基于16S rRNA基因序列鉴定秘鲁茎柔鱼和澳洲双柔鱼

通过对秘鲁茎柔鱼及澳洲双柔鱼的线粒体16S rRNA基因片段进行聚合酶链式反应（polymerase chain reaction,PCR）扩增和序列比对,确定2 种柔鱼品种的特异性位点,并由此设计品种特异性引物。利用多重PCR体 系对秘鲁茎柔鱼和澳洲双柔鱼进行快速、准确地鉴定和区分,并对该体系的灵敏度进行检验。结果表明：在多重 PCR体系下,秘鲁茎柔鱼和澳洲双柔鱼分别扩增出长度400、229 bp的品种特异性条带,2 种柔鱼的混合样品则同时 扩增出长度400、229 bp的条带;且该多重PCR体系可以检测澳洲双柔鱼中1%秘鲁茎柔鱼的掺入,检测限为0.1 ng。     

多重位点特异性PCR快速检测牛肉中常见掺假动物源性成分

目的:基于动物线粒体cytb基因的多态性位点,建立一种特异性多重PCR体系检测牛肉、猪肉和鸡肉的方法。方法:提取肉类的基因组DNA,利用不同物种mtDNA cytb基因序列的SNP位点的差异,设计特异性引物。进行多重PCR扩增,利用扩增产物片段大小不同,检测牛肉中常见的掺假动物源性成分。通过灵敏性试验,确定最低检测量。结果:实验设计的引物特异性良好,在同一反应体系中,在同一退火温度52℃条件下,牛肉DNA扩增后产生149 bp的特异性条带,猪肉DNA扩增片段为261 bp,鸡肉DNA扩增片段为554 bp,未发生非特异性扩增。且检测的最低浓度达到100 pg/μL,具有高度的灵敏性和适用性。结论:根据动物线粒体cytb基因的差异性位点,开发的多重PCR体系,可一次性地同时检测牛肉、猪肉和鸡肉,可快速、灵敏、高通量地分析食品中掺假动物成分的来源。     

基于lolB和toxR基因的水产品中霍乱弧菌双重PCR检测方法

选择lolB和toxR两种基因序列设计2对特异性引物,建立一种针对霍乱弧菌所有生物型菌株的双重PCR检测方法,扩增目的片段大小分别为519bp和779bp。结果表明:在同步扩增中,仅霍乱弧菌模板可同时扩增出2种基因片段,4株对照菌模板无任何扩增条带;敏感性检测结果显示,该双重PCR最低能检测3.42×103CFU/mL菌落数的霍乱弧菌。所建立的基于lolB和toxR两种基因的双重PCR检测方法特异性强、敏感性高、方法简单、用时短,可用于霍乱弧菌检测。     

新西兰柔鱼与北太平洋柔鱼的品种特异性聚合酶链式反应鉴定

利用聚合酶链式反应（polymerase chain reaction,PCR）技术扩增新西兰柔鱼和北太平洋柔鱼的线粒体细胞色素C氧化酶亚基Ⅰ（cytochrome C oxidase subunit Ⅰ,COⅠ）基因,通过多重序列比对检测2 种鱿鱼之间的多态性位点。根据2 种鱿鱼的特异性位点分别设计2 种鱿鱼的品种特异性引物,并利用多重PCR实现2 种鱿鱼品种的特异性鉴定和区分。结果表明：建立的品种特异性PCR鉴别体系对新西兰柔鱼和北太平洋柔鱼分别扩增出214 bp和339 bp的品种特异性条带,该方法的检测限低至0.1 ng,且能检测出新西兰柔鱼中1%的北太平洋柔鱼掺入,并能有效检测市场上2 种鱿鱼及其复配鱼糜制品的原料来源。     

纳豆芽孢杆菌和嗜酸乳杆菌融合子的多重PCR鉴定

为建立纳豆芽孢杆菌和嗜酸乳杆菌融合子快速鉴定的多重聚合酶链式反应(polymerase chain reaction,PCR)方法,根据芽孢杆菌的芽孢形成早期因子基因spo0A和13株乳酸菌β-半乳糖苷酶基因的保守序列分别设计引物对P1/P2和P3/P4,以亲本菌株DNA为模板,优化每对引物在适宜退火温度条件下的PCR体系,在最优体系下的特异性分析结果表明P1/P2能特异性扩增出spo0A基因片段(308 bp),而7株乳酸菌全部呈阴性;P3/P4可扩增出所有受试乳酸菌和经表型鉴定确定的阳性融合子中的目标基因片段(576 bp),而对芽孢杆菌无扩增。多重PCR结果表明在P1/P2的最优体系中只有308 bp片段的扩增,而在P3/P4的最优体系中可实现2个片段的共扩增,对融合后获得的50个菌株的鉴定结果与单重PCR和表型鉴定结果100%吻合。该体系中模板DNA 1 ng/μL,每条引物0.4μmol/L,脱氧核糖核苷三磷酸0.25 mmol/L,Taq酶0.06 U/μL;PCR时94℃预变性5 min;每个循环(共30个)94℃30 s、53℃30 s、72℃60 s,产物末端72℃延伸7 min。该方法简便、快速、特异性好,适用于芽孢杆菌、乳酸菌以及二者融合子的快速鉴定。     

基于blaCARB-17和toxR基因的副溶血弧菌双重PCR检测方法的建立

建立一种针对副溶血弧菌bla_CARB-17和toxR基因的双重PCR检测方法。按照副溶血弧菌的bla_CARB-17和toxR基因序列设计并合成引物,于同一反应体系中进行PCR扩增,优化退火温度、退火时间和引物比例,确定双重PCR的特异性和灵敏性,最后用实验室分离鉴定的副溶血弧菌分离株验证。结果表明:建立的双重PCR最佳退火条件是52℃ 30 s,引物比例1:1,在同一体系中特异地扩增出副溶血弧菌的bla_CARB-17和toxR基因的303、350 bp片段,其它对照菌株均无扩增,表明该方法特异性良好。双重PCR的最低检测限达1×102 CFU/mL,对实验室鉴定的56个副溶血弧菌分离株验证均为阳性。建立的双重PCR方法操作简单、高效快速、特异性强,适用于副溶血弧菌的检测。     

转基因水稻潮霉素标记基因PCR检测方法的建立及其在基因水平转移研究中的应用

为建立用于基因水平转移研究, 尤其是DNA经加工和消化后稳定性研究的针对转基因水稻潮霉素标记基因hpt（hygromycin phosphotransferase）的定性和实时定量PCR体系,设计针对hpt的上游通用引物多个片段定性PCR扩增体系,以植物叶绿体基因rbcl为内对照,PCR扩增产物经测序验证.将定性PCR中最小片段（236 bp）连接到质粒载体pUC18-pMD T载体上,提取质粒经验证后做外标.应用TaqMan-MGB荧光探针和引物,建立定量的外标校正曲线法,并评价方法的精密度.建立的定性PCR体系能稳定扩增出236 bp～910 bp不同大小的5个hpt片段,并经测序验证.实时定量PCR的线性范围为105～10拷贝（R^2=0.998）,最低能检出10拷贝,重复性好.本研究已成功建立了用于转基因水稻标记基因hpt基因水平转移研究的定性和定量PCR系统。     

添加扩增内标的单增李斯特菌PCR检测方法的建立与应用

以单增李斯特菌inl A基因为靶基因,设计一对特异性引物,以16S rRNA为扩增内标对照,建立了一种含有扩增内标的单增李斯特菌PCR检测方法。优化了PCR反应体系,并对PCR检测方法的特异性、灵敏度、人工污染样品及食品样品检测效果进行了测试。对2株单增李斯特菌、1株英诺克李斯特菌以及19株非李斯特菌菌株进行PCR检测,结果显示,只有2株单增李斯特菌能被检出大小为826 bp的特异性片段,其余20株细菌只能检出1 500 bp的扩增内标片段。灵敏度实验结果显示,基因组DNA和纯培养物的最低检出限分别为1.80×10~2fg/μL和1.21×10~2CFU/m L。当人工污染牛乳样品中单增李斯特菌在最低接种量为0.48 CFU/m L时,经8 h增菌培养后可被该方法检出。采用该研究建立的检测方法对36种食品样品进行检测,证实了该检测方法可以指示检测过程中出现的假阴性现象。综上所述,该研究建立的PCR检测方法能特异性的检测单增李斯特菌,并可有效排除检测过程中出现的假阴性现象,提高检测的准确性。     

多重PCR检测原料乳中沙门氏菌、无乳链球菌和金黄色葡萄球菌的研究

目的:建立快速检测沙门氏菌、无乳链球菌和金黄色葡萄球菌的多重PCR方法。方法:根据沙门氏菌(Salmonella)组氨酸转运操纵子基因、无乳链球菌(Streptococcus agalactiae)的STRA-Agl-23-1D基因和金黄色葡萄球菌(Staphylococcus aureus)的耐热核酸酶nuc基因分别设计引物,进行PCR扩增及反应条件的优化,建立这3种菌的多重PCR检测方法。结果:通过建立多重PCR方法,3对引物同时特异性地扩增出495 bp、360 bp和279 bp目的片段;3种菌的检测限:沙门氏菌1.9×102 cfu/mL、无乳链球菌2.0×102 cfu/mL、金黄色葡萄球菌2.9×102 cfu/mL;3种菌DNA含量的(最低)检出限分别为3.86、25.5、3.47pg。结论:本文建立的多重PCR检测方法,简单、快速、灵敏度高,具有很好的应用前景。     

科氏滑柔鱼和剑尖枪乌贼的多重PCR鉴定

目的:利用单核苷酸多态性(SNP)分子标记设计特异性引物,建立多重PCR体系,实现鱿鱼品种剑尖枪乌贼及其混淆品科氏滑柔鱼的分子鉴定。方法:利用PCR扩增2种鱿鱼品种的细胞色素c氧化酶亚基I(Cytochrome Oxidase Subunit I,COI)的核苷酸序列,通过多重序列比对,发掘了2种鱿鱼品种的单核苷酸多态性位点(SNP)。设计各自的位点特异性引物,并建立了鉴定2种鱿鱼品种的多重PCR体系。结果:在多重PCR体系中,科氏滑柔鱼产生了419 bp的特异性条带,剑尖枪乌贼产生了233 bp的特异性条带,两者的混合品产生了清晰的419 bp和233 bp条带。结论:建立的多重PCR体系不仅可以对科氏滑柔鱼和剑尖枪乌贼进行准确的分子鉴定,而且可以实现对两者混合品的有效鉴定。     

晒烟品种塘蓬烟抗白粉病基因的等位性测定和序列分析

[背景和目的]广东连江县晒烟品种"塘蓬烟"是我国特有的烟草隐性遗传白粉病抗性种质资源,但到目前为止对其抗病机制未见深入报道.[方法]利用塘蓬烟花粉与抗白粉病烟草品种Kutsaga E1及感病品种K326杂交进行抗病基因的等位性检测;利用等位基因分子标记检测塘蓬烟中感白粉病基因NtMLO1/2的突变情况;利用PCR克隆技...     

应用多重PCR检测食品中SARS病毒靶基因的初步研究

以分子生物学方法——聚合酶链式反应(PCR)技术为基础,初步探讨了食品中SARS病毒靶基因片段的多重PCR检测技术。根据GenBank公开发表的SARS病毒基因组cDNA序列,人工合成克隆特异性靶基因DNA片段,以此片段作为阳性对照,并将其添加到罐装蘑菇、牛肉干、大豆、鱼干等食品的cDNA中作为阳性样品,再根据世界卫生组织推荐的引物序列合成引物,进行单PCR与多重PCR检测分析。结果表明:以单PCR法获得了121bp、182bp及302bp3条靶基因片段;以二重PCR法获得了121bp+182bp、121bp+302bp与182bp+302bp的靶基因片段组合;以三重PCR法获得了121bp+182bp+302bp的靶基因片段组合。检测灵敏度的实验表明:182bp片段的模板DNA量在0.003ng以上时,单PCR都能扩出清晰的条带,而121bp的靶基因片段只有模板DNA量在0.03ng以上时,单PCR才能扩出清晰的条带。它们的二重PCR分析的灵敏度与相应的单PCR分析灵敏度相同。     

A pilot study on PCR-based detection of four foodborne pathogenic microorganisms

To establish PCR-based detection methods for Staphylococcus aureus, Shigella, Pasteurella multocida and Pseudomonas aeruginosa, the nuc, ipah, ptfa and oprl genes were amplified by singleplex PCRs and multiplex PCR using specific primers that were designed according to the DNA sequences retrieved from GenBank. Then the annealing temperature was optimized, accompanied by a study of the specificity and sensitivity of the singleplex PCRs and multiplex PCR. The results showed that DNA fragments of 280, 474, 150 and 331 bp were specifically amplified from the four pathogenic bacteria mentioned above. No target DNA fragments were obtained from other pathogenic bacteria, including Salmonella typhimurium, Campylobacter jejuni, Clostridium perfringens and pathogenic Escherichia coli. The sensitivity of the singleplex PCRs were 100, 1, 1 and 10 pg/μL respectively. The detection limits of the four pathogenic bacteria in the multiplex PCR were 100, 1, 10 and 10 pg/µL respectively. These results showed that singleplex PCRs and multiplex PCR have good specificity and sensitivity. In conclusion, this experiment has laid a foundation for further research on rapid detection methods against these four pathogenic bacteria in food.     

A multiplex PCR assay based on 16S rRNA and hly for rapid detection of L. monocytogenes in Milk

A multiplex PCR assay was developed by targeting ‘16S rRNA’ and ‘hly’ genes for detection of Listeria or Listeria monocytogenes in dairy foods on the basis of amplification of 1200 and 713 bp products, respectively. The assay conditions were optimized to make it truly rapid and to cut down the cost. The authenticity of the multiplex PCR was ascertained by using Nested PCR targeted against internal region of ‘hly’ gene that produced an amplified product of 188 bp. The multiplex PCR assay was found to be specific for detection of L. monocytogenes only since none of the non-listerial cultures gave positive signal. The sensitivity of the multiplex PCR was limited to 10 ng pure DNA and 1–10 cells of L. monocytogenes after 4–6 h enrichment in Listeria enrichment broth. When applied to 20 raw milk and 10 pasteurized milk samples, L. monocytogenes could not be detected in any of the samples by the multiplex PCR assay. This assay could find potential application in dairy industry for monitoring dairy foods for this high risk food pathogen on routine basis.     

Degradation and possible carry over of feed DNA monitored in pigs and poultry

A possible carry over of foreign food DNA into the body after consumption was examined. After feeding pigs with conventional and recombinant (Bt-) maize, different body samples were investigated using DNA-extraction followed by PCR procedures to detect chloroplast genes of different length (199 bp and 532 bp), a maize-specific gene (zein) and a specific transgene present in Bt-maize (cryIa). Initially, a time-dependent degradation of feed DNA in the gastrointestinal tract of pigs was analysed within the juices from stomach and three parts of the small intestine (duodenum, jejunum, ileum). Subsequently, a possible transfer of residual chloroplast specific DNA as well as recombinant Bt-maize DNA fragments into different pig organs (blood, muscle, liver, spleen and lymph nodes) was examined. The suitability of the introduced DNA extraction procedure was verified through amplification of a universal gene (ubiquitin) demonstrating the successful PCR analysis within a range of 189-417 bp long DNA. Short chloroplast DNA fragments (199 bp) could be successfully amplified from the intestinal juices of pigs up to 12 h after the last feeding. In contrast, chloroplast-specific DNA was not found in any pig organ investigated so far. Specific gene fragments from the transgene maize (Bt-maize) were never detected in any pig sample. A field study examining supermarket poultry samples (leg, breast and wing muscle, stomach) led to frequent detections of the short chloroplast DNA fragment (199 bp). Furthermore, faint signals for the maize specific zein gene fragment were detected in these poultry tissues. Additional PCR examinations using unhatched chicken embryos provided the first indication that neither chloroplast nor maize genes are present endogenously within the wild-type poultry genome. Therefore, a transient transfer of short forage DNA into most poultry organs can be suspected.     

多重PCR快速检测三种食品病原菌

建立一种快速、经济、实用的可以同步检测食品中金黄色葡萄球菌、沙门氏菌、小肠结肠炎耶尔森氏菌的多重聚合酶链式反应(polymerase chain reaction,PCR)的方法。根据金黄色葡萄球菌的nuc基因,沙门氏菌的invA基因,小肠结肠炎耶尔森氏菌的ail基因,分别设计了三对引物,对单个基因PCR和单管多重PCR扩增进行特异性、灵敏性实验以及优化反应体系。三对引物能特异性扩增出236、475、127bp的目的条带。建立的多重PCR同时对3种食源性致病菌进行检测具有较高的灵敏度,灵敏度金黄色葡萄球菌为102CFU/mL、沙门氏菌为102CFU/mL、小肠结肠炎耶尔森氏菌为102CFU/mL。初步建立能同步、简便、快速、灵敏地检测食品中金黄色葡萄球菌、沙门氏菌和小肠结肠炎耶尔森氏菌的三重PCR方法。     

多重PCR快速检测婴幼儿奶粉中的病原菌

为了建立同时检测婴幼儿奶粉中阪崎肠杆菌、金黄色葡萄球菌、蜡样芽孢杆菌的多重PCR方法,根据阪崎肠杆菌ompA基因、金黄色葡萄球菌nuc基因、蜡样芽孢杆菌hblA基因分别设计3对引物进行多重PCR扩增,并对反应条件进行优化。结果多重PCR扩增出长度为514、156、235bp的特异性目的条带。不增菌的情况下,多重PCR同时检测3种病原菌的灵敏度是103cfu/mL,3种病原菌在奶粉中的检出限是104cfu/g。建立的多重PCR反应准确、快速、高效,为同时检测婴幼儿奶粉中的阪崎肠杆菌、金黄色葡萄球菌、蜡样芽孢杆菌提供了新方法。     

Design of PCR primers and gene probes for the general detection of bacterial populations capable of degrading aromatic compounds via catechol cleavage pathways

For the general detection of bacterial populations capable of degrading aromatic compounds, two PCR primer sets were designed which can, respectively, amplify specific fragments from a wide variety of catechol 1,2-dioxygenase (C12O) and catechol 2,3-dioxygenase (C23O) genes. The C12O-targeting primer set (C12O primers) was designed based on the homologous regions of 11 C12O genes listed in the GenBank, while the C23O-targeting one (C23O primers) was designed based on those of 17 known C23O genes. Oligonucleotide probes (C12Op and C23Op) were also designed from the internal homologous regions to identify the amplified fragments. The specificity of the primer sets and probes was confirmed using authentic bacterial strains known to carry the C12O and/or C23O genes used for the primer and probe design. Various authentic bacterial strains carrying neither C12O nor C23O genes were used as negative controls. PCR with the C12O primers amplified DNA fragments of the expected sizes from 5 of the 6 known C12O-carrying bacterial strains tested, and positive signals were obtained from 4 of the 5 amplified fragments on Southern hybridization with the C12Op. The C23O primers amplified DNA fragments of the expected size from all the 11 tested C23O-carrying bacterial strains used for their design, while the C23Op detected positive signals in the amplified fragments from 9 strains. On the other hand, no DNA fragments were amplified from the negative controls. To evaluate the applicability of the designed primers and probes for the general detection of aromatic compound-degrading bacteria, they were applied to wild-type phenol- and/or benzoate-degrading bacteria newly isolated from a variety of environments. The C12O and/or C23O primers amplified DNA fragments of the expected sizes from 69 of the 106 wild-type strains tested, while the C12Op and/or C23Op detected positive signals in the amplified fragments from 63 strains. These results suggest that our primer and probe systems can detect a considerable proportion of bacteria which can degrade aromatic compounds via catechol cleavage pathways.     

5 类致泻性大肠埃希氏菌多重荧光PCR检测方法的建立

目的：建立快速检测和鉴别5?类致泻大肠埃希氏菌的多重实时荧光聚合酶链式反应（polymerase chain reaction,PCR）方法。方法：根据5?类致泻大肠埃希氏菌的11?组毒力基因eae、stx1、stx2、ipaH、uidA、estla、estlb、aggR、pic、elt、astA建立多重实时荧光PCR方法,该方法包括A、B?2?个反应体系,可分别检测肠致病性大肠埃希氏菌、肠出血性大肠埃希氏菌、肠侵袭性大肠埃希氏菌、肠产毒大肠埃希氏菌、肠聚集性大肠埃希氏菌,优化反应体系的引物、探针浓度以及PCR反应条件,并对该方法的灵敏度和特异性进行检测。结果：11?组基因的探针和引物均可特异性地扩增相应的基因片段,A体系检测下限可达到2.6×104?copies/反应,B体系检测下限可到达2.2×104?copies/反应。结论：本研究所建立的多重实时荧光PCR方法可以对同一样本同时采用A、B?2?个反应体系进行检测,不但可以确定待测样本是否为大肠埃希氏菌,而且还能判定其致病型别。为大肠埃希氏菌的临床检测、疾病预防以及爆发溯源等提供参考。