a model food that usually has a high incidence of contamination with this pathogen. Results with pure cultures and artificially contaminated chicken meat samples indicate that the PCR assay was highly specific and sensitive. The melting point analysis of the 160 bp amplified DNA fragment was different for L. monocytogenes isolates of the two major phylogenetic divisions of the species, 1 and 2. The assay was then used to survey retail ground chicken meat for contamination with L. monocytogenes. Thirty-seven samples were enriched according to the United States Department of Agriculture culture assays to detect L. monocytogenes on meat. The use and efficiency of PCR assay was examined following both primary and secondary enrichments, which were also plated on chromogenic agar for enumeration of L. monocytogenes and nonpathogenic Listeria spp. to investigate the discrepancies between culture and PCR. Overall, L. monocytogenes was detected in 75% of the samples. Primary enrichment yielded detection rates of 70% and 37% for culture and PCR, respectively. The corresponding rates for secondary enrichment were 54% and 70%, respectively. Test sensitivity is therefore influenced by the type of enrichment and is probably related not only to the limited growth of L. monocytogenes in the primary enrichment media (false-negative PCR results), but also to the high populations of nonpathogenic Listeria spp. in the secondary enrichment broths (false-negative culture results). The main challenge of rapid PCR-based detection of L. monocytogenes from food is the poor sensitivity of primary enrichment media. The improvement of enrichment conditions may help increase assay sensitivity.     

秦皇岛市首次从豆制品等食品中检出单核细胞增生李斯特菌

[目的]对秦皇岛市食品中单核细胞增生李斯特菌的污染状况进行监测与分析。[方法]用LB增菌液增菌后,接种青岛海博显色培养基进行分离,动力试验、溶血实验及API生化鉴定。[结果]从120份食品中检出单核细胞增生李斯特菌9株,总检出率为7.5%,其中从豆制品中检出2株,检出率为20%,速冻米面食品中检出4株,检出率为20%,水产品中检出3株,检出率为15%,其他样品未检出。[结论]我市首次从豆制品水产品速冻米面食品中检出单核细胞增生李斯特菌,且超市食品中该菌污染严重,应对超市食品进行重点监测。     

四种食源性致病菌多重PCR检测方法的建立

目的建立一种多重PCR检测方法同时检测食品中沙门菌、志贺菌、金黄色葡萄球菌和单增李斯特菌。方法参考沙门菌侵袭蛋白A(invA)基因序列,志贺菌侵袭性质粒抗原(ipaH)基因序列,金黄色葡萄球菌引物设计参考耐热核酸酶(nuc)基因序列,单增李斯特菌引物设计参考溶血素蛋白(hly)基因序列设计引物,通过多重PCR对4种食源性致病菌的目的基因进行扩增,并对反应体系进行优化。结果对15株目标菌和17株非目标菌的检测未出现假阳性和假阴性结果,产物分子量与预期一致;4种菌的检测灵敏度分别至少达到10pg/μl;对产物的测序分析表明所得序列与目的基因序列吻合;对319份样品的检测结果显示,其中4份生鲜乳中检出金黄色葡萄球菌,1份生猪肉中检出沙门菌。结论该方法具有良好的检测特异性,可供食源性致病菌的快速检测。     

Detection of Escherichia coli O157:H7 and Listeria monocytogenes in beef products by real-time polymerase chain reaction

Rapid methods for the detection of Escherichia coli O157:H7 and Listeria monocytogenes in food products are important to the food industry and for public health. Conventional microbiological methods and newly developed molecular-based techniques such as polymerase chain reaction (PCR)-based methods are time consuming. In this study, a faster method based on utilization of a hybridization probe with real-time PCR, was developed and applied for detection of E. coli O157:H7 and L. monocytogenes from artificially contaminated raw ground beef and fully cooked beef hotdogs. Target genes for E. coli O157:H7 and L. monocytogenes were rfbE and hylA, respectively. An analysis of 169 bacterial strains showed that the chosen primers and probes were specific for detection of E. coli O157:H7 and L. monocytogenes by real-time PCR. The assay was positive for nine of 10. E. coli O157:H7 strains, and all L. monocytogenes (7/7) strains evaluated. Bacterial strains lacking these genes were not detected by these assays. Detection limits of real-time PCR assays ranged from 10(3) to 10(8) colony forming units (CFU)/ml for E. coli O157:H7 in modified tryptic soy broth and 10(4) to 10(8) CFU/mL for L. monocytogenes in Fraser Broth. Detection sensitivity ranged from 10(3) to 10(4) CFU/g of raw ground beef or hotdog without enrichment for E. coli and L. monocytogenes. Approximately 1.4-2.2 CFU/g of E. coli O157:H7 in raw ground beef were detected following an enrichment step of 4 h. Approximately 1.2-6.0 CFU/g of L. monocytogenes in beef hotdogs were detected following an enrichment step of 30 h. The real-time PCR assays for detection of E. coli O157:H7 and L. monocytogenes in raw ground beef and beef hotdogs were specific, sensitive and rapid.