Use of the polymerase chain reaction and oligonucleotide probes for the rapid detection and identification of Carnobacterium species from meat.

The polymerase chain reaction (PCR) was used selectively to amplify specific rDNA sequences of Carnobacterium divergens, C. mobile, C. piscicola and C. gallinarum in purified DNA extracts, crude cell lysates and food samples. The PCR products were visualized by agarose gel electrophoresis and identified, at species level, by hybridization reactions with three specific oligonucleotide probes for C. divergens, C. mobile and C. piscicola/C. gallinarum designed from 16S rRNA sequence data. The PCR was sufficiently sensitive to amplify DNA from a single bacterium to detectable levels after 30 cycles of amplification. Both radioactive (32P) and non-radioactive alkaline phosphatase labelled probes was able to detect the PCR products. Detection was highly specific and the probes did not hybridize with DNA samples from any other of the bacterial species tested. These methods enabled the rapid and specific detection and identification of carnobacteria from pure cultures and samples of meat.     

Use of PCR analysis for detecting low levels of bacteria and mold contamination in pharmaceutical samples

PCR assays were developed and compared to standard methods for quality evaluation of pharmaceutical raw materials and finished products with low levels of microbial contamination. Samples were artificially contaminated with less than 10 CFU of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Aspergillus niger. Bacterial DNA was extracted from each enrichment broth by mild lysis in Tris-EDTA-Tween 20 buffer containing proteinase K while mold DNA was extracted by boiling samples in Tris-EDTA-SDS buffer for 1 h. A 10-microl aliquot of extracted DNA was added to Ready-To-Go PCR beads and specific primers for E. coli, S. aureus, and P. aeruginosa. However, 50-microl aliquots of extracted mold DNA were used for amplification of specific A. niger DNA sequences. Standard methods required 6-8 days while PCR detection of all microorganisms was completed within 27 h. Low levels of microbial contamination were detected in all raw materials and products using PCR assays. Rapid quality evaluation of pharmaceutical samples resulted in optimization of product manufacturing, quality control, and release of finished products.     

Use of the polymerase chain reaction and oligonucleotide probes for the rapid detection and identification of Carnobacterium species from meat

J.L. BROOKS, A.S. MOORE, R.A. PATCHETT, M. D. COLLINS AND R.G. KROLL. 1992. The polymerase chain reaction (PCR) was used selectively to amplify specific rDNA sequences of Carnobacterium divergens, C. mobile, C. piscicola and C. gallinarum in purified DNA extracts, crude cell lysates and food samples. The PCR products were visualized by agarose gel electrophoresis and identified, at species level, by hybridization reactions with three specific oligonucleotide probes for C. divergens, C. mobile and C. piscicola/C. gallinarum designed from 16S rRNA sequence data. The PCR was sufficiently sensitive to amplify DNA from a single bacterium to detectable levels after 30 cycles of amplification. Both radioactive (³²P) and non-radioactive alkaline phosphatase labelled probes was able to detect the PCR products. Detection was highly specific and the probes did not hybridize with DNA samples from any other of the bacterial species tested. These methods enabled the rapid and specific detection and identification of carnobacteria from pure cultures and samples of meat.     

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。     

大仓鼠DNA 指纹谱探针的筛选

采用一种简便提取高质量DNA 的方法, 从大仓鼠肝脏组织中提取其总DNA , 分别以人工合成的微卫星核心序列(GTG)₅和(CA)₈做单一引物, 进行特异引物PCR 反应。电泳检测后回收15 条特异性片段。与被标记过的大仓鼠基因组DNA 反向杂交结果表明, 15 个片段中(GTG)_5-8 、(CA)_8-1b和(CA_{) 8-5b}产生了较强的阳性信号。我们依据3 个片段的测序结果设计适合DIG标记的探针, 该探针得到的大仓鼠不同地理种群个体的指纹图谱有较高的个体特异性和种群多态性, 而且与传统的来源于其它生物重复序列的探针如33.6 和33.15 形成的指纹图谱相比得到的变异适中, 便于统计。     

利用微分离黑麦1R染色体的体外扩增产物进行染色体着染研究

首先对显微分离出的黑麦（ＳｅｃａｌｅｃｅｒｅａｌｅＬ．）１Ｒ染色体进行了两轮Ｓａｕ３Ａ连接接头介导的ＰＣＲ扩增（ＬＡ＿ＰＣＲ）。经Ｓｏｕｔｈｅｒｎ杂交证实这些染色体扩增片段来源于基因组ＤＮＡ之后,再利用１Ｒ染色体的第二轮扩增产物、黑麦基因组ＤＮＡ、ｒＤＮＡ基因为探针,与其根尖细胞中期分裂相进行染色体原位杂交,发现微分离的１Ｒ染色体体外扩增产物中包含大量的非该染色体特异性重复序列,而其信息量却较黑麦总基因组少;当以适量的黑麦基因组ＤＮＡ进行封阻时,微分离染色体的体外扩增产物成功地被重新定位在中期分裂相的一对１Ｒ染色体上,说明微分离１Ｒ染色体的ＰＣＲ扩增产物中的确包含了该染色体特异性的片段。此外,以从１Ｒ染色体微克隆文库中筛选出的一单、低拷贝序列和一高度重复序列分别为探针,染色体原位杂交检测发现,这一高度重复序列可能为端粒相关序列;而单、低拷贝序列却未检测到杂交信号。这些结果从不同侧面反映出染色体着染技术是证实微分离、微切割染色体的真实来源及筛选染色体特异性探针的有利工具。建立了可供参考的植物染色体着染实验体系,为染色体微克隆技术在植物中的进一步应用提供了便利。     

Construction of a DNA library from chromosome 4 of rice （Oryza sativa） by microdissection

A simple method to create a chromosome-specific DNA librqary of rice,including microdissection,amplification,charterization and cloning,is described.Rice chromosome 4 from a metaphase cell has been isolated and amplified by the Linker Adapter PCR (LA-PCR).The PCR products were labeled as probes with DIG-11-dUTP using the random priming method.Southern blot analysis with rice genomic DNA and specific RFLP markers demonstrated that the PCR products were derived from rice chromosome 4.A large library comprising over 100,000 recombinant plasmid microclones from rice chromosome 4 was constructed.Colony hybridization showed that 58% of the clones contained single or low-copy sequences and 42% contained repetitive sequences.The size of inserts generated by PCR ranged from 140bp to 500bp.This method will facilitate cloning of the specific chromosome DNA markers and important genes of rice.     

PCR amplification of chromosome-specific alpha satellite DNA: definition of centromeric STS markers and polymorphic analysis.

Alpha satellite DNA is a tandemly repetitive DNA family found at the centromere of every human chromosome. Chromosome-specific subsets have been isolated for over half the chromosomes and have prove useful as markers for both genetic and physical mapping. We have developed specific oligonucleotide primer sets for polymerase chain reaction (PCR) amplification of alpha satellite DNA from chromosomes 3, 7, 13/21, 17, X, and Y. For each set of primers, PCR products amplified from human genomic DNA are specific for the centromere of the target chromosome(s), as shown by somatic cell hybrid mapping and by fluorescence in situ hybridization. These six subsets represent several evolutionarily related alpha satellite subfamilies, suggesting that specific primer pairs can be designed for most or all chromosomal subsets in the genome. The PCR products from chromosome 17 directly reveal the polymorphic nature of this subset, and a new DraI polymorphism is described. The PCR products from chromosome 13 are also polymorphic, allowing in informative cases genetic analysis of this centromeric subset distinguished from the highly homologous chromosome 21 subset. These primer sets should allow placement of individual centromeres on the proposed STS map of the human genome and may be useful for somatic cell hybrid characterization and for making in situ probes. In addition, the ability to amplify chromosome-specific repetitive DNA families directly will contribute to the structural and functional analysis of these abundant classes of DNA.     

Oligo-chip based detection of tick-borne bacteria

We have developed an oligonucleotide-chip based assay for detection of 16S ribosomal PCR products from tick-borne bacteria. This chip contains 14 specific probes, which target variable regions of 16S rDNA of tick-borne bacteria including Borrellia spp., Rickettsia spp., Anaplasma spp., Coxiella burnetii and Francisella tularensis. The specificity of these probes was tested by hybridization of the chip with fluorescently labeled PCR products amplified from the genomic DNA of selected tick-borne bacteria. The assay was also tested for detection of tick-borne bacteria in single ticks.     

DETECTION OF BOTULINAL TOXIN GENES: TYPES A AND E OR B AND F USING THE MULTIPLEX POLYMERASE CHAIN REACTION

Multiple primer sets were combined with the polymerase chain reaction (PCR) and examined for use in the amplification of toxin gene fragments from four Clostridium botulinum types (A, B, E, and F). Vegetative cells obtained from overnight cultures were used directly in the PCR analyses without purification of chromosomal DNA. Gene fragments were amplified from the different botulinal toxin genes that code for types A, B, E and F toxins using a single PCR protocol. Toxin gene fragments were amplified from types B and F toxigenic organisms using the PCR and specific primer sets for these types in a single PCR tube. Type A and E toxin genes were also examined using type A and E specific primers in a separate PCR tube. The PCR-amplified products were separated by electrophoresis on agarose gels containing ethidium bromide. The identity of the PCR products were confirmed by DNA hybridization using type specific probes. We conclude that this method is useful for the rapid and direct identification of toxigenic botulinal organisms that code for the toxin types A, B, E, or F.     

Diagnosis of alpha 1-antitrypsin deficiency by enzymatic amplification of human genomic DNA and direct sequencing of polymerase chain reaction products.

We have compared sequencing of cloned "polymerase chain reaction" (PCR) products and the direct sequencing of PCR products in the examination of individuals from six families affected with alpha 1-antitrypsin (AAT) deficiency. In families where paternity was in question we confirmed consanguinity by DNA fingerprinting using a panel of locus-specific minisatellite probes. We demonstrate that direct sequencing of PCR amplification products is the method of choice for the absolutely specific diagnosis of AAT deficiency and can distinguish normals, heterozygotes and homozygotes in a single, rapid and facile assay. Furthermore, we demonstrate the reproducibility of the PCR and a rapid DNA isolation procedure. We have also shown that two loci can be simultaneously amplified and that the PCR product from each locus can be independently examined by direct DNA sequencing.     

DNA microarray probe preparation by gel isolation nested PCR

To develop a simplified method that can rapidly prepare DNA microarray probes in a massive scale, a lambda phage genomic DNA-fragments library was constructed for the microarray-probes collection. Four methods of DNA band recovery from the first PCR products were tested and compared. The DNA microarray probes were collected by a novel method of nested PCR that was mediated by gel isolation of the first PCR products. This method was named GIN-PCR. The probes that were prepared by this GIN-PCR technique were used as subjects to fabricate a DNA microarray. The results showed that a wooden toothpick was superior to the other 3 methods, since this technique can steadily transfer the DNA bands as the template of the second PCR after the first PCR. A group of probes were successfully collected and DNA microarrays were constructed using these probes. Hybridization results demonstrated that this technique of DNA recovery and probe preparation was rapid, efficient, and effective. We developed a cost-effective and less labor-intensive method for DNA microarray probe preparation by nested PCR that is mediated by wooden toothpick transfer of the DNA bands in the gel after electrophoresis.     

谷类作物半粒种子的PCR分析及其在标记辅助选择育种中的应用

建立了适于种子nNA分析的快速简便的PCR方法。水稻、小麦和玉米的半粒种子用提取缓冲液处理,产生的提取液可用于PCR和RAPD分析。水稻半粒种子的DNA和来自叶片的DNA用专一的PCR引物扩增后可以产生同样的PCR产物。含有胚的另半粒种子可以正常发芽。将半粒种子的PCR分析方法用于水稻白叶枯病抗性基因型的鉴定,证明是植物育种和遗传学研究中一种非常实用的方法。     

Comparison of different labeling methods for the production of labeled target DNA for microarray hybridization

Different labeling methods were studied to compare various approaches to the preparation of labeled target DNA for microarray experiments. The methods under investigation included a post-PCR labeling method using the Klenow fragment and a DecaLabel DNA labeling kit, the use of a Cy3-labeled forward primer in the PCR, generating either double-stranded or single-stranded PCR products, and the incorporation of Cy3-labeled dCTPs in the PCR. A microarray that had already been designed and used for the detection of microorganisms in compost was used in the study. PCR products from the organisms Burkholderia cepacia and Staphylococcus aureus were used in the comparison study, and the signals from the probes for these organisms analyzed. The highest signals were obtained when using the post-PCR labeling method, although with this method, more non-specific hybridizations were found. Single-stranded PCR products that had been labeled by the incorporation of a Cy3-labeled forward primer in the PCR were found to give the next highest signals upon hybridization for a majority of the tested probes, with less non-specific hybridizations. Hybridization with double-stranded PCR product labeled with a Cy3-labeled forward primer, or labeled by the incorporation of Cy3-labeled dCTPs resulted in acceptable signal to noise ratios for all probes except the UNIV 1389a and Burkholderia genus probes, both located toward the 3' end of the 16S rRNA gene. The comparison of the different DNA labeling methods revealed that labeling via the Cy3-forward primer approach is the most appropriate of the studied methods for the preparation of labeled target DNA for our purposes.     

两种DNA探针杂交检测结核分支杆菌方法的研究

为改进结核杆菌DNA探针的特异性与实用性,研制了以生物素标记的两种对结核分支杆菌特异的DNA探针:一个5’端标记的20bp的寡核苷酸探针和一个采用PCR方法合成的188bp长链探针。两种探针分别与结核分支杆菌的全染色体DNA,以及基因组上IS6110序列的一段317bp的PCR扩增产物进行斑点杂交,以碱性磷酸酶(AP)催化的染色反应检测,测试了两个探针的敏感性和特异性。系统地比较研究了两种探针杂交检测条件:探针的浓度选择,杂交温度与洗膜温度的选择,以及杂交与洗膜温度对检测的敏感性与特异性的影响。寡核苷酸探针和188bp探针杂交检测纯化结核分支杆菌基因组DNA的敏感性分别为100ng与6ng,杂交检测PCR产物的敏感性分别是400pg与50pg。两探针的最佳杂交浓度均为40～160ng/ml,最佳杂交温度分别是42℃与68℃,最佳洗膜温度分别是60℃与60～68℃之间。两种探针均仅与结核分支杆菌及BCG有杂交信号,而与其它受试分支杆菌及非分支杆菌杂交结果都呈阴性。它们的特异性都很强,但188bp探针的敏感性约是寡核苷酸探针的7～16倍,而且188bp探针检测本底较低,是检测结核分支杆菌的较佳选择     

Vector PCR.

A strategy employing PCR technology to facilitate the amplification of DNA segments inserted in plasmid vectors is described. Nine oligonucleotide primers specific for vector sequences bracketing cloning sites in seven commonly used vectors were designed. We used these primers for the amplification of 25 different inserts ranging in size from 0.4-4.8 kb. Vector PCR-generated products used as radiolabeled DNA probes in Southern hybridization compared favorably with conventionally prepared probes. This strategy was successfully applied to single colonies of bacteria containing recombinant plasmids for direct amplification of the plasmids insert from the bacterial lysate. Vector PCR enabled the production of microgram quantities of DNA from limited amounts of starting material without the time-consuming steps required for bacterial culture and purification of plasmid DNA. The amplification reaction is independent of the DNA segment to be amplified, rendering the method universally applicable.     

SIMULTANEOUS PCR DETECTION OF BACTERIA AND MOLD DNA SEQUENCES IN PHARMACEUTICAL SAMPLES BY USING A GRADIENT THERMOCYCLER

A gradient thermocycler, the Stratagene RoboCycler 96-Gradient, was evaluated for the simultaneous PCR amplification of microbial genes which indicated the presence of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Aspergillus niger in pharmaceutical samples. Suspensions of pharmaceutical products were inoculated with pure cultures of bacteria and mold. After a 24 h incubation, bacterial DNA was extracted from each enrichment broth using a mild lysis in Tris-EDTA-Tween 20 buffer containing proteinase K while mold DNA was extracted by boiling the samples in Tris-EDTA-SDS buffer for 1 h. A 10 μL aliquots of extracted DNA was added to Ready-To-Go PCR beads and specific primers for E. coli, S. aureus, and P. aeruginosa. However, 50 μL aliquots of extracted mold DNA were used for amplification of specific A. niger DNA sequences. The individual samples were loaded into the RoboCycler 96-Gradient thermocycler. Simultaneous detection of all microbial genes was performed by using a gradient profile that allowed the use of DNA primers with different annealing temperatures. Standard methods for quality control evaluation of pharmaceutical products required 6–8 days while simultaneous PCR detection of bacteria and mold DNA sequences was completed within 27 h.