Sensitive detection of mycoplasmalike organisms in field-collected and in vitro propagated plants of Brassica, Hydrangea and Chrysanthemum by polymerase chain reaction

A polymerase chain reaction (PCR) protocol, previously designed for amplification of a DNA fragment from aster yellows mycoplasmalike organism (MLO), was employed to investigate the detection of MLO DNA in field-collected and in vitro micropropagated plants. PCR with template DNA extracted from symptomatic, naturally-infected samples of Brassica, Chrysanthemum and Hydrangea, each yielded a DNA band corresponding to 1.0 Kbp. However, no DNA product was observed when either infected Ranunculus (with phyllody disease) or Gladiolus with (symptoms of ‘germs fins’) was used as source of template nucleic acid for PCR; further experiments indicated absence of target DNA in the case of Ranunculus and the presence of substances in Gladiolus which inhibited the PCR. The MLO-specific DNA was detected by PCR using less than 95 pg of total nucleic acid (equivalent to total nucleic acid from 1.9, ug tissue) in the case of field-collected Hydrangea and less than 11.4 pg of nucleic acid (equivalent to total nucleic acid from 19 ng of tissue) in the case of field-collected Brassica. The findings illustrate highly sensitive detection of MLOs in both field-grown and in vitro micropropagated infected plants.     

印度尼西亚输华植物产品截获的有害生物情况分析

【目的】印度尼西亚与我国有着多元素的农产品贸易往来,通过统计印度尼西亚输华植物及植物产品所携带的有害生物,分析其对我国农产品进出口的影响,能为应对印度尼西亚技术性贸易措施提供思路,并为我国农产品企业"走出去"提供参考。【方法】经动植物检验检疫信息资源共享服务平台查询,统计印度尼西亚输华植物及植物产品所携带的有害生物类别、截获途径及截获地区。【结果】我国在印度尼西亚输华植物及植物产品中检出有害生物共计2512种96958批,鉴定至种且检出超过1000批次的有害生物共计14种,检疫性有害生物共计72种5286批。昆虫类有害生物占比达78.5%,检疫性昆虫达65.2%。【结论】在首要做好木材类有害生物检疫的同时,也需警惕一些非检疫性的仓储害虫,保持并加强南方各省份港口的有害生物查验力度,加强印度尼西亚输华货物的检疫。     

Amplification of DNA bound on clay minerals

DNA adsorbs and binds on clay minerals, which provides protection to the DNA against degradation by nucleases but does not eliminate the ability of bound DNA to transform cells. These observations support the concept that 'cryptic genes' can persist in the environment when bound on particles and that the genes could subsequently be expressed if an appropriate host was transformed. The polymerase chain reaction (PCR) was used to amplify free and bound DNA from Bacillus subtilis and calf thymus. DNA bound on montmorillonite, but not on kaolinite, was amplified. However, amplification occurred when kaolinite was pretreated with sodium metaphosphate. DNA was not released from the clays during the amplification procedure. The type of clay (e.g. its structure and charges) affected amplification. Because DNA bound on clay is protected against biodegradation, the ability to amplify DNA bound on clay by the PCR has palaeontological, archaeological, and anthropological implications for the detection of 'ancient' DNA, as well as for monitoring the persistence of recombinant DNA introduced to the environment in genetically modified organisms.     

应用PCR技术检测病人血液标本中斑点热群立克次体DNA

本文以聚合酶链反应（PCR）,采用二次扩增法直接检测蜱传斑点热病人血液标本中斑点热群立克次体DNA,结果从7份标本中检出阳性1份,进一步的限制性片段多态性分析证明和斑点热群黑龙江立克次体基因型相同。实验证明：黑龙江立克次体对人具有致病性,同时也证明PCR技术快速、简单、敏感,用于斑点热群立克次体的早期诊断是可行的,并可作出分型鉴定。     

Amplification and analysis of human DNA present in mosquito bloodmeals

Abstract. DNA fingerprinting should permit the identification of individual human hosts of haematophagous arthropods, providing epidemi-ologically useful information, for example, the biting rates on different people and the impact of insecticide-impregnated bednets.
Investigations reported here demonstrate that it is possible to extract, amplify and fingerprint human DNA from the bloodmeals of individual female Anopheles gambiae mosquitoes kept at 24^oC for up to 10–15 h post-ingestion.     

DNA分析技术及其在植物研究中的应用

从DNA/DNA杂交、RFLP分析、DNA的限制酶图谱和核苷酸序列分析、PCR技术、DNA指纹技术、RAPD分析等六个方面详细描述DNA分析技术在植物学研究中的应用 ,并讨论了DNA分析技术与植物系统学的关系。     

Proboscidean DNA from Museum and Fossil Specimens: An Assessment of Ancient DNA Extraction and Amplification Techniques

Applications of reliable DNA extraction and amplification techniques to postmortem samples are critical to ancient DNA research. Commonly used methods for isolating DNA from ancient material were tested and compared using both soft tissue and bones from fossil and contemporary museum proboscideans. DNAs isolated using three principal methods served as templates in subsequent PCR amplifications, and the PCR products were directly sequenced. Authentication of the ancient origin of obtained nucleotide sequences was established by demonstrating reproducibility under a blind testing system and by phylogenetic analysis. Our results indicate that ancient samples may respond differently to extraction buffers or purification procedures, and no single method was universally successful. A CTAB buffer method, modified from plant DNA extraction protocols, was found to have the highest success rate. Nested PCR was shown to be a reliable approach to amplify ancient DNA templates that failed in primary amplification.     

Amplification efficiency of thermostable DNA polymerases

The amplification efficiencies of several polymerase chain reaction (PCR) enzymes were compared using real-time quantitative PCR with SYBR Green I detection. Amplification data collected during the exponential phase of PCR are highly reproducible, and PCR enzyme performance comparisons based upon efficiency measurements are considerably more accurate than those based on endpoint analysis. DNA polymerase efficiencies were determined under identical conditions using five different amplicon templates that varied in length or percentage GC content. Pfu- and Taq-based formulations showed similar efficiencies when amplifying shorter targets (<900 bp) with 45 to 56% GC content. However, when amplicon length or GC content was increased, Pfu formulations with dUTPase exhibited significantly higher efficiencies than Taq, Pfu, and other archaeal DNA polymerases. We discuss the implications of these results.     

Amplification of Nucleic Acids by Polymerase Chain Reaction (PCR) and Other Methods and their Applications

Abstract

The in vitro replication of DNA, principally using the polymerase chain reaction (PCR), permits the amplification of defined sequences of DNA. By exponentially amplifying a target sequence, PCR significantly enhances the probability of detecting target gene sequences in complex mixtures of DNA. It also facilitates the cloning and sequencing of genes. Amplification of DNA by PCR and other newly developed methods has been applied in many areas of biological research, including molecular biology, biotechnology, and medicine, permitting studies that were not possible before. Nucleic acid amplification has added a new and revolutionary dimension to molecular biology. This review examines PCR and other in vitro nucleic acid amplification methodologies—examining the critical parameters and variations and their widespread applications—giving the strengths and limitations of these methodologies.     

Stable marker on flagellin gene sequences related to arabinose non-assimilating pathogenic Burkholderia pseudomallei

Using PCR-based isolation and sequence analysis of the flagellin gene from two distinct biotypes of Burkholderia pseudomallei, a 15-bp deletion was found within the variable domain of the gene in isolates capable of assimilating arabinose (Ara+). This finding led to the development of a PCR-based method in order to differentiate and identify pathogenic B. pseudomallei for epidemiological study. A pair of specific primers was designed covering the 15-bp deletion region at the variable domain. PCR-amplification products of 176 and 191 bp in size were detected from 41 Ara+ isolates and 39 Ara - isolates of B. pseudomallei, respectively. Moreover, flagellin gene fragments of other bacterial species tested in this study were not amplified using these primers. The results suggest that the flagellin gene sequences of both B. pseudomallei biotypes in this region are stable and distinct. This method can be applied and useful for the epidemiological study of B. pseudomallei.     

人头发DNA的提取及其基因扩增

头发是犯罪现场最容易得到的材料之一。本文报道从人的头发中提取DNA的方法以及利用PCR(聚合酶链反应)技术,从少量人发DNA扩增大量拷贝数的基因片段。对基因扩增得到的大量基因片段进行进一步的基因分析,将为法医物证学提供客观证据,具有重要价值。     

Identification of Single Meloidogyne Juveniles by Polymerase Chain Reaction Amplification of Mitochondrial DNA

Polymerase chain reaction (PCR) was used to amplify a specific 1.8-kb sequence of mitochondrial DNA from single juveniles and eggs from 17 populations of Meloidogyne incognita, M. hapla, M. javanica, and M. arenaria. Approximately 2 μg amplified product were produced per reaction. Restriction digestion of the amplified product with HinfI permitted discrimination of clonal lineages of the four species. Meloidogyne javanica, however, could not be separated from M. hapla by the enzymes used in these experiments. Various amplification conditions and nematode lysis procedures were examined in order to optimize the speed and quality of identifications.     

Expression of the linear DNA plasmid pRS64 in the plant pathogenic fungus Rhizoctonia solani

The plant pathogenic isolate RI-64 of anastomosis group 4 of Rhizoctonia solani possesses three linear DNA plasmids (pRS64-1, -2, and -3). Unique poly(A)^– RNA, 0.5 kb in length and hybridizable with the pRS64 DNAs was found in mycelial cells of the isolate RI-64. The overall homology at the nucleotide level between pRS64-1, -2, and -3, and the cDNA prepared from the poly(A)^– RNA was 100%, 73%, and 84%, respectively. The open reading frames found in pRS64-1, -2, and -3 (ORF1-1, ORF2-1, and ORF3-1) are 68 amino acids long. The amino acids sequence showed no significant homology with known proteins. Extracts from Escherichia coli cells expressing ORF1-1 contain a specific protein of 7 kDa. Antisera raised against the ORF1-1 product obtained from E. coli cells cross-reacted with the specific proteins found in the mycelia. The results indicate that the DNA plasmids found in R. solani contain a sequence that encodes a specific protein which may be involved in determination of plant pathogenicity.     

用DREAM技术进行全长质粒快速定点突变

利用“设计限制酶辅助突变”(Designed Restriction Enzyme Assisted Mutagenesis, DREAM)进行全长质粒快速定点突变。根据突变位点附近氨基酸靶序列, 以简并密码子进行逆向推导, 这样在不改变氨基酸序列的前提下可以得到数目巨大的隐性突变体(Silent mutants), 这些突变体中包含大量的限制性酶切位点, 选择合适的酶切位点设计引物, 用Phusion超保真DNA聚合酶扩增全长质粒的DNA序列, 得到的PCR产物用T4多聚核苷酸激酶添加5￠磷酸基团后进行平末端连接, 转化大肠杆菌受体菌后用设计的酶切位点进行快速筛选。本研究用该方法成功地纠正了长约8 kb的质粒pcDNA3.1-pIgR中的突变碱基, 从而获得了多聚免疫球蛋白受体(pIgR)的野生型氨基酸序列。以上结果表明: 利用DREAM技术将限制性酶切位点引入目的基因而不改变目的蛋白质的氨基酸序列, 使突变体的筛选简单化; 配合使用高保真和高效率的Phusion DNA聚合酶可以进行长达8 kb的全长质粒的快速突变; 该方法无需使用定点突变试剂盒和特殊的受体菌, 同时避免了核酸杂交以及同位素的使用。     

Detection of pathogenic Entamoeba histolytica DNA in liver abscess fluid by polymerase chain reaction.

, , and 1992. Detection of pathogenic Entamoeba histolytica DNA in liver abscess fluid by polymerase chain reaction. International Journal for Parasitology 22: 1193–1196. A sensitive method for detection of pathogenic Entamoeba histolytica DNA in drained fluids from liver abscess patients, using the polymerase chain reaction (PCR), has been developed. The PCR employs oligonucleotide primers specific for the gene encoding the 30 kDa molecule of pathogenic E. histolytica. Liver abscess fluids (19 samples), from 14 patients with a presumptive amebic liver abscess, were examined microscopically and by the PCR method. Only two of the 19 samples were positive microscopically, whereas all 19 samples tested positive by PCR. This technique can be used to confirm the diagnosis of amebic liver abscess.