食品中单增李斯特菌PCR检测方法建立与评价

目的建立单核细胞增生李斯特菌（Listeria monocytogenes，LM）快速、敏感、特异的PCR诊断方法。方法采用聚合酶链式反应技术（PCR）特异性扩增单核细胞增生李斯特菌溶血素基因（hlyA），并评价该方法的特异性与敏感性。结果在706bp处出现nuc基因的目的片断，只有单增李斯特菌的目的片段获得扩增，其他菌种扩增均呈阴性；该方法可以检测到3．3ng／L的DNA。结论PCR方法比传统细菌检测方法更特异、快速、灵敏和简便。为食品中单增李斯特菌的快速检测提供了新的手段。     

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。     

Taq Man-MGB探针Real-timePCR快速检测单增李斯特菌的研究

目的：建立TaqMan—MGB探针Real—time荧光PCR快速检测单增李斯特菌技术。方法：在对单增李斯特菌invA序列进行分析、比较基础上，设计一对特异性TaqMan—MGB探针法引物及探针，通过Real—timePCR反应条件和反应体系的优化，实现对单增李斯特菌的快速检测；用克隆到pMD18-T载体上的李斯特菌invA基因阳参片段及不同菌株、食品标本验证方法的特异性和敏感性。结果：方法的灵敏性高，其循环阈值与模板浓度的对数值具有很好的对应关系，最低可检测57个拷贝数，经18h增菌，可检测低至4．5cfu／ml的细菌；特异性强，检测6株单增李斯特菌标准株和100株单增李斯特菌样品分离株的PCR循环域值（cT值）均小于25，而90株威氏李斯特菌、英诺克李斯特菌、绵羊李斯特菌以及非李斯特菌PCR循环域值（CT值）大于35；快速，最快1h可出结果，实际样品20h可出结果，而常规细菌培养法需要一周以上；对103份速冻米面食品进行检测，13份阳性，与常规方法无显著性差异（P〉0．05）。结论：TaqMan—MGB探针的单增李斯特菌Real—time荧光PCR检测方法具有特异性强，敏感性高，易操作等优点，可推广应用。     

李斯特菌属荧光定量PCR检测方法的建立

目的采用荧光定量PCR技术,建立可以同步检测李斯特菌属与单增李斯特菌的快速检测方法。方法选取单增李斯特菌hlyM基因和李斯特菌属23Sr DNA基因为靶基因,分别设计引物和探针,在构建二者的阳性重组质粒基础上,对李斯特菌属和单增李斯特菌进行荧光定量PCR的检测。结果建立的李斯特菌属和单增李斯特菌单重荧光定量PCR检测方法与双重荧光定量PCR的检测方法敏感度一致,分别为18.6拷贝/μl和23.2拷贝/μl。结论建立的双重荧光定量PCR方法可以同步检测李斯特菌属与单增李斯特菌。     

利用胶体金免疫层析技术快速定量检测单增李斯特菌方法的建立

〔目的〕建立胶体金免疫层析技术快速定量检测单增李斯特菌方法。〔方法〕利用胶体金标记和双抗体夹心免疫层析技术,建立单增李斯特菌的快速检测方法,评价其特异性和敏感性,并拟合标准曲线进行定量检测;在牛奶、橙汁、果冻等即食食品样品中添加单增李斯特菌模拟污染样品,评价该方法对半固体、液体等即食食品、可疑生物恐怖样品的检测能力。〔结果〕该法可在10min内完成定性和半定量检测,定量检测灵敏度为3.5×103cfu/ml,线性范围3.5×105～3.5×108cfu/ml,回收率在99%～101.7%之间。〔结论〕建立的检测单增李斯特菌的胶体金免疫层析方法,能快速、灵敏、特异、准确地对样品中的单增李斯特菌进行定性、定量检测。     

2000～2004年浙江省食品中产单核李斯特菌污染状况调查

目的：掌握浙江省食品中产单核李斯特菌污染状况。以及内化素基因（Inl）和李氏溶血素O（Hly）两种致病基因携带情况。方法：按GB4789．30方法分离单增生李斯特菌，采用PCR技术检测李斯特菌的两种致病基因。结果：2000～2004年从2282份食品中共分离产单核李斯特菌163株，总污染率为7．14％（163／2282）。生肉类、熟肉及水产品的污染率分别为12．80％、7．67％和2．24％；114份生食蔬菜中1份标本分离到产单核李斯特菌；285份乳及乳制品、59份冰激凌均未分离到产单核李斯特菌；163株产单核李斯特菌有155株同时检测到内化素基因和李氏溶血素O基因。结论：我省存在发生李斯特菌食物中毒的潜在危险。     

套式PCR快速检测单增李斯特氏菌

根据GenBank数据库的单增李斯特氏菌iap基因设计两对引物,建立了套式PCR快速检测单增李斯特氏菌的方法。两对引物分别扩增出约1500bp和500bp片段,与预期大小一致。对菌液、模拟样品的检测表明,本方法能有效地克服食品基质、培养基成分和杂菌对PCR检验的干扰作用。套式PCR方法具有很好的特异性;灵敏性实验检测极限为101CFU／ml;人工污染猪肉检测极限为103CFU／ml;可在7h内完成检测,很适宜于进出口食品中单增李斯特氏菌的快速检测。     

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。     

奶液模拟标本中单增李斯特菌real-time PCR检测方法的建立

目的建立了基于TaqMan探针的real-time PCR技术针对奶液模拟标本中单增李斯特菌的快速检测方法。方法用单增李斯特菌hlyO基因的部分片段作为靶基因,制作标准曲线,定量检测奶液中的单增李斯特菌。结果通过对不同李斯特菌及一些较为常见的致病菌的DNA进行扩增,只有单增李斯特菌能够产生扩增曲线,其余菌株均不产生扩增曲线。单增李斯特菌的检测灵敏度可以达到9copies/反应体系。结论该方法特异性好,灵敏度高,整个实验可在1.5h内完成,可用于食品中单增李斯特菌的快速检测和疫情暴发时的相关病原调查。     

应用Taqman实时PCR法检测猪肉中单核细胞增生李斯特菌

目的建立敏感快速的检测猪肉中单核细胞增生李斯特菌的实时PCR方法。方法以hlyA基因为靶标,建立并验证实时PCR法的特异性。选用单核细胞增生李斯特菌CMCC 54004,制备不同浓度的纯菌液,用实时PCR进行检测,制作标准曲线并计算扩增效率。进行人工染菌实验,染菌量分别为每25g猪肉样本1.3×100、1.3×101、1.3×102、1.3×103、1.3×104、1.3×105和1.3×106CFU。分别在增菌0、4、8、12、18、24、30、36和46 h取1 ml培养液,提取DNA进行实时PCR检测,并用PCR和传统方法进行检测,比较3种方法检测的敏感性和特异性。采集24份市售猪肉样本,用这3种方法进行检测,进一步比较三者的阳性检出率。结果建立的实时PCR法特异性好,对纯菌液的检出限为1.3×103CFU/ml。人工染菌样本增菌24 h后,实时PCR检出限1.3 CFU/25 g,PCR及传统方法达到这一检出限需要增菌46 h。根据增菌24 h的检测结果,建立实时PCR样本标准曲线。24份猪肉样本,实时PCR检出17份阳性,阳性率70.83%(17/24),与PCR和传统方法的阳性率一致。根据所得的样本标准曲线,对检测样本进行定量分析,确定了阳性样本中初始含量菌。结论所建立的实时PCR具有快速简便、敏感特异等优点,整个操作可在27 h内完成,适用于猪肉中单核细胞增生李斯特菌的快速定量检测。     

Microbiological surveillance of a bovine raw milk farm through multiplex real-time PCR

Raw milk is increasingly appreciated by consumers but can be contaminated by a variety of zoonotic pathogens. Therefore, preventive measures, such as on-farm hazard analysis critical control point (HACCP) programs, must be applied to protect consumers. The aim of the present study was the comparison of a multiplex real-time polymerase chain reaction (PCR) assay with a culture-based approach in an on-farm quality assurance program for the detection of Escherichia coli O157, Salmonella spp., and Listeria monocytogenes in bulk tank milk, in-line milk filters, manure, and feces. Results revealed that the real-time PCR was more sensitive in detecting E. coli O157 than the culture method in filters (48% vs. 4% positive), manure (93% vs. 7% positive) and feces (60% vs. 4% positive). The two methods were equally efficient in detecting L. monocytogenes (8% of filters), while Salmonella spp. was not detected in any sample. In conclusion, the real-time PCR, by reducing analysis time to two working days, can be proposed as a useful tool in the raw milk primary production setting as a rapid and user-friendly screening method.     

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.     

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.     

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.     

Prevalence and antimicrobial resistance of foodborne pathogens isolated from food products in China

This study was undertaken to investigate the prevalence of Salmonella, Listeria monocytogenes, Staphylococcus aureus, Vibrio parahaemolyticus, and Escherichia coli O157:H7 in Chinese food products. The prevalence of these pathogens was 3.46%, 5.79%, 7%, 0.24%, and 0%, respectively. Raw meats were mainly contaminated with Salmonella (39/365, 10.7%), L. monocytogenes (26/365, 7.1%), and S. aureus (40/365, 11%), while cooked food products were mainly contaminated with L. monocytogenes (45/384, 11.7%) followed by S. aureus (12/384, 3.1%), and raw milk was mainly contaminated with S. aureus (34/209, 16.3%) and Salmonella (4/209, 1.9%). Antimicrobial resistance was evaluated in Salmonella, L. monocytogenes, and S. aureus. Antimicrobial resistance for L. monocytogenes was most frequently observed for cefotaxime (51/72, 70.8%) followed by furazolidone (40/72, 55.6%). Multiple resistance (resistance to >or=2 antibiotics) was observed for 63.9% (46/72) of L. monocytogenes isolates. Resistance of Salmonella was most frequently observed to amoxicillin (11.6%), ticarcilline (11.6%), cephalothin (11.6%), and cefuroxime (11.6%). Multiple resistance was observed for 16.3% (7/43) of the Salmonella isolates. Staphylococcus aureus was resistant to penicillin (93.1%) followed by tetracycline and oxacillin COAG (49.4% and 37.9%, respectively). About 79% (69/87) of S. aureus isolated demonstrated multiple resistance. The data showed that raw meat, cooked food products, and raw milk were most commonly contaminated with foodborne pathogens and many pathogens were resistant to different antibiotics. The study provided useful information for assessment of the possible risk posed to Chinese consumers, which has significant public health impact in China.     

实时荧光定量PCR法与常规PCR法及细菌培养法检测单增李斯特菌的比较

目的：比较实时荧光定量PCR法、常规PCR法及细菌培养法检测单核细胞增生性李斯特菌的灵敏度与特异性。方法：采用建立的实时荧光定量PCR、常规PCR及传统细菌培养法3种方法,同时对单核细胞增生性李斯特菌等细菌进行检测。结果：实时荧光定量PCR检测的灵敏度可达19 cfu/ml,且有很高的特异性,对英诺克李斯特菌等10种相关细菌均无交叉反应,从细菌核酸提取至完成检测仅需3 h左右。结论：实时荧光定量PCR检测由于在密封环境中进行,避免了产物与环境间的交叉污染,且是3种方法中最为快速敏感的方法,适用于公共卫生应急疫情的实验室快速诊断。