用PCR快速筛选递次截短的DNA克隆

目的为了建立一种快速、简便而准确地筛选递次截短的ＤＮＡ克隆的技术，加速对基因全长ｃＤＮＡ或ＤＮＡ大片段的测序和鉴定。方法在载体多克隆位点两侧设计引物，直接ＰＣＲ扩增插入片段，以此为基础选择系列缺失亚克隆，并与常规酶切法作比较。结果根据ＰＣＲ产物大小排序，准确地鉴定出系列缺失亚克隆，并可将ＰＣＲ产物直接用于ＰＣＲ循环测序，比酶切法分辨率高，操作步骤简化，筛选时间短。结果所建立的ＰＣＲ快速筛选法不仅是一种行之有效的鉴定方法，而且可以显著地节省时间和实验材料。该方法也可适用于其它各种质粒载体。     

单核细胞增生性李斯特菌iap基因克隆和PCR条件优化

目的 构建单核细胞增生性李斯特菌Listeria monocytogenes 54002-4株p60蛋白iap基因原核克隆载体.方法 利用自行设计的引物通过梯度PCR优化扩增条件,扩增出单核细胞增生性李斯特菌54002-4株的iap基因.在iap基因的5'端和3'端分别引入BamH Ⅰ和Xho Ⅰ 2个酶切位点,琼脂糖凝胶电泳分析、回收PCR产物,并将回收纯化的PCR产物与pMD 18-T载体进行连接.将该重组质粒转化人大肠杆菌JM109感受态细胞,经1 mmol/L IPTG诱导4～6 h后,观察转化效果.结果 培养无色菌落细菌,提取质粒,经PCR鉴定和核苷酸序列测定后确定获得阳性重组质粒pMD18-T-Iap.结论 通过梯度PCR摸索出最佳反应条件,建立PCR优化条件和方法,经转化获得iap基因克隆载体.     

重组PCR构建bcr—abl mRNA竞争RT—PCR内标物

目的竞争ＰＣＲ可用于ｍＲＮＡ的定量测定，为了得到白血病中ｂｃｒ－ａｂｌｍＲＮＡ定量ＰＣＲ的竞争内标物。方法利用重组ＰＣＲ技术，实施对ｂ３ａ２型ｂｃｒ－ａｂｌｃＤＮＡ中一２７６ｂｐ片段的缺失法定点突变。结果经ＤＮＡ序列分析证实，重组后的ｃＤＮＡ片段与重组前相比，５′和３′端大部分序列相同，仅中间５５ｂｐ的部分序列缺失，同时引入１９ｂｐ外源ＤＮＡ片段，即净缺失３６ｂｐ，得到２４０ｂｐ的重组ＰＣＲ产物。较ｂ２ａ２型ｂｃｒ－ａｂｌｃＤＮＡ相应的２０１ｂｐ扩增片段长３９ｂｐ，使之适于作为ｂ３ａ２和ｂ２ａ２型两类ｂｃｒ－ａｂｌｍＲＮＡ定量ＰＣＲ的通用内标物。结论表明重组ＰＣＲ是一种获得靶基因的竞争性内标物的简便而可靠的方法     

肠道致病菌PCR检测与鉴定研究

目的 为了实现对腹泻等病原菌的快速、准确检测与鉴定，对引起感染性腹泻的暴发与流行的常见肠道致病菌进行检测与鉴定方法研究。方法 将常规ＰＣＲ与半套式ＰＣＲ及随机引物扩增ＤＮＡ多态性分析（ＲＡＰＤ）等技术相结合，对常见肠道致病菌，如志贺菌、沙门菌和致病性大肠杆菌Ｏ１５７：Ｈ７等进行检测与鉴定。结果 ｕｉｄＡ引物可特异扩增沙门菌、志贺菌及大肠杆菌，而３种特异性引物则只扩增相应的致病菌；第一次ＰＣＲ敏感性     

PCR产物的克隆方法

ＰＣＲ产物的克隆方法肖翠英张思仲聚合酶链反应（ｐｏｌｙｍｅｒａｓｅｃｈａｉｎｒｅａｃｔｉｏｎ，ＰＣＲ）已经成为分子生物学的强有力工具，并应用于核酸检测、序列分析、克隆、突变研究和现代分子生物学的许多其它方面。现今，克隆前扩增ＤＮＡ已经成为日常的操作。...     

构建T载体快速克隆丙型肝炎病毒5′端非编码区扩增产物

为探讨自行构建Ｔ载体的简便方法，克隆丙型肝炎病毒（ＨＣＶ）５′端非编码区（５′ＮＣＲ）扩增产物，并为制备ＨＣＶ检测探针作准备。以ＳｍａⅠ消化质粒ｐＧＥＭ３Ｚｆ（＋），酶切产物纯化后在仅含有脱氧腺苷酸的聚合酶链反应（ＰＣＲ）缓冲液中７０℃作用２小时，构建成Ｔ载体。根据ＰＣＲ扩增产物３′端存在一个非模板依赖的脱氧腺苷酸原理，将扩增产物直接克隆入Ｔ载体。同时以ＳｍａⅠ消化的质粒ｐＧＥＭ３Ｚｆ（＋）与ＰＣＲ产物平端连接作为对照。限制性酶切长度多肽性分析，ＰＣＲ及ＤＮＡ序列测定等均证实克隆构建成功。ＡＴ克隆的连接效率约为平端连接的４２倍。以上结果表明，构建Ｔ载体经济、简便。ＡＴ克隆快速、有效。所构建的克隆可用作ＰＣＲ实验对照模板或制备探针。     

PCR技术在单克隆抗体cDNA的筛选及标记同位素探针上的应用

以往对cDNA克隆鉴定过程中,经斑点杂交筛选出的克隆数较多,在进一步的鉴定中不可避免地要进行质粒扩增、抽提、纯化酶解、片段分离等许多步骤。本文对这一步的鉴定采用了PCR技术,这样便可省去酶解及片段分离等繁琐步骤。且由于扩增片段纯净,故可直接用于Southern blot鉴定及DNA序列分析。 本文另一新的探索是利用PCR法标记探针,尤其是小片段DNA探针,其不同于常用的缺口平移和随机引物延伸标记。由于它在扩增过程中只有位于两个引物间的片段才能标上同位素,所以标记后的探针量大、比放射性强,加之PCR合成仪的出现,使此法更加高效、简便安全。     

TA克隆及双链DNA：测序：介绍一种快速克隆及分析PCR产物的方法

将人酸性纤维母细胞生长因子’（ａＦＧＦ’）ｃＤＮＡ的ＰＣＲ产物以ＴＡ连接方式克隆入ｐＣＲ￣（ＴＭ）ＩＩ质粒，然后采用Ｔ７和Ｓｐ６启动子特异性引物对克隆的片段以双脱氧未端终止法进行双链ＤＮＡ测序。结果表明这项技术是一种快捷而可靠的克隆及分析ＰＣＲ产物的方法     

双抗体夹心免疫PCR—检测神经肽Y

建立了一种双抗体夹心免疫ＰＣＲ和神经肽Ｙ单克隆抗体包被；多克隆抗体作为夹心抗本，生物素标记的羊抗免ＩｇＧ和游离的亲合素格为连接分子；生物素化的腺病毒六邻本基因重组质粒ＤＮＡ作为指示分子；用腺病毒六邻体基因的特异引物扩增指标分子。     

EB病毒BHRF1基因的克隆及其表达产物抑制细胞凋？…

目的 ＥＢ病毒ＢＨＲＦ１基因克隆，其表达产物抑制ＣＨＯ细胞凋亡功能的研究。方法 以Ｂ９５－８细胞株ＥＢＶ ＤＮＡ为模板设计一对引物，采用ＰＣＲ技术扩增ＢＨＲＦ１基因，用目的基因内的两个单酶切点ＢａｍＨⅠ、ＢｇｌⅠ进行酶切鉴定。用引物上所引入的两个酶切位点ＮｃｏⅠ、ＳｃｌⅠ酶切目的基因后定向连接到ｐＢＶ２２１载体上，转入宿主菌ＤＨ５α经质粒抽提，单、双酶切鉴定，从所获克隆中筛选重组质粒克隆。温控诱导     

16S rDNA快速鉴定血液制品中污染细菌的测序方法的建立

目的 建立基于16S rDNA快速鉴定细菌的PeR测序方法(PCR-SBT).方法 通过一组16S rDNA通用引物扩增得到基因组全长,PCR产物经纯化后直接测序分析.利用BLAST软件从GenBank数据库中搜索相关菌株的16S rDNA全序列,采用Clustal X软件进行多序列比对和同源性分析,确定细菌的种属,并与常规生化鉴定结果比较.利用大肠杆菌基因组一系列稀释度标本进行PeR扩增,检测方法的敏感性.结果 实验利用多对引物建立了16S rDNA全长序列分析方法,13个标准菌株通过PCR-SBT方法获得约1400 bp的全长序列.比对分析13个标准菌株测序结果与预期标准序列完全一致,证实建立的PCR-SBT方法结果可靠.利用建立的PCR-SBT法,对实验室从血小板制品和脐带血中分离得到不同未知菌株进行鉴定,成功确定了这些菌株的种属.以大肠杆菌DNA为模板,方法的最低检测限为反应体系DNA含量0.2 ng.结论 建立的基于16S rDNA的PCR-SBT方法是可行的,可快速准确地检测及鉴定细菌种类,尽早发现细菌污染血制品,对污染血制品输注后的针对性治疗方面具有潜在的应用价值.     

多重PCR检测肺炎链球菌血清型方法及临床应用

目的:建立一种可检测常见肺炎链球菌(SP)重要血清型的多重PCR(mPCR)方法,并初步应用于临床。方法:根据SP荚膜多糖基因序列(cps)的保守序列cpsA,设计8种SP血清型特异性引物,优化PCR条件后,对SPDNA进行扩增,通过电泳分离出型特异性条带,用于检测8种已知血清型和126株未知血清型SP菌株,并与产物纯化克隆后的测序结果比对。结果:8种已知血清型SP的mPCR分型结果与传统方法分型结果及测序结果一致;mPCR可一次检测出87.50%的血液来源SP血清型和31.81%呼吸道来源SP血清型。结论:mPCR方法可快速检测8种重要SP血清型,对初筛血液来源SP血清型有较高应用价值。     

Duchenne/becker muscular dystrophy carrier detection using quantitative PCR and fluorescence-based strategies

Dystrophin gene deletions account for up to 68% of all Duchenne (DMD) and Becker (BMD) muscular dystrophy mutations. In affected males, these deletions can be detected easily using multiplex PCR tests which monitor for exon presence. In addition, quantitative dosage screening can discriminate female carriers. We previously analyzed multiplex PCR products by gel electrophoresis and quantitation of fluorescently labeled primers with the Gene Scanner? in order to test carrier status. These multiplex PCR protocols detect DMD gene deletions adequately, but require up to 18 pairs of fluorochrome-labeled primers. We previously described two alternative fluorescent labeling strategies, each with approximately 1,000-fold greater sensitivity than ethidium bromide staining, which can be used to quantify the products of multiplex PCR. The first method uses the DNA intercalating thiazole orange dye TOTO-1 to stain PCR products after 20 cycles. In the second method, fluorescein-12,2′-dUTP is incorporated into products during PCR as a fluorescent tag for subsequent quantitative dosage studies. Both methods label all multiplexed exons including the 506 bp exon 48 fragment that is difficult to detect and quantify by standard ethidium bromide staining. Using this approach, we determined DMD/BMD carrier status in 24 unrelated families using a fluorescent fragment analyzer. Analysis of fluorochrome-labeled PCR products facilitates quantitative multiplex PCR for gene-dosage analysis. © 1993 Wiley-Liss, Inc.     

PCR for detection and identification of Streptococcus sobrinus

Oligonucleotide primers were designed based upon a comparison of the dextranase gene (dex) sequences from Streptococcus sobrinus and S. mutans. The primers amplified a 1610-bp long DNA fragment on the dex gene by a PCR. The pair of primers was specific to S. sobrinus as the other members of the mutans streptococci - S. mutans, S. downei, S. cricetus, S. rattus, S. macacae and S. ferus - gave no PCR products. Other gram-positive oral bacteria (15 strains of 10 species of cocci and 18 strains of 12 species of rods) and gram-negative oral bacteria (3 strains of 3 species of cocci and 31 strains of 22 species of rods) also gave negative results in the PCR. The PCR procedure was able to detect as little as 100 fg of purified chromosomal DNA or as few as 9 cfu of S. sobrinus NIDR6715. Seven clinical isolates of S. sobrinus were also positive in the dex PCR. This laboratory developed the S. mutans-specific PCR (dexA PCR) method with the primers specific for a portion of the dextranase gene of S. mutans Ingbritt. Primers for the dex and dexA PCR methods detected two species exclusively from the mutans streptococci. Furthermore, these two species were effectively differentiated by the species-specific amplicons with different lengths. The application of the PCR method to human dental plaque showed that the prevalence of S. sobrinus (83%) in oral cavities was higher than currently supposed (0-50%). These results suggest that the described PCR method is suitable for the specific detection and identification of human cariogenic bacteria, S. sobrinus and S. mutans.     

Microarray-based identification of bacteria in clinical samples by solid-phase PCR amplification of 23S ribosomal DNA sequences

The rapid identification of the bacteria in clinical samples is important for patient management and antimicrobial therapy. We describe a DNA microarray-based PCR approach for the quick detection and identification of bacteria from cervical swab specimens from mares. This on-chip PCR method combines the amplification of a variable region of bacterial 23S ribosomal DNA and the simultaneous sequence-specific detection on a solid phase. The solid phase contains bacterial species-specific primers covalently bound to a glass support. During the solid-phase amplification reaction the polymerase elongates perfectly matched primers and incorporates biotin-labeled nucleotides. The reaction products are visualized by streptavidin-cyanine 5 staining, followed by fluorescence scanning. This procedure successfully identified from pure cultures 22 bacteria that are common causes of abortion and sterility in mares. Using the on-chip PCR method, we also tested 21 cervical swab specimens from mares for the presence of pathogenic bacteria and compared the results with those of conventional bacteriological culture methods. Our method correctly identified the bacteria in 12 cervical swab samples, 8 of which contained more than one bacterial species. Due to the higher sensitivity of the on-chip PCR, this method identified bacteria in five cervical swab samples which were not detected by the conventional identification procedure. Our results show that this method will have great potential to be incorporated into the routine microbiology laboratory.     

耐甲氧西林葡萄球菌荧光定量PCR检测方法的实验研究

目的建立一种简便、特异的mecA基因荧光定量PCR检测方法，用于耐甲氧西林葡萄球菌（MRS）的快速鉴定。方法以煮沸法快速制备DNA模板，采用SYBRGreenI随机参入法，建立mecA基因的实时荧光定量PCR检测体系。并对检测体系的敏感性、特异性和灵敏度进行评价。结果本法对纯菌落的检测敏感性和特异性分别为98．5％和96．9％，检测灵敏度可达10^1CFU／ml，最小检菌量约为3个菌／反应体系。结论本实验所设计的荧光定量PCR方法用于MRS的检测具有快捷、高敏感性、高特异性和高灵敏度的特点。适用于MRS的快速检测。     

单核细胞增生李斯特菌与志贺菌双重实时PCR检测技术的建立

目的 建立一种快速、特异、灵敏、准确定量的单核细胞增生(单增)李斯特菌(Listeriamonocytogenes)与志贺菌(Shigella)同步检测方法.方法 分别根据单增李斯特菌溶血素O基因hly与志贺菌侵袭性质粒抗原H基因ipaH设计合成引物和探针.构建重组质粒pGEM-T-hly与pGEM-T-ipaH,并以EcoR I单酶切使环状重组质粒线性化作为标准品.优化反应体系,分析特异性.双重荧光定量PCR对人工污染的脱脂灭菌乳进行检测.结果 成功构建了重组质粒标准品,并运用5'、3'端分别标记FAM、TAMRA的hly基因探针和5'、3'端分别标记HEX、TAMRA的ipaH基因探针成功建立了单增李斯特菌与志贺菌同步荧光定量PCR检测方法.结论 建立的方法有较强的特异性,线性范围好(105～101copies/μl,R2≥0.998),灵敏度为10 copies/PCR,同步检测人工污染脱脂灭菌乳的灵敏度为102CFU/ml.     

Quantitation of encapsidated recombinant adeno-associated virus DNA in crude cell lysates and tissue culture medium by quantitative, real-time PCR

Recombinant AAV vectors are produced by transient transfection of mammalian cells. The virus is usually purified from a combination of lysed cells and spent culture medium by HPLC. We have developed a quantitative, real-time PCR assay for quantifying encapsidated single-stranded viral DNA (i.e. DNA-containing virions) in cell lysates and the spent culture medium. This requires extensive DNaseI digestion to reduce the amount of AAV replicative DNA, as well as plasmid and cellular DNA, to negligible amounts. To demonstrate the utility of this assay, we produced recombinant AAV in HeLa cells and five different types of 293 cells. We used primers to the EGFP transgene to detect the production of a recombinant AAV. We assayed the cell lysates and media by both our quantitative PCR assay and a functional transduction assay. The quantitative PCR assay data correlated well with the transduction assay data. Because this assay only requires standard PCR primers and SYBR Green I dye to detect the amplification of the PCR template, it will readily adapt to any target DNA sequence within the recombinant AAV genome. The recombinant AAV vector does not need to express a reporter gene, such as EGFP or beta-galactosidase in order to assay the amount of virus produced.     

Comparison of a LightCycler-based real-time PCR for quantitation of Epstein-Barr viral load in different clinical specimens with semiquantitative PCR

Measurement of viral load is important in predicting and monitoring of Epstein-Barr virus (EBV)-associated diseases especially in immunocompromised patients. The objectives of this study were the development of a LightCycler-based real-time PCR assay using primers and probes which recognize the virus capsid antigen p23-encoding region and its comparison to the semiquantitative PCR. The LightCycler protocol shows a high degree of specificity and inter- and intra-assay reproducibility. Concerning sensitivity, a good correlation between both methods was demonstrated for standard plasmid DNA, reference DNA isolated from the EBV-genome containing Namalwa cell line, and DNA extracted from plasma/cerebrospinal fluid (CSF). The detection limit was determined with 1 copy/microl eluate for the standard plasmid DNA and with 500 copies/ml plasma or CSF. For DNA derived from peripheral blood mononuclear cells (PBMCs), a decrease of sensitivity by factor 10-100 was found when larger amounts of background DNA (500 and 100 ng) were used presuming an inhibitory effect of cellular DNA. This was supported by running dilutions of the plasmid standard carried out with EBV-negative Ramos cell DNA. Thus, the cut-off level was estimated with 100-500 copies/10(5) PBMCs, when 50 or 10 ng total DNA were tested. The results indicate that the real-time PCR described here is a first line tool for the determination of viral load in plasma and CSF. Semiquantitative nested PCR is used for screening of PBMCs viral load. Positive specimens containing more than 500 copies/10(5) cells are measured for exact values by real-time PCR. To circumvent inhibitory effects of cellular DNA, measurements should be carried out generally with 50-10 ng DNA.