DNA探针在呼吸机相关性肺炎病原学检测中的诊断价值

目的建立一种早期、快速检测呼吸机相关性肺炎致病菌的分子生物学方法。方法采用聚合酶链反应合成铜绿假单胞菌、甲氧西林耐药金黄色葡萄球菌、大肠杆菌、肺炎克雷伯杆菌和流感嗜血杆菌这5种细菌的特异DNA探针和细菌16SrRNA的通用探针,分别与生物素标记的细菌、病毒和真菌DNA杂交。杂交法和培养法同时检测100份痰液标本。结果所合成的DNA探针具有高度特异性,与其他细菌、病毒、真菌间无交叉反应。该方法可检测出1 ng细菌DNA。杂交法阳性率显著高于培养法。结论所合成的DNA探针敏感性高,特异性强,具有较高的应用价值,可应用于临床标本检测。     

Epidemiology and diagnosis of African trypanosomiasis using DNA probes

The accurate identification of trypanosome species has been a challenging problem in the epidemiology of African trypanosomiasis, both human and animal. The last 10 years have seen great progress through the application of deoxyribonucleic acid (DNA) probe technology, although this has also revealed greater complexity than supposed. While a single repetitive DNA probe can identify all members of the subgenus Trypanosoma (Trypanozoon), including the human pathogens T. brucei gambiense and T. b. rhodesiense as well as the non-tsetse-transmitted trypanosomes T. evansi and T. equiperdum, at least 6 probes are needed to distinguish members of the subgenus Nannomonas, in which only 2 species, T. congolense and T. simiae, were previously recognized. Similarly, the subgenus Duttonella appears to consist of more than one species. Use of this battery of DNA probes to identify the trypanosomes carried by tsetse flies in the field has yielded some surprises about the accuracy (or inaccuracy) of previous identification methods. An unexpectedly high prevalence of mixed infections has been found in all the field studies carried out so far. The large number of infections that remain unidentified by the available probes suggests the existence of other, as yet unknown, trypanosome species. Limited use of the polymerase chain reaction has been made for diagnosis of human and animal trypanosomiasis, due to its high cost.     

Identification of Leishmania from mucosal leishmaniasis by recombinant DNA probes.

Three cases of mucosal leishmaniasis are described. Parasites isolated from mucosal lesions were identified by Southern blot analysis of their genomic deoxyribonucleic acids (DNAs) using recombinant DNA probe pDK20. Parasites from 2 patients were identified as Leishmania donovani s.l. One of the patients had pure mucosal lesions, while in the second patient there was dissemination of the parasite to other organs. The spectrum of the disease caused by L. donovani is discussed. The parasite from the third patient was identified as L. major.     

Phylogenetic and epidemiological analysis of Neisseria meningitidis using DNA probes.

The genetic relationships between various serotypes and serogroups of meningococcal strains were investigated by restriction fragment-length polymorphism (RFLP) analysis using a number of random DNA probes and a probe containing a truncated copy of the meningococcal insertion sequence IS1106. The data were used to estimate genetic distance between all pairs of strains and to construct phylogenetic trees for meningococcal strains. B15:P1.16R strains isolated from cases of systemic meningococcal disease in two health districts with a high incidence of disease were clonal in contrast to similar strains from cases occurring in other parts of the UK. Strains from these areas, which contain a similar genomic deletion, were found to be derived from two distinct lineages within the B15:P1.16R phylogenetic group. RFLP data demonstrated that present serological typing systems for the meningococcus do not necessarily reflect true genetic relationships.     

DNA改组及其在疫苗研制中的应用

DNA改组 (DNAshuffling)是上世纪 90年代中期发展起来的一种全新的分子水平上的人工定向进化技术。它模拟自然界进化的机制 ,改变了传统的进化途径 ,通过体外重组来改造靶基因 ,并定向筛选具有预期性状的突变体 ,从而大大加速了蛋白质的进化进程。该技术自建立以来 ,已在生物工程各领域得到了越来越广泛的应用。本文总结了DNA改组的基本原理、技术路线、特点、改进与发展 ,并综述了其在疫苗研制方面的研究进展。     

Quick blots and nonradioactive detection of DNA probes for the identification of mosquitoes.

The quick blot protocol is an improved technique for preparing crude insect homogenates for hybridization to nucleic acid probes. Individual insects are ground in wells of a microtiter plate and transferred to a dot blot manifold. This allows preparation of multiple filters and provides uniformity and an orderly arrangement of samples. The high background detection resulting from use of crude insect homogenates with nonradioactive detection systems was eliminated by incubating quick blot filters in a laundry stain remover containing proteases. We used mosquito species-specific DNA probes to demonstrate the effectiveness of nonradioactive DNA labeling systems with quick blots.     

Detection of novel trimethoprim resistance determinants in the United Kingdom using biotin-labelled DNA probes

Two collections of trimethoprim R plasmids, isolated from strains of Escherichia coli during 1978-83 and 1987-8 respectively, were retrospectively screened with specific biotinylated DNA probes for the presence of genes encoding particular DHFR enzymes. The results confirmed that the type I DHFR gene was the predominant plasmid-encoded gene conferring trimethoprim resistance in strains of E. coli from the Nottingham area of the UK, but indicated that genes encoding the more recently recognized types of DHFR enzymes had appeared in the bacterial gene pool and could be recognized with increased frequency in the latter plasmid collection. This was particularly true of the type IIIa and type VII enzymes which together accounted for 27% of the trimethoprim R plasmids examined in 1987-8.     

The use of DNA probes for malaria diagnosis: Memorandum from a WHO Meeting

Current progress in the development and potential application of DNA probes for malaria diagnosis was reviewed at an informal WHO Consultation in Geneva in October 1985. The development and use of such probes for malaria diagnosis is based on the premise that within any organism there are unique DNA sequences which differentiate that organism from closely related organisms. DNA probes specific for Plasmodium falciparum have been developed in several laboratories. Their major characteristic is that they are highly repeated within the P. falciparum genome. Their reported sensitivity in laboratory studies of from 5-10 pg to 1 ng DNA, which is equivalent to 10²-10⁴ ring-stage parasites in a single sample, appears to be well within the range of that obtained by standard microscopical diagnosis. The technique, therefore, apparently has potential for operational use. A test based on use of the complete genome of P. falciparum has also been developed, and studies have recently been initiated on the diagnostic application of RNA probes and DNA probes specific for the human malaria parasites.     

Recent applications of DNA microarray technology to toxicology and ecotoxicology

Gene expression is a unique way of characterizing how cells and organisms adapt to changes in the external environment. The measurements of gene expression levels upon exposure to a chemical can be used both to provide information about the mechanism of action of the toxicant and to form a sort of "genetic signature" for the identification of toxic products. The development of high-quality, commercially available gene arrays has allowed this technology to become a standard tool in molecular toxicology. Several national and international initiatives have provided the proof-of-principle tests for the application of gene expression for the study of the toxicity of new and existing chemical compounds. In the last few years the field has progressed from evaluating the potential of the technology to illustrating the practical use of gene expression profiling in toxicology. The application of gene expression profiling to ecotoxicology is at an earlier stage, mainly because of the the many variables involved in analyzing the status of natural populations. Nevertheless, significant studies have been carried out on the response to environmental stressors both in model and in nonmodel organisms. It can be easily predicted that the development of stressor-specific signatures in gene expression profiling in ecotoxicology will have a major impact on the ecotoxicology field in the near future. International collaborations could play an important role in accelerating the application of genomic approaches in ecotoxicology.     

PNA与DNA探针对漏声表面波传感器特异性的影响

目的探讨以PNA作为杂交探针与普通的DNA探针相比,在漏声表面波生物传感器生物杂交反应中的优越性。方法用巯基固定法将PNA、DNA两种探针分别固定在漏声表面波生物传感器的金膜表面,分别与完全匹配、1个及2个碱基错配的靶序列进行杂交反应,观察两种不同的探针与靶序列反应所引起的相位变化及反应平衡时间。结果与DNA探针相比,PNA探针识别碱基错配的能力显著提高,且杂交平衡时间更短。结论 PNA探针可提高漏声表面波生物传感器的检测特异性。     

The importance of transmission electron microscopy analysis of spermatozoa: Diagnostic applications and basic research

This review is aimed at discussing the role of ultrastructural studies on human spermatozoa and evaluating transmission electron microscopy as a diagnostic tool that can complete andrology protocols. It is clear that morphological sperm defects may explain decreased fertilizing potential and acquire particular value in the field of male infertility. Electron microscopy is the best method to identify systematic or monomorphic and non-systematic or polymorphic sperm defects. The systematic defects are characterized by a particular anomaly that affects the vast majority of spermatozoa in a semen sample, whereas a heterogeneous combination of head and tail defects found in variable percentages are typically non-systematic or polymorphic sperm defects. A correct diagnosis of these specific sperm alterations is important for choosing the male infertility’s therapy and for deciding to turn to assisted reproduction techniques. Transmission electron microscopy (TEM) also represents a valuable method to explore the in vitro effects of different compounds (for example drugs with potential spermicidal activity) on the morphology of human spermatozoa. Finally, TEM used in combination with immunohistochemical techniques, integrates structural and functional aspects that provide a wide horizon in the understanding of sperm physiology and pathology.Abbreviations: transmission electron microscopy: TEM; World Health Organization: WHO; light microscopy: LM; motile sperm organelle morphology examination: MSOME; intracytoplasmic morphologically selected sperm injection: IMSI; intracytoplasmic sperm injection: ICSI; dysplasia of fibrous sheath: DFS; primary ciliary dyskinesia: PCD; outer dense fibers: ODF; assisted reproduction technologies: ART; scanning electron microscopy: SEM; polyvinylpirrolidone: PVP; tert-butylhydroperoxide: TBHP     

Differential diagnosis of Taenia saginata and Taenia solium infections: from DNA probes to polymerase chain reaction

The objective of this work was the rapid and easy differential diagnosis of Taenia saginata and T. solium. First, a T. saginata size-selected genomic deoxyribonucleic acid (gDNA) library was constructed in the vector lambda gt10 using the 2-4 kb fraction from the parasite DNA digested with EcoR1, under 'star' conditions. After differential screening of the library and hybridization analysis with DNA from T. saginata, T. solium, T. taeniaeformis, T. crassiceps, and Echinococcus granulosus (bovine, porcine, and human), 2 recombinant phages were selected. They were designated HDP1 and HDP2. HDP1 reacted specifically with T. saginata DNA, and HDP2 recognized DNA from both T. saginata and T. solium. The 2 DNA probes were then sequenced and further characterized. HDP1 was a repetitive sequence with a 53 bp monomeric unit repeated 24 times in direct tandem along the 1272 bp fragment, while the 3954 bp HDP2 was not a repetitive sequence. Using the sequencing data, oligonucleotides were designed and used in a polymerase chain reaction (PCR). The 2 selected oligonucleotides from probe HDP1 (PTs4F1 and PTs4R1) specifically amplified gDNA from T. saginata, but not T. solium or other related cestodes, with a sensitivity of < 10 pg of T. saginata gDNA, about the quantity of DNA in one taeniid egg. The 3 oligonucleotides selected from the HDP2 sequence (PTs7S35F1, PTs7S35F2, and PTs7S35R1) allowed the differential amplification of gDNA from T. saginata, T. solium and E. granulosus in a multiplex PCR, again with a sensitivity of < 10 pg. These diagnostic tools have immediate application in the differential diagnosis of T. solium and T. saginata in humans and in the diagnosis of dubious cysts in the slaughterhouse. We also hope to apply them to epidemiological surveys of, for example, soil and water in endemic areas.     

Diagnostic x rays,DNA repair genes and childhood acute lymphoblastic leukemia

A model for childhood leukemia proposes that characteristic chromosomal translocations can arise in utero and that for most cases a second hit occurring postnatally will be necessary. Possible causal mechanisms for leukemias are environmental factors such as ionizing radiation from x rays and inherited susceptibility from polymorphisms in DNA repair genes. We performed a case-control study of childhood acute lymphoblastic leukemia measuring reported postnatal x rays in 701 cases aged 0-14 y and in as many population-based controls matched on age and sex. In addition we performed a case-only study in 207 cases to evaluate the interaction between x ray exposure and polymorphisms in DNA repair genes. There was an increase in risk of leukemia with number of x rays: the adjusted odds ratio for two or more x rays vs. none was 1.48 (95% confidence interval: 1.11-1.97). That risk was slightly higher among girls (odds ratio = 1.67). A polymorphism in the APE gene (ex 5) involved in the base excision repair system was suggestive of an increased risk among boys and a reduced risk among girls. HMLH1 (ex 8), a mismatch repair gene, was associated with reduction of risk among girls. Results from the genetic data are still preliminary and must be interpreted with caution especially because of the relatively small number of genotyped cases. However, ionizing radiation from x rays as well as polymorphisms in DNA repair genes are plausible risk factors for childhood leukemia and should be studied more.