应用xMAP液念芯片多重快速检测四种病原微生物的研究

目的:建立一种多重、快速、特异性好、灵敏度高的病原微生物检测方法。方法:根据GenBank数据库中的小肠结肠炎耶尔森氏菌、单核细胞增生性李斯特菌、产气荚膜梭菌、鼠疫耶尔森氏菌基因序列,分别针对ail、hly、cpe、3a基因设计4对引物和4条探针。通过重叠PCR扩增各目的基因并构建重组质粒,以该重组质粒DNA为模板,通过多重PCR同时扩增上述4个基因,建立xMAP液态芯片检测技术,在此基础上对标准菌株基因组DNA进行检测并验证该方法的特异性和敏感性。结果:xMAP液态芯片对质粒DNA和标准菌株基因组DNA的检测结果与多重PCR结果一致。该方法能在3.5 h内同时完成对4种病原菌的检测,特异性好,且敏感性要高于PCR方法,灵敏度最高可达200CFU/ml。结论:xMAP液态芯片技术是病原微生物的多重快速检测的新方法,具有很好的应用价值和前景。     

PCR快速鉴定鼠疫耶尔森氏菌

建立一种简便、快速、特异的PCR检测方法,用于鼠疫耶尔森氏菌的快速鉴定。针对鼠疫耶尔森氏菌特异的一段染色体序列3a设计引物,扩增-276bp片段的鼠疫标识序列。应用该PCR反应体系,对我国17个生态型及1个待定的生态型共计275株鼠疫耶尔森氏菌及48株相关菌株的PCR扩增结果表明,实验菌株均扩增出预期的276bp片段产物带,48株相关菌株均阴性,其检测灵敏度为100pg DNA。说明该方法用于鼠疫耶尔森氏菌的检测鉴定简便、快捷,具有很高的特异性和敏感性。     

沙门菌、志贺菌、副溶血性弧菌多重PCR检测方法的研究

建立快速检测沙门菌、志贺菌和副溶血性弧菌的多重PCR方法[1-4].根据沙门菌hilA基因、志贺菌ipaH基因及副溶血性弧菌TDH基因设计特异性PCR引物[5-6],被检样品经4 h振荡培养后金属浴裂解制备DNA模板,使用全自动毛细管电泳核酸检测系统分析PCR扩增产物.在580、423和245 bp处分别出现预期的特异性DNA条带,且无非特异扩增条带出现.敏感性试验显示沙门菌在模拟标本中的检测灵敏度为101-2cfu/mL、志贺菌为101cfu/mL、副溶血性弧菌为102cfu/mL.该方法操作方便、分析时间短、特异性和灵敏度高,可用于公共卫生突发事件食源性病原菌的快速检测.     

多种食源性致病菌检测的多重PCR 方法的研究

目的:利用多重PCR技术,建立可以同时检测多种食源性致病菌的多重PCR方法。方法:分别选择沙门氏菌invA基因,志贺氏菌的ipaH基因,单核细胞增生李斯特氏菌的hlyA基因,大肠杆菌O157:H7的eaeA基因,副溶血弧菌的toxR基因,设计多重PCR引物,建立多重PCR检测体系,并对该体系进行特异性和灵敏度实验。结果:通过对19株菌株进行实验,所有的目标菌株均为阳性,而其余菌株为阴性。对多重PCR体系的灵敏度进行考察,沙门菌的灵敏度为5000 CFU/mL;志贺氏菌的灵敏度为5500CFU/mL;单核细胞增生李斯特氏菌的灵敏度为5200 CFU/mL;O157:H7的灵敏度为5000CFU/mL;副溶血弧菌的灵敏度为6300CFU/mL。结论:建立的多重PCR体系能实现多种致病菌同时检测。     

鮰爱德华氏菌外膜微孔蛋白N基因PCR快速检测方法的建立

根据Gen Bank中鮰爱德华氏菌Edwardsiella ictaluri外膜微孔蛋白N(porin N)基因序列(Gen Bank No:NC_012779.2)设计了1对引物,预计目的片段大小为381 bp。通过对反应体系和条件的优化,并进行特异性试验、敏感性试验及人工感染组织样品检测,建立了一种快速检测鮰爱德华氏菌的PCR方法。结果表明,在所检测的鮰爱德华氏菌、迟缓爱德华氏菌、嗜水气单胞菌、温和气单胞菌、杀鲑气单胞菌、豚鼠气单胞菌、嗜麦芽寡养单胞菌、鲁氏耶尔森氏菌、海豚链球菌、不动杆菌、产气肠杆菌、大肠杆菌、拟态弧菌、荧光假单胞菌、弗氏柠檬酸杆菌15种细菌中仅鮰爱德华氏菌扩增出特异性条带;敏感性试验结果显示,该方法最小核酸检出量为9.35×10-3ng·μL-1;同时对人工感染的病料肝脏、细菌基因组DNA、细菌菌液及菌落进行扩增,结果显示4种材料均能检测出大小为381 bp的基因片段。本研究所建立的方法特异强、灵敏度高,适用于鮰爱德华氏菌感染病例的高效、快速检测。     

应用肽核酸探针检测鼠疫耶尔森氏菌

目的:利用特异的肽核酸(PNA)探针、链霉亲和素包被的磁珠和cy5纳米颗粒,通过荧光扫描技术,建立一种特异、快速、准确地检测鼠疫耶尔森氏菌的方法。方法:针对鼠疫耶尔森氏菌pMT1质粒上的caf1基因设计并合成一对特异PNA探针,经生物素标记后,分别与链霉亲和素包被的磁珠和cy5纳米颗粒结合;将探针与待测鼠疫耶尔森氏菌的基因组DNA杂交后,利用荧光扫描技术进行检测。探讨了多个实验因素对测定的影响,并进行了特异性和灵敏度检测。结果:建立并优化了利用PNA探针检测鼠疫耶尔森氏菌的方法,得到较好的线性关系;检测的灵敏度为0.9μg/mL(待测DNA)。结论:PNA探针与靶基因的结合不易受杂交液离子强度的影响,结合后具有较高的稳定性。本研究建立的分析方法能够灵敏、特异、稳定地对鼠疫耶尔森氏菌进行定量检测,为鼠疫的监控、诊断提供了有力手段。     

基因芯片技术检测3种食源性致病微生物方法的建立

建立一种运用多重PCR和基因芯片技术检测和鉴定志贺氏菌、沙门氏菌、大肠杆菌O157的方法, 为3种食源性致病菌的快速检测和鉴定提供了准确、快速、灵敏的方法。分别选取编码志贺氏菌侵袭性质粒抗原H基因(ipaH)、沙门氏菌肠毒素(stn)基因和致泻性大肠杆菌O157志贺样毒素(slt)基因设计引物和探针, 进行三重PCR扩增, 产物与含特异性探针的芯片杂交。对7种细菌共26株菌进行芯片检测, 仅3种菌得到阳性扩增结果, 证明此方法具有很高的特异性。3种致病菌基因组DNA和细菌纯培养物的检测灵敏度约为8 pg。对模拟食品样品进行直接检测, 结果与常规细菌学培养结果一致, 检测限为50 CFU/mL。结果表明：所建立的基因芯片检测方法特异性好, 灵敏度高, 为食源性致病菌的检测提供了理想手段, 有良好的应用前景。     

多重PCR技术检测微生物肥料中巨大芽孢杆菌和蜡样群芽孢杆菌的研究与应用

巨大芽孢杆菌是微生物肥料生产中的常用菌种, 与之形态上相似的蜡样群芽孢杆菌(蜡样芽孢杆菌、苏云金芽孢杆菌、蕈状芽孢杆菌)则是产品中常见的污染菌, 传统方法区分两者费时费力, 有必要建立检测这两类芽孢杆菌的PCR方法。本文利用已登录的spoOA基因序列分别设计和筛选了上述两个种(群)的特异引物, 并建立了多重PCR检测技术。使用该方法对巨大芽孢杆菌、蜡样群芽孢杆菌和其他芽孢菌共3属13种24株标准菌株的基因组DNA进行扩增, 以检验其特异性。结果显示, 巨大芽孢杆菌、蜡样群芽孢杆菌基因组DNA分别产生大小不同的唯一产物, 其他芽孢杆菌均为阴性。该多重PCR检测方法的灵敏度经测定为105 CFU/mL。同时对10株待测菌株和8个微生物肥料产品进行检测, 其鉴定结果与常规鉴定结果一致。以上结果表明, 本文建立的多重PCR方法具有较高的特异性和灵敏度, 可快速、准确鉴定巨大芽孢杆菌和蜡样群芽孢杆菌, 在微生物肥料检测方面有良好的实用前景。     

弗氏柠檬酸菌甘油脱水酶基因在大肠杆菌中的克隆和表达

以弗氏柠檬酸菌（Citrobacter freundii）基因组DNA为模板,通过PCR得到甘油脱水酶（glycerol dehydratase）基因dhaB、dhaC、dhaE,克隆到表达载体pSE380上,得到重组质粒pSn-dhaBCE。将此重组质粒转化到E．coli JM109中,重组菌株SDS-PAGE结果显示有明显的61kD、22kD、16kD三条特异性蛋白条带出现。重组菌株经诱导表达,酶活力为11．59U／mL。     

鼠伤寒沙门氏菌多重PCR检测方法的研究

分别根据沙门氏菌16S rRNA、质粒毒力基因spvC、致病基因invB、fimA序列设计4对引物,对沙门氏菌株及非沙门氏株菌基因组DNA进行多重PCR检测。结果该方法能检测出6.3×10² 个cfu/ml纯培养的沙门氏菌,人工染菌食品模拟检测结果显示,熟鸡肉初始含菌量为17cfu/g、全脂奶粉为11cfu/g、生牛肉为13.6cfu/g,经过8h增菌,PCR检测为阳性。该体系能鉴定产生多种毒力因子的沙门氏菌,特异性强、敏感性高,为检测和鉴定沙门氏菌株提供了一个新方法。     

One multiplex control for 29 cystic fibrosis mutations

A simple approach is described to synthesize and clone an inexhaustible supply of any homozygous and/or heterozygous controls diluted with yeast genomic DNA to mimic human genome equivalents for use throughout the entire multiplex mutation assay. As a proof of principle, the 25 cystic fibrosis mutation panel selected by the American College of Medical Genetics and four additional mutant sequences were prepared as a single control mixture. The 29 CFTR mutations were incorporated into 17 gene fragments by PCR amplification of targeted sequences using mutagenic primers on normal human genomic DNA template. Flanking primers selected to bind beyond all published PCR primer sites amplified controls for most assay platforms. The 17 synthesized 433-933-bp CFTR fragments each with one to four homozygous mutant sequences were cloned into nine plasmid vectors at the multiple cloning site and bidirectionally sequenced. Miniplasmid preps from these nine clones were mixed and diluted with genomic yeast DNA to mimic the final nucleotide molar ratio of two CFTR genes in 6 x 10(9) bp total human genomic DNA. This mixture was added to control PCR reactions prior to amplification as the only positive control sample. In this fashion >200 multiplex clinical PCR analyses of >4,000 clinical patient samples have been controlled simultaneously for PCR amplification and substrate specificity for 29 tested mutations without cross contamination. This clinically validated multiplex cystic fibrosis control can be modified readily for different test formats and provides a robust means to control for all mutations instead of rotating human genomic controls each with a fraction of the mutations. This approach allows scores of additional mutation controls from any gene loci to be added to the same mixture annually.     

多重PCR快速检测化妆品中三种致病菌的研究

利用多重PCR技术建立快速检测化妆品中三种致病菌的方法。根据已报道的大肠杆菌phoA基因、铜绿假单胞菌外膜蛋白基因oprL和金黄色葡萄球菌特异性序列SmaI选择特异性引物,对人工染菌化妆品进行多重PCR检测。结果显示,三种致病菌的基因组DNA均可与各自引物特异性结合,扩增产物大小分别为622 bp、504 bp和426 bp。该方法用于人工污染的化妆品中,大肠杆菌的检出限浓度为103 CFU/mL,铜绿假单胞菌和金黄色葡萄球菌的检出限浓度为105 CFU/mL。作者建立的多重PCR方法可同时快速、特异地对化妆品中三种致病菌进行检测,在化妆品行业具有较大的应用价值。     

Electrical microarrays for highly sensitive detection of multiplex PCR products from biological agents

For the sensitive detection of amplicons derived from diagnostic PCR, a novel electrical low-density microarray is applied and compared to state-of-the-art quantitative real-time PCR. The principle of the electrochemical method and the effective use for analysis are described. Interdigitated array gold electrodes (IDA-E) embedded into a silicon chip are the core technology of the fully automated compact biosensor system, basing on enzyme coupled electrochemical detection. The biointerface is built up with thiol-modified capture oligonucleotides on gold and mediates the specific recognition of hybridised target DNA amplified with uniplex or multiplex PCR. In here we show the potential of the designed electrical microarray to function as an advanced screening method for the parallel detection of a panel of the four pathogens Bacillus anthracis, Yersinia pestis, Francisella tularensis and ortho pox viruses (genus), which are among the most relevant biowarfare agents. PCR products, generated from 10 to 50 gene equivalents, have been detected reproducibly. The experiments with varying pathogen amounts showed the good reliability and the high sensitivity of the method, equivalent to optical real-time PCR detection systems. Without PCR the total assay time amounts to 27 min. The advantage of the combination of multiplex-PCR with electrical microarray detection avoiding intensive PCR probe labelling strategies is illustrated.     

食源性致病菌多重PCR快速检测方法建立与应用

利用PCR技术,建立多组多重食源性致病菌PCR快速检测方法。设计受试菌特异性引物,反应体系中加入多对引物和多种DNA模板,采用正交试验优化PCR反应条件,进行特异性引物的PCR扩增。建立了多组多重食源性致病菌PCR快速检测方法,方法中所检测受试菌株和模拟样品均出现特异性扩增条带,结果与实际相符。所建立多组多重PCR快速检测体系符合设计要求,可以应用于食源性突发公共卫生事件的应急检测和日常样品检测工作。     

Rapid detection of Yersinia pestis with multiplex real-time PCR assays using fluorescent hybridisation probes

The objective of the present study was to establish a system of real-time polymerase chain reactions (PCRs) for the specific detection of Yersinia pestis using the LightCycler (LC) instrument. Twenty-five strains of Y. pestis, 94 strains of other Yersinia species and 33 clinically relevant bacteria were investigated. Assays for the 16S rRNA gene target and the plasminogen activator gene (resides on the 9.5-kb plasmid) and for the Y. pestis murine toxin gene and the fraction 1 antigen gene (both on the 100-kb plasmid) were combined for the use in two multiplex assays including an internal amplification control detecting bacteriophage lambda-DNA. Applying these multiplex assays, Y. pestis was selectively identified; other bacteria yielded no amplification products. The lower limit of detection was approximately 0.1 genome equivalent. Rat or flea DNA had no inhibitory effects on the detection of Y. pestis. The results obtained using the multiplex real-time assays showed 100% accuracy when compared with combinations of conventional PCR assays. We developed and evaluated a highly specific real-time PCR strategy for the detection of Y. pestis, obtaining results within 3 h including DNA preparation.     

基因芯片技术检测3种肠道病原微生物方法的建立

目的:建立一种运用多重PCR和基因芯片技术检测和鉴定伤寒沙门氏菌、痢疾杆菌和单核细胞增生利斯特菌的方法。方法:分别选取伤寒沙门氏菌染色体ViaB区域中编码调控Vi抗原表达的基因(vipR)、痢疾杆菌编码侵袭质粒抗原H基因(ipaH)和单核细胞增生利斯特菌溶血素基因(hlyA)设计引物和探针,探针3'端进行氨基修饰,下游引物标记荧光素Cy3。在优化的PCR和杂交反应条件下,进行三重PCR扩增,产物与包括3种致病菌特异性探针的基因芯片杂交。在评价基因芯片的特异性和灵敏度之后,对临床样本进行检测。结果:只有3种目的致病菌的PCR产物在相应探针位置出现特异性信号,其他阴性细菌均无信号出现;3种致病菌的检测灵敏度均可达到103CFU/mL;检测30例临床样本的结果与常规细菌学培养结果一致。结论:所建立的可同时检测伤寒沙门氏菌、痢疾杆菌和单核细胞增生利斯特菌的基因芯片方法快速、准确,特异性高,重复性好,为3种肠道致病菌的快速检测和鉴定提供了新方法和新思路。     

Multiplex PCR assay for detection of bacterial pathogens associated with warm-water Streptococcosis in fish

A multiplex PCR-based method was designed for the simultaneous detection of the main pathogens involved in warm-water streptococcosis in fish (Streptococcus iniae, Streptococcus difficilis, Streptococcus parauberis, and Lactococcus garvieae). Each of the four pairs of oligonucleotide primers exclusively amplified the targeted gene of the specific microorganism. The sensitivity of the multiplex PCR using purified DNA was 25 pg for S. iniae, 12.5 pg for S. difficilis, 50 pg for S. parauberis, and 30 pg for L. garvieae. The multiplex PCR assay was useful for the specific detection of the four species of bacteria not only in pure culture but also in inoculated fish tissue homogenates and naturally infected fish. Therefore, this method could be a useful alternative to the culture-based method for the routine diagnosis of warm-water streptococcal infections in fish.     

利用PCR方法鉴定hiTAIL-PCR扩增产物中的质粒骨架片段

T-DNA突变体是研究基因功能的重要资源。高效热不对称交错PCR (hiTAIL-PCR)是克隆突变体中T-DNA插入位点侧翼序列的常用方法。然而我们发现, 利用hiTAIL-PCR克隆到的一些侧翼序列并不对应于宿主的染色体DNA序列, 而是质粒的骨架DNA片段。通过设置1组RB-S4/AC1或者LB-A4/AC1对照反应, 用PCR方法鉴定了hiTAIL-PCR扩增产物中位于T-DNA侧翼的质粒骨架片段。在后续分析中, 通过排除这些片段, 提高了利用hiTAIL-PCR获得宿主染色体DNA片段的效率。同时, 通过调整反应程序, 使得整个PCR的反应时间也大为缩短。在拟南芥(Arabidopsis thaliana) T-DNA突变体drf1侧翼序列的克隆实例中, 对照反应的引入将hiTAIL-PCR中需鉴定的22条扩增产物降至4条, 效率提高了81.8%。