荧光定量PCR技术及其在动物传染病定量检测中的应用

荧光定量PCR是近年发展起来的一 种新的实时定量检测特定核酸技术,它是核 酸探针技术、荧光共振能量传递技术和PCR 技术的有机结合,而荧光探针是荧光定量 PCR的核心。目前报道的荧光探针主要有 TaqMan、Amplisensor、分子信标、Lightcycler 以及在这4种探针基础上发展起来的其他荧 光探针。各种荧光探针在定量PCR中的作 用是识别和报告特定核酸。这些荧光探针与 相应的靶分子杂交时经历一个自发荧光形成 或消失的构象变化,只有与完全互补的靶核 酸杂交时才会出现这种变化,当靶核酸存在 碱基错配或缺失时,荧光探针就不会与之杂 交,也不会出现荧光的变化。在检测时根据 这种荧光变化来确定检测中特定核酸的存在 和量的多少。荧光探针用于定量PCR不但 提高了PCR的检测灵敏度,还能在同一封闭 管中对模板进行准确定量检测,特别适合特 定核酸扩增的实时监测。荧光定量PCR用 于动物传染病的诊断,不但能定量检测病原 体感染的强弱和在机体内的分布,还具有灵 敏、快速、省力的特点。     

Accelerated digestion of nucleic acids by pepsin from the stomach of chicken

1. Nucleic acids have become an important nutritional supplement in poultry feed; however, the digestion of nucleic acids in poultry is unclear. The objective of this study was to investigate the digestion of nucleic acids by chicken pepsin in vitro.

2. The extracted pepsinogen from the stomach of the chicken was purified to homogeneity. Upon activation at pH 2.0, chicken pepsinogen was converted to its active form.

3. Nucleic acids, including λ-DNA, salmon sperm DNA and single-strand DNA (ssDNA), can be used as substrates and digested into short-chain oligonucleotides by pepsin.

4. Interestingly, the digestion of the nucleic acids was inhibited when pepsin was treated by alkaline solution (pH 8.0) or pepstatin A. Also, the digestion of the nucleic acids was not affected by the addition of haemoglobin or bovine serum albumin.

5. The results suggested that nucleic acids could be digested by chicken pepsin. Thus pepsin may have a role in digesting nucleic acids in vivo. Nucleic acids added to poultry fed may be digested, starting from the stomach.

     

General aspects of transmissible spongiform encephalopathies and hypotheses about the agents

Summary

This article reviews the shared characteristics of the group of transmissible spongiform encephalopathies (SEs), both human and animal, and the major theories regarding the nature of the agents involved.

All transmissible SE diseases share two striking characteristics: the degenerative changes including vacuolation in the central nervous system, and the assumption that these disorders are caused by unconventional, transmissible agents. This article examines the major hypotheses that have been postulated about these agents: the virus theory, the virino theory, the prion theory, and the recently proposed ‘unified theory’. Both the virus and the virino hypotheses assume that a small nucleic acid is involved as part of the agent, while the prion hypothesis does not.

The prion model obviates the need for a role of a nucleic acid in the propagation and replication of the agent, but does not explain the existence of strain variation. Nucleic acids in a micro‐organism, as proposed in the virino and the virus hypotheses, could explain this variation. However, to date, no disease‐specific nucleic acids have been identified. The ‘unified’ theory tries to reconcile the essentials of the virino and prion theories.

The article also describes the discovery of the so‐called prion protein (PrP), its isoforms, and the coding host gene, the PrP gene. It goes on to discuss the results of experiments with transgenic animals, indicating that mutations in the PrP gene may play a decisive role in the pathogenesis of at least some SEs. Finally, two different models, both involving the conversion of normal PrP^C into PrP^Sc as part of the pathogenesis of SE, are discussed.     

基于CRISPR/Cas系统的现场核酸检测技术

核酸检测技术在病原鉴定中发挥了至关重要的作用.传统核酸检测主要依赖聚合酶链式反应(polymerase chain reaction,PCR)技术,但该技术难以满足便携式现场核酸检测需求.近年来发明的CRISPR/Cas生物传感技术可实现高灵敏度、高特异性、快速准确的现场核酸检测.本文重点探讨了CRISPR/Cas生物...     

The role of serum and biliary antibodies and cell-mediated immunity in the clearance of S. typhimurium from chickens

The development of three parameters of immunity in response to a non-lethal infection of $\text{Salmonella typhimurium}$ in adult chickens has been examined. Intravenous inoculation of 1 × 10⁶ organisms established infection in the liver, spleen and intestinal tract of all birds; the organism persisted in these sites until day 9 of the infection, after which it was cleared rapidly from all sites. High levels of agglutinating and haemagglutinating antibodies were found in serum and bile 5 days after infection; they peaked at days 7 to 10, and detectable antibody was still present in both fluids 6 weeks after infection. The presence of this antibody did not appear to cause a significant reduction in organism numbers in any of the sites examined. Cell mediated immunity was detected at day 14. It is suggested that cell mediated immunity is responsible for clearance of the organisms from the tissues.     

新疆伊犁州首起牛结节性皮肤病疫情的诊断及处置

2019年8月,在新疆伊犁州察布查尔县、伊宁市、霍城县的牛群中发现牛皮肤上长满大小不等的肿胀结节,伴有体温升高等临床症状,并有1头牛死亡。随后专家赶赴现场开展临床诊断和流行病学调查。根据病牛临床症状和剖检变化,初步判定为牛结节性皮肤病(LSD);采集发病牛样品,包括各种皮肤结节和血清,并采集肺脏、脾脏、淋巴结等脏器,送至省级实验室开展病原学、血清学检测,同时送至国家外来动物疫病研究中心确诊。省级实验室荧光PCR检测显示,发病牛样品为LSD病毒核酸阳性;ELISA检测显示,有16份牛血清LSD病毒抗体阳性。经国家外来动物疫病研究中心检测,送检的疑似病料确诊为LSD病毒核酸阳性,这是我国首起LSD疫情。通过做好疫源追踪、消毒灭源、疫情解封风险评估,同时在新疆伊犁州使用山羊痘疫苗紧急免疫防治LSD,后期持续临床监测显示没有新的疫情发生。     

The effect of exogenous purine supply on the endogenous excretion of purine derivatives in the urine of growing lambs

An experiment was made to determine the absorption of purine metabolites in dietary nucleic acids through the digestive tract, and also to determine the utilization of nucleic acids absorbed in the body, using growing lambs. Two pairs of 120‐ and 180‐day‐old twin female lambs with a bodyweight of 18.2–19.0 kg were kept in metabolism crates and fed on purine‐free milk replacement (MR) with supplements of exogenous purine (purine base or purine nucleoside) at a level of 0.2 mmol/BW^0.75/d for 5 consecutive days, and thereafter they were maintained in the crates for 4 days. The daily amount of exogenous purine supply was calculated based on the urinary excreted purine derivatives (PD) in lambs fed on milk replacement alone. A urine sample was collected daily for 9 consecutive days, and the urinary excretion of PD was determined daily. Urinary PD excretion opened to increase within 24 h after the dose of purine bases, and the level was recovered on 3 days after ceasing the exogenous purine supply. The recovery of PD in the urine was about 70% of the purine supplement. When purine nucleosides were added to the feed, urinary PD excretion was initiated within 24 h after dosing, and the values were recovered after ending the purine nucleoside supply. The recovery rate of PD in the urine was only 30% of the supplemented purine. The plasma allantoin levels were almost similar after feeding purine bases and purine nucleosides, and the values were mostly in the range (40–60 µmol/L). These findings indicate that an exogenous purine can be directly incorporated into the body, and the purine as nucleoside is more effectively utilized for the synthesis of nucleic acids than as a purine base in the body of growing lambs.     

Die Rolle des Lymphsystems sowohl bei der Abwehr als audi bei der Ernährung

The role of the lymphatic system in the defense as well as in the nutrition of the body The subepithelial lymphatic tissue of the digestive tract taken as a whole amounts to six and one half times that of all the other lymphatic tissues of the body combined. And in the subepithelial lymphatic tissue of the digestive tract there are three times a many lymphocytes as in the circulating blood. This concentration of lymphatic tissue in the digestive tract suggests participation in the animal's metabolism. Despite reutilization (up to 80%) of its own nucleic acids and macromolecular protein substances, the mammalian body has to extract the remaining 20% from the environment This phylogenetically increasing demand for outside substances forced the mammalian organism not only to be able to distinguish nutritious from toxic substances, but also brought about the manufacture of substances that are harmless to the body from those that were harmful when ingested. This underscores the dual function of the lymphatic tissue in both immune and metabolic reactions.     

非洲猪瘟病毒核酸定性检测方法比对

为提升兽医实验室非洲猪瘟病毒核酸检测能力和质量控制水平,积累检测经验,以更好地开展非洲猪瘟检测,2020年7月,参加了北京市农业农村局组织的“非洲猪瘟实验室检测能力比对”项目。本次比对同时选用《非洲猪瘟检疫技术规范》(SNT 1559—2010标准)中的普通PCR方法、荧光定量PCR方法以及商品化试剂盒,对5份验证样品开展非洲猪瘟病毒核酸检测和结果比对。比对发现,3种检测方法结果一致,样品检测结果与预期一致,结果满意。此次比对确认了实验室现有检测方法能够满足非洲猪瘟病毒核酸的日常检测需求,同时提升了实验室人员的检测能力,积累了检测经验,可为非洲猪瘟防控提供有力的技术支撑。     

Sensitive and specific polymerase chain reaction detection of Toxoplasma gondii for veterinary and medical diagnosis.

A polymerase chain reaction (PCR) method was developed for the detection of Toxoplasma gondii. A universal- and a T. gondii-specific primer was used to amplify a region of the small subunit ribosomal RNA gene. This approach allows for a theoretical detection limit of 0.01 zoite of T. gondii per sample assayed. Experiments showed that this PCR method could detect 0.1 pg of T. gondii DNA, which represents about one organism. Polymerase chain reaction tests using DNAs of cat, dog, swine, cattle, human, Sarcocystis cruzi, Eimeria ahsata, E. vermiformis, and Escherichia coli indicated no cross-reaction with nucleic acids of hosts, related coccidia, or bacteria. Data on the sensitivity and specificity suggest that this PCR assay could be extremely useful for the diagnosis of toxoplasmosis in human and veterinary medicine, as well as for food safety surveys.     

Free radical biology and pathology

A free radical is defined as any species capable of independent existence (hence the term “free”) that contains one or more unpaired electrons. The following are examples of free radicals²: superoxide radical O₂^.-, hydroxy radical OH, hydrogen peroxide H₂O₂ (if two hydroxy radicals ever meet, they can join their unpaired electrons and make oxygen-oxygen covalent bonds giving H₂O₂. However, if one extra electron is added to H₂O₂, it makes OH⁻ and with the addition of an extra e⁻ we have an hydroxy radical). Ultraviolet radiation produces hydroxy radicals.Other molecules of concern are oxides of nitrogen, ozone and singlet oxygen (rearrangement of electrons that produces very rapid oxidation). So we are stuck with the conundrum of having essential life giving chemicals in themselves causing toxicity-oxidation. Hemoglobin makes superoxide radicals — oxyhemoglobin slowly releases superoxide radical (O₂^.-) and forms ferric hemoglobin (methemoglobin) which is unable to bind and transport O₂. This release of O₂^.- may happen only one in a thousand cycles of O₂ binding and release, but there is a large mass of hemoglobin in the body and so the total body O₂^.- produced by this mechanism is significant — in the human body it has been estimated that up to 3% of hemoglobin releases O₂^.-.Unpaired electrons make for very unstable, highly reactive atoms and/or molecules. Paired electrons, by the way of contrast, are the characteristic of a far more stable state.This is a very hazardous, unnatural and unstable state, because electrons normally come in pairs. This odd, unpaired electron in a free radical causes it to collide with other molecules so it can steal an electron from them, which changes the structure of these other molecules and causes them to also become free radicals. This can create a self perpetuating chain reaction in which the structure of millions of molecules are altered in a matter of nano-seconds wreaking havoc with genetic material (DNA), protein molecules, enzymes and cells.Free radicals are “capable of reversibly or irreversibly damaging compounds of all biochemical classes, including nucleic acids, protein and free amino acids, lipids and lipoproteins, carbohydrates, and connective tissue macromolecules. These species may (also) have an impact on such cell activities as membrane function, metabolism, and gene expression.” In the human it is now recognized that free radicals are contributing causes to a large number of disease entities.     

番鸭细小病毒和鹅细小病毒生化及基因组特性的比较

番鸭细小病毒莆田株（MPV-P）和鹅细小病毒莆田株（GPV-P）分别感染的番鸭胚尿囊液经氯仿处理、PEG沉淀和Sepharose4B柱纯化。纯化样品在电镜下观察，均见到实心和空心2种病毒粒子。病毒粒子呈圆形，立体对称，无囊膜，直径为20-24nm。经SDS-PAGE分析，MPV和GPV均呈现3条结构蛋白带。MPV结构蛋白的相对分子质量约为VP1 89000、VP2 78000和VP3 61000，其中VP3为主要结构蛋白。GPV要相对分子质量与MPV有微小差别。MPV和GPV核酸均为单链DNA，长度约为51000bp。分别以MPV核酸和GPV核酸为模板，加到无引物的DNA聚合酶Ⅰ大片段合成体系中，合成产物经1%琼脂糖凝胶电泳，均可见长度约为5600bp的双链DNA带，证明MPV和GPV核酸的3'末端具有发夹结构。对这2种病毒核酸及经Klenow合成的双链核酸进行限制性内切酶分析，两者的酶切结果明显不同，表明MPV和GPV核酸在一级结构上存在明显差异。以上结果证明，MPV和GPV不但在致病性上有显著养异，而且在核酸结构上也有明显差异。     

PCR结合斑点杂交技术检测禽多瘤病毒方法的建立及应用

鹦鹉幼雏病是由禽类多瘤病毒(APV)引起的多种鹦鹉雏鸟死亡的急性病毒性传染病,严重危害鹦鹉养殖业的健康发展.为提高分子生物学方法检测APV的敏感性和特异性,对APV基因片段进行克隆和序列分析,设计合成1对特异性引物,以VP1基因为模板,经PCR扩增获得731 bp的核苷酸DNA,并用DIG标记,制备用于检测APV的特异...     

Agricultural education and research in New Zealand

Extract

The Presidential address provides an opportunity for the president of organizations such as ours to express publicly his ideas and opinions on any matter with somewhat more weight than they might otherwise carry. This privilege must therefore be taken with due humility and circumspection, for it is not unusual for statements to be weighed solely on the standing of the person making them, without reference to his special qualifications for doing so; nor, unfortunately, is it rare for people well qualified in certain spheres to make seemingly authoritative statements on things about which they know very little.     

Peste des petits ruminants virus detected in tissues from an Asiatic lion (Panthera leo persica) belongs to Asian lineage IV

In this study, peste des petits ruminants virus (PPRV) was detected in frozen pooled tissue samples from a dead Asiatic lion (Panthera leo persica). The samples were negative for canine distemper virus and positive for PPRV nucleic acids when tested with one-step RT-PCR using the appropriate virus-specific primers. Subsequent amplification, cloning, and sequencing of the partial nucleocapsid, matrix, and fusion genes confirmed the presence of PPRV nucleic acid. Comparative sequence and phylogenetic analyses of the structural genes of the isolated virus confirmed that the virus belonged to Asian lineage IV and was closely related to PPRV circulating in India.     

荧光定量PCR技术及其应用研究进展

荧光定量PCR是近年发展起来的一种新的实时定量检测特定核酸技术,它是核酸探针技术、荧光共振能量传递技术和PCR技术的有机结合,而荧光探针是荧光定量PCR的核心.荧光定量PCR具有特异性强、灵敏度高、重复性好、定量准确、速度快、全封闭反应争特点,是对原有PCR技术的革新,扩大了PCR的应用范围.论文综述了FQ-PCR技术的原理、FQ-PCR实时定量检测系统及其应用.     

Nitrogen metabolism and its control mechanisms

N intake in the form of protein has neither got an upper nor a lower limit for agricultural working animals within a diet and there is no control mechanism for it. A high surplus of certain amino acids results in a reduction of feed intake. N excretion in faeces depends on 1) the excretion of N containing indigestible feedstuffs, 2) bacterial nitrogen synthesis in the large intestine and 3) the excretion of true endogenous N containing substances (digestion enzymes, intestinal epithelium, N containing endogenous secretion). There are no other control mechanisms for N excretion in faeces. N excretion in urine mainly comprises the nitrogen from the degeneration of amino acids and nucleic acids. The interrelations between urea, NH3, allantoin, creatine and creatinine, uric acid and hippuric acid depend on the species (monogastric or ruminants), on the nitrogen and N amount consumed and on the recycling ratio of the amino acids. The absolute amount of N excretion is not subject to any control mechanism, it depends on the intake of protein and NPN substances, the interim stages, however, which lead to the formation of excretory products, are intermediately controlled. The most important interim stage is protein biosynthesis, which is a fixed, intermediately controlled value in maintenance level. Under growth conditions only, the protein synthesis quota can exceed the protein degradation quota of the total organism (positive N balance). The control mechanisms of protein biosynthesis have, according to current knowledge, the following structure: Stimulation: 1) growth hormone (STH) stimulates protein synthesis by means of somatomedins; 2) hormones of the thyroid gland (T4 and T3) are controlled by the hormone stimulating the thyroid gland (TSH); 3) insulin. Inhibition: 1) somatostatin inhibits STH, TSH and insulin; 2) cortisol directly inhibits protein synthesis and stimulates protein degradation. The control mechanisms of protein turnover in addition to genetic coding and proteolysis extend in the framework of evolution over the period of 3,400 million years from the existence of the bacterial cell to the development of mammals, which is 74% of the age of the earth and approximately 90% since the existence of the first traces of life. The control mechanisms of protein turnover in mammals do not permit gene manipulation in protein synthesis as in bacterial cells since the control mechanisms mentioned are missing there.     

The Development of a Rapid and Sensitive,High-Through-Put Protocol for RNA:DNA Ratio Analysis

Abstract

We present a new technique for determining RNA:DNA ratios in larval and juvenile fish that relies on the rapid homogenization of samples with salts, detergents, and proteinase K and the detection of DNA and RNA with specific fluorescent dyes. Samples are not purified before assay of nucleic acids. The technique was designed for use in 96 or 384 well plates and required only 50 μL of sample per well. The detection of RNA and DNA is linear over 500 ng and is sensitive to a nucleic acid content as little as that within 50 μg of tissue. The new technique was used to characterize the RNA:DNA ratio in the white muscle of juvenile mummichogs Fundulus heteroclitus and larval winter flounder Pleuronectes americanus. This technique was compared with other techniques that use fluorescent dyes to determine the RNA:DNA ratio in fish samples.     

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.