核酸杂交技术及其在分子病理学研究中的应用

核酸杂交技术最初是在1961年建立的。Hall等利用核酸能变性与复性的原理使核酸探针与靶序列在液相中杂交,然后通过平衡密度梯度离心分离到杂交体建立了核酸杂交技术。随后,于1962年Bautz等将糖苷化DNA吸附在硝化纤维素柱上,Bolton等将变性的单链DNA固定在琼脂中,建立     

几种分子生物学技术在兽医领域中的应用

第四讲核酸的分子杂交技术及其应用１概述核酸的分子杂交（ｍｏｌｅｃｕｌａｒｈｙｂｒｉｄｉｚａｔｉｏｎ）技术是目前生物化学和分子生物学研究中应用最广泛的技术之一,是定性或定量检测特异ＲＮＡ或ＤＮＡ序列片段的有力工具。它是利用核酸分子的碱基互补原则而发展起...     

化学修饰法制备B组轮状病毒生物素核酸探针及应用研究

用亚硫酸氢钠—乙二胺溶液对B组轮状病毒的ssRNA胞嘧啶修饰和转氨基作用，与生物素e—氨基己酸N—羟基琥珀酰亚胺酯反应，制备探针与样品在硝酸纤维膜上进行点杂交，经亲合素—碱性磷酸酶系统显色，可测出106.25－212.5pg的RNA靶序列，特异性试验表明：B组生物素探针只能与B组核酸杂交，而与无关核酸无杂交信号，用B组探针检测45份仔猪腹泻粪样，检出B组阳性5份，检出率11．1％，明显高于PAGE法，实验结果表明，生物素核酸探针制备简单并且有特异、灵敏、稳定的优点，可用于各组轮状病毒的检测。     

鹅细小病毒VP1-VP3非重叠序列地高辛探针的制备和应用

根据GPV H1株核苷酸序列，设计了扩增VP1-VP3基因非重叠序列的1对引物，对其结构蛋白VP1与VP3非重叠核苷酸序列进行PCR扩增，将PCR产物纯化、回收后制备出GPV VP1-VP3基因DIG标记核酸探针，其标记效率达到0．1pg／μl。特异性检测结果表明，该探针能与GPV不同毒株核酸发生特异性杂交，而与对照的DPV、GPMV等病毒的核酸杂交反应均为阴性；敏感性检测结果表明该探针对GPV的最低检出量为0．032ng。上述试验结果表明该探针可以用于GPV感染临床病料的检测。     

核酸适体在乳及乳制品检测上的运用分析

我国乳制品行业发展迅速,检测需求逐渐增加。随着食品安全相关法律法规完善和监管力度加强,传统乳制品检测方法存在诸多不足。核酸适体在食品和医疗领域应用广泛,具有良好生物相容性、低免疫原性、高稳定性和高特异性等特点,有利于检测体系构建。核酸适体是一种以指数形式存在的单链DNA或RNA分子。利用适配体的特异性识别能力,可通过双链DNA或RNA杂交得到荧光信号,用于检测蛋白质及各种酶等生物材料。与传统检测手段相比,核酸适体具有可设计性强、合成简便快速、成本较低等优势。     

用核酸探针检测新城疫病毒的研究

用光敏生物素标记鸡新城疫ｃＤＮＡ制备核酸探针，经斑点杂交和碱性磷酸酶显色后，探针同该ｃＤＮＡ的ＰＣＲ产物，ＰＣＲ产物重组子和新城疫强，弱毒株呈现阳性反应，与ＩＢＶ，ＩＬＴ，ＭＧ和正常尿囊液等均呈阴性杂交反应，田间病料的杂交试验也表明该ｃＤＮＡ探针是且特异的检测方法。     

DNA—RNA杂交法检测新城疫病毒的研究

用光敏生物素标记鸡新城疫ｃＤＮＡ，制备核酸探针，经斑点杂交和碱性磷酸酶显色后，探针同该ｃＤＮＡ的ＰＣＲ产物、ＰＣＲ产物重组子和新城疫强弱毒株呈现阳性反应，与ＩＢＶ、ＩＬＴ、ＭＧ和正常尿囊液等均呈阴性杂交反应，田间病料和杂交试验也表明该ｃＤＮＡ探针是敏感且特异的检测方法。     

用核酸探针检测新城疫病毒的研究

用光敏生物素标记鸡新城疫ｃＤＮＡ，制备核酸探针，经斑点杂交和碱性磷酸酶显色后，探针同该ｃＤＮＡ的ＰＣＲ产物、ＰＣＲ产物重组子和新城疫强、弱毒株呈现阳性反应，与ＩＢＶ、ＩＬＴ、ＭＧ和正常尿囊液等均呈阴性杂交反应，田间病料的杂交试验也表明该ｃＤＮＡ探针是敏感且特异的检测方法     

核酸检测技术在水产动物病毒感染诊断中的应用

目前,应用于水产动物病毒感染诊断中的核酸检测技术主要包括核酸杂交、聚合酶链反应、聚舍酶链反应与分子杂交联用、聚合酶链反应与免疫学联用、限制性酶切技术、环介导的等温扩增、基因芯片及测序等技术。这些技术发展迅速,并在原有基础上根据实际的需求产生出很多相应的衍生技术。它们已被广泛应用在水产动物病毒感染诊断中并发挥着巨大的作用。核酸检测技术以其无法比拟的优点得到了快速的推广和应用,有着广阔的发展空间。     

核酸探针技术及其在大肠杆菌诊断中的应用

核酸探针又称基因探针,是随着分子生物学及分子遗传学的深入发展而产生的一种新的检测技术。与常规的实验室检测方法相比,核酸探针技术具有高度特异、敏感、快速、简便等优点。近几年,基于探针只能和靶细菌等的 DNA 杂交,而不与其它 DNA 杂交的原理,已将探针技术作为一种有效的检测手段,应用于细菌、病毒、疟原虫、钩端螺旋体等的鉴定和分类,并收到一定效果。     

Probe seeks gene: nucleic acid hybridization as a current contribution to virologic diagnosis

Molecular nucleic acid hybridization is based on the ability of single-stranded DNA/RNA to form hybrids with complementary labeled nucleic acids. This review shortly describes the components of this technique and presents the most important hybridization methods (spot-, Southern blot-, in situ-hybridization). The (potential) applications of nucleic acid hybridization as a diagnostic tool are discussed (e.g., for viruses which grow insufficiently or not at all in cell culture; virus latency; viruses with labile or antigenically variable envelope proteins, resp.; for virus classification) and yet existing limitations are indicated. An impetus for this technique in means of diagnostic application is expected to result from the Polymerase Chain Reaction (PCR) in the next future.     

In situ hybridization for the detection of transmissible gastroenteritis virus in pigs and comparison with other methods.

Archived formalin-fixed, paraffin-embedded tissues from 25 pigs naturally infected with transmissible gastroenteritis virus (TGEV) were examined by in situ hybridization for TGEV nucleic acid using a nonradioactive digoxigenin-labeled cDNA probe that targeted the nucleocapsid sequence of TGEV strains. The results of in situ hybridization for the detection of TGEV were compared with virus isolation (VI), a fluorescent antibody test (FAT), and transmission electron microscopy (TEM). VI, FAT, and TEM were tested over a course of time before the in situ hybridization was performed. Positive hybridization signals were detected in duodenal, jejunal, and ileal enterocytes from 21 pigs. Hybridization signals were confined to the cytoplasm. Intestinal specimens from 25 piglets were evaluated by 4 tests. Twenty-one of 25 were positive by in situ hybridization. Of these 21 samples, 5 (24%) were positive for TGEV by all 4 tests, 15 (71%) were positive by FAT, 14 (67%) were positive by VI, and 6 (29%) were positive by TEM. In situ hybridization for the detection of TGEV in formalin-fixed, paraffin-embedded tissues provides a rapid means of confirmation of a histopathological diagnosis of TGEV without virus isolation, or when only formalin-fixed intestinal specimens were available.     

In situ hybridization for the detection and localization of swine Chlamydia trachomatis

Gnotobiotic piglets were inoculated intralaryngeally with swine Chlamydia trachomatis strain R33 or orally with swine C. trachmatis strain R27. Archived formalin-fixed, paraffin-embedded tissues from piglets euthanatized 4-7 days postinoculation were examined by in situ hybridization for C. trachomatis nucleic acid using a nonradioactive digoxigenin-labeled DNA probes that targeted specific ribosomal RNA or omp1 mRNA molecules of the swine C. trachomatis strains. Positive hybridization signals were detected in bronchial epithelial cells, bronchiolar epithelial cells, pneumocytes, alveolar and interstitial macrophages, and jejunal and ileal enterocytes. Chlamydia-infected cells had a strong signal that was confined to the intracytoplasmic inclusions. Positive hybridization signals were not detected in tissue sections from an uninfected control piglet or in C. psittaci-infected sheep placenta. The morphology of host cells was preserved despite the relatively high temperature required in parts of the incubation procedure. The data indicate that in situ hybridization can be used to detect swine C. trachomatis in formalin-fixed, paraffin-embedded tissue specimens.     

Detection of bovine viral diarrhea virus by spot hybridization with probes prepared from cloned cDNA sequences

A cytopathic strain of bovine viral diarrhea virus (BVDV) was purified from infected cell culture fluids by isopycnic density-gradient centrifugation. Genomic RNA was extracted and tailed with adenine residues at the 3' end with poly-A polymerase. Double-stranded complementary DNA (cDNA) was synthesized, using the poly-A-tailed RNA as a template and oligo-dT as a primer, and then cloned into the pUC9 plasmid. Virus-specific cDNA sequences, varying in length from 0.5 to 2.5 kilobases (kb), were obtained. One BVDV-specific sequence of cloned cDNA, 1.1 kb in length and with an internal Pst I restriction endonuclease cleavage site, was selected for use as a probe. The cloned cDNA insert was removed from the plasmid either with or without flanking plasmid sequences and labeled with 32P-nucleotides by nick translation for use as hybridization probes for BVDV. The performance of probes of smaller fragments of the insert was compared to that of the intact sequence in hybridization assays. In addition, 2 methods of specimen preparation were compared to establish optimum parameters for hybridization. The hybridization assay was 10-100 times more sensitive than infectivity assays for BVDV in infected cell cultures. Freezing of specimens reduced by 10-fold the sensitivity of hybridization for BVDV target sequences. The probes prepared from the cloned cDNA hybridized with all cytopathic and noncytopathic BVDV strains tested but not with uninfected cell cultures, cellular ribosomal RNA, bovine coronavirus, bluetongue virus, or bovine adenovirus 3. Probes prepared with native plasmid DNA did not hybridize with BVDV or uninfected cell cultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

In situ hybridization for the detection of the apxIV gene in the lungs of pigs experimentally infected with twelve Actinobacillus pleuropneumoniae serotypes

The detection of the apxlV gene in lung tissues from pigs experimentally infected with the 12 major A. pleuropneumoniae serotype (1 to 12) reference strains was studied by in situ hybridization using a non-radioactive digoxigenin-labeled DNA probe. In situ hybridization produced a distinct positive signal in all pigs inoculated with the 12 A. pleuropneumoniae serotypes. Positive hybridization typically exhibited a dark-brown to black reaction product in intracellular and extracellular locations, without background staining. A strong hybridization signal was seen in degenerated alveolar leukocytes ("oat cells") adjacent to the foci of coagulative necrosis and in the alveolar spaces. The in situ hybridization methodology developed for the detection of the apxIV gene is a valuable tool for the diagnosis of porcine pleuropneumonia caused by A. pleuropneumoniae when only formalin-fixed tissues are submitted for diagnosis.     

The landscape of North American Rangeland Social Science: A Systematic Map

Rangeland scientists have made substantial progress in understanding ecological dynamics of rangelands, but the social factors have received less attention in North America. A body of North American rangeland social science has developed over the past 4 decades, with the number of studies increasing each decade. However, these works have not been systematically reviewed to assess the state of rangeland social science in North America or to identify research gaps. We developed a systematic map to characterize this literature by 1) the research objectives and questions; 2) who was studied; 3) where research was conducted; 4) which theories, methodologies, and methods were applied; and 5) how these research characteristics have changed from 1970 to 2017. We found that most (81%) North American rangeland social science has studied ranchers, farmers, and/or landowners, with limited consideration of other stakeholders (e.g., ranch workers, youth). Although age (43% of the studies) and education (40%) are often considered, other attributes/identities, such as gender (28%) and race or ethnicity (18%), are less frequently included. The most commonly used research method is surveys (52%), and much of rangeland social science does not make explicit connections to either specific methodological or theoretical frameworks. The limited application of theories, methodologies, and a lack of diverse methods has potentially constrained who and what have been studied in North America. The limited consideration of gender and race in rangeland social science is echoed in the limited number of studies that have accounted for the effects of social identities and power relationships on people’s connection to and management of rangelands. This review highlights the need for more North American research that 1) is informed by social theory, 2) applies a diversity of methods, 3) considers a broader diversity of stakeholders, and 4) draws from multiple social science disciplinary traditions.     

Detection and localization of ApxI, -II and -III genes of Actinobacillus pleuropneumoniae in natural porcine pleuropneumonia in natural porcine pleuropneumonia by in situ hybridization

In situ hybridization techniques that employed a nonradioactive digoxigenin-labeled probe were used to detect and localize ApxI, II and III genes in tissue sections of pneumonic lung naturally infected with Actinobacillus pleuropneumoniae. In pigs infected with either serotype 2 or 6, a hybridization signal for apxIICA, apxIIICA, apxIBD, and apxIIIBD was detected, and in pigs infected with serotype 5, a hybridization signal for apxICA, apxIICA, and apxIBD was detected in the pneumonic lesions. A hybridization signal for apxIICA and apxIBD was detected in pigs infected with serotype 7. A strong hybridization signal for apx genes was seen in streaming degenerate alveolar leukocytes bordering zones of coagulative necrosis. Simultaneous detection of hybridization signals for the apxCA and apxBD genes provided scientific evidence that the expression of the apx genes could be potential indicators of the production of corresponding Apx toxins. This study demonstrates the expression of ApxI, II, and III genes in pneumonic lesions caused by A. pleuropneumoniae.     

Contributions of Citizen Scientists and Habitat Volunteers to Monarch Butterfly Conservation

Volunteers can contribute to wildlife conservation by protecting and restoring habitat, or by collecting citizen science data. Much remains unknown about how citizen scientists contribute to conservation beyond data collection, or the extent that volunteering with citizen science or habitat conservation is associated with increased participation in other forms of conservation. We surveyed citizen science and habitat volunteers from monarch butterfly (Danaus plexippus) programs. Both types of volunteers conducted conservation outreach and created and managed monarch habitat. Habitat volunteers were more likely to create new habitat for monarchs in urban or suburban areas, whereas citizen scientists were more likely to maintain existing habitat in rural areas. Most volunteers increased their participation in conservation after joining a formal monarch project. Our results provide information about the capacity for habitat volunteers to engage in conservation, as well as evidence of an unexplored benefit of citizen science, the creation and protection of habitat.     

Comparison between two in situ methods for Mycobacterium avium subsp. paratuberculosis detection in tissue samples from infected cattle

Paratuberculosis or Johne's disease is a chronic infectious disorder caused by Mycobacterium avium subsp. paratuberculosis (Map). The disease produces diarrhea and weight loss in cattle and other animal species, and it is characterized by granulomatous enteritis and lymphadenitis. Histopathology and in situ techniques can be used as a diagnostic test, but the performance of these methods was not previously compared. The aim of this paper was to evaluate the ability of immunohistochemistry and in situ hybridization to detect Map in formalin-fixed tissue samples from infected cattle. Samples (ileum or ileocecal lymph node) from four animals that had positive Map culturing, lesions and detectable acid fast bacilli, as well as from two control animals, were tested by immunohistochemistry and in situ hybridization. Immunostaining and positive hybridization were observed in areas with lesions from infected animal samples, inside the cytoplasm of macrophages, epithelioid and giant cells. Immunostaining was intense in three samples and weak in one, while hybridization was weak in all cases. In situ hybridization was positive in negative areas of tissues analyzed by immunohistochemistry, which could be related to spheroplast detection as it was previously described for this method. Control samples resulted negative by these two methods. Both techniques were able to detect Map in formalin fixed and paraffin embedded tissues, however immunohistochemistry produced higher intensity staining and was easier to perform. Therefore, we believe that immunohistochemistry and in situ hybridization to be useful for the post-mortem diagnosis and research of Paratuberculosis.     

Comparison of DNA-spot hybridization, cell culture and direct immunofluorescence staining for the diagnosis of avian chlamydiae

DNA-spot hybridization, cell culture and direct immunofluorescence staining were compared for the detection of avian Chlamydia psittaci strains in cell culture dilutions and in routine samples submitted for diagnosis. With dilutions of infected cell culture material, growth in BGM cells was by far the most sensitive technique, detecting 0.01 infected cells (20 elementary bodies) ml-1. DNA-spot hybridization and direct immunofluorescence staining were of approximately equal sensitivity, both detecting 16 infected cells (3.2 x 10(4) elementary bodies) per ml-1. When 27 avian liver and spleen samples were assayed, all 3 tests performed similarly (13 positive and 12 negative by all 3 tests). This suggests that in most avian samples presented for diagnosis, sufficient numbers of chlamydiae are present to allow any of the test to the be used. Thus, the direct immunofluorescence staining method is currently the test of choice for routine diagnosis since it is available in kit form, is relatively simple and quick to perform, and like DNA-spot hybridization, detects non-viable as well as viable organisms. However, if low levels of chlamydiae are to be effectively detected, such as in carrier birds or birds with recently acquired infections, then cell culture should be used.