RNA病毒重组及发生机制
重组在RNA病毒中很常见,但有关重组的许多关键问题目前尚无法解释。本文主要针对RNA重组的发生机制及其影响因素,解释重组频率发生变化的原因,阐述RNA重组未来仍需解决的问题。RNA重组是RNA病毒特殊的遗传信息交换方式,其频率在不同的RNA病毒中显著不同。目前,研究者普遍认为重组发生的机制可分为复制型、非复制型重组以及重配,并且借助新方法和新技术,逐渐了解了影响该过程的病毒和细胞因子。有研究表明,RNA病毒中RNA的重组和重配率非常高,这与RNA依赖性RNA聚合酶(RdRp)的保真性相关。因此,深入了解病毒重组机制并发现其中的规律,可实现对新型病毒以及新疫情的预警预报,从而有预见性地控制疫病的发生和流行。Recombination and the Mechanism of RNA VirusesRecombination is widely observed in RNA viruses,but many key issues therein could not be explained till now. In the paper,the reasons for the change of recombination frequency were explained based on RNA recombination mechanism and the influencing factors,and relevant issues to be addressed in the future were stated. RNA recombination was a special way to exchange genetic information by RNA viruses,with different frequency in various RNA viruses. At present,it was generally believed by researchers that,the mechanism of recombination could include replication,non-replication and reassortment,and the viruses and cytokines that might affect the process were gradually recognized with the help of new methods and technologies. It was studied that the recombination and reassortment rate of RNA were extremely high in RNA viruses,which was related to the fidelity of dependent RNA polymerase(RdRp). Therefore,the new virus or outbreak could be early warned and reported through further recognition of the mechanism and rules of virus recombination,so as to control the occurrence and prevalence of animal diseases in a predictable way.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202102018&v=2vpJqQNi66HOy5JCBsWddjPx5O5qhK2daInInEe2DUKETJQ0PrIidWOYKlD%25mmd2Fju4I
2021-02-26
鸭圆环病毒PCR检测方法的建立与应用
鸭圆环病毒病是由鸭圆环病毒(duck circovirus,DuCV)引起的一种重要免疫抑制病,影响着我国养鸭业的发展。为建立针对DuCV的PCR快速检测方法,对GenBank中登录的DuCV全基因组序列进行遗传进化分析,通过对两种基因型的DuCV全基因组序列进行比对,在共同保守区筛选出1对引物,建立了可以同时检测DuCV两种基因型的PCR快速检测方法,并对该方法的敏感性和特异性进行评价。结果显示,该方法具有良好的敏感性,检测下限达1.0 fg;特异性良好,对其他常见鸭病毒核酸检测均为阴性;利用该方法对20份临床发病鸭样品进行检测,发现有6份呈DuCV阳性,序列分析表明均属于DuCV-1。该PCR检测方法的建立为鸭圆环病毒病的快速诊断和防控提供了重要手段。Development and Application of a PCR Assay for Duck CircovirusDuck circovirus(DuCV)disease is a serious immunosuppressive disease caused by DuCV,posing a threat to the duck farming in China. In order to establish a rapid PCR assay for DuCV,the genetic evolution analysis was performed for the whole genome sequence of two DuCV genotypes that were registered in GenBank and subsequently compared with each other,then a pair of primers were screened in their common conservative area to establish a rapid PCR assay that could detect the two genotypes simultaneously. Then the sensitivity and specificity of the method were evaluated. The results showed that,by the method,the lower detection limit could reach 1.0 fg due to its good sensitivity;all nucleic acid detection for other common duck viruses were negative due to its good specificity;6 out of 20 samples of clinically infected ducks were positive for DuCV,which fell into DUCV-1 as indicated by sequence analysis. In conclusion,the method could rapidly diagnose,prevent and control DuCV as an important tool.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202101021&v=2vpJqQNi66GwD6p6B%25mmd2FdPDgcZ9km9zF8R7QPpJ2wJj%25mmd2BAqhBLbS7YWcN4DvUX%25mmd2FKpLt
2021-01-14
鉴别H5亚型两种分支禽流感病毒双重实时荧光RT-PCR方法的建立及应用
为准确区分H5亚型禽流感病毒2.3.2.1分支和2.3.4.4分支的毒株,通过对GenBank和GISAID数据库中发表的以及本实验室保存的H5亚型禽流感病毒HA基因序列进行比对,设计1对特异性引物和2条探针,建立了区分H5亚型禽流感病毒不同分支毒株的实时荧光RT-PCR方法,并对该方法的反应体系和反应参数进行了优化,同时开展了特异性和敏感性试验,以及临床应用检测。结果显示:该方法具有良好的特异性,与其他亚型禽流感病毒及常见禽病病毒无交叉反应;对H5亚型禽流感病毒2.3.2.1分支和2.3.4.4分支毒株的检测灵敏度分别为495.0 copies/μL和22.3 copies/μL;100份临床家禽拭子禽流感病毒检测结果与常规RT-PCR和病毒分离检测结果一致,符合率为100%。结果表明,该方法特异、敏感、准确、耗时少,同时还可区分不同分支,可应用于H5亚型禽流感病毒的准确、快速检测。Establishment and Application of a Duplex Real-time RT-PCR Assay for Identifying Two Branches of H5 Subtype Avian Influenza VirusIn order to accurately distinguish the strains of two evolutionary clades including 2.3.2.1 and 2.3.4.4 of H5 subtype avian influenza virus(AIV),a pair of specific primers and two TaqMan probes were designed by comparing HA gene sequences of H5 subtype AIV registered in Genbank and GISAID with the ones reserved in our laboratory,a duplex real-time RT-PCR assay was established to distinguish different clades of the virus,and its reaction system and parameters were optimized. Meanwhile,the specificity,sensitivity and its clinical application were tested. The results showed that,the specificity of the method was good with no any cross-reaction with other subtypes AIV or common avian viruses;the detection limits of clades 2.3.2.1 and 2.3.4.4 were 495.0 and 22.3 copies/μL,respectively;100 clinical poultry swab samples were tested by the established method,and the results were consistent with those by traditional RT-PCR and virus isolation,with the coincidence rate of 100%. As a conclusion,the established method was specific,sensitive,accurate and convenient,which could be used to distinguish different clades of H5 subtype AIV.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202101020&v=2vpJqQNi66HpC2DtPpxmZ7MbcnGvT1y1caIfZvmjLYrGOkLUTMOCulgxxUIz4kWz
2021-01-14
牛源多杀性巴氏杆菌PlpE蛋白的原核表达及多克隆抗体制备
为体外表达牛源多杀性巴氏杆菌(Pasteurella multocida)PlpE基因及制备抗PlpE蛋白多克隆抗体,根据PlpE基因核酸序列设计了1对特异性引物,通过PCR扩增PlpE基因,构建重组质粒pET-32a-PlpE;摇菌提取质粒,进行双酶切鉴定,鉴定正确后将重组质粒pET-32a-PlpE转化至BL21(DE3)感受态细胞,加入IPTG进行诱导,对诱导蛋白进行SDS-PAGE和Western Blot鉴定;使用纯化蛋白PlpE免疫北京大耳白兔,制备多克隆抗体,并进行间接ELISA和Western Blot分析。结果显示,扩增的PlpE基因大小为1 050 bp,在原核细胞中诱导表达的PlpE蛋白约56 kDa。间接ELISA结果显示,所制备多克隆抗体效价可达1:512 000;Western Blot结果显示,所制备多克隆抗体反应性良好。PlpE蛋白的成功表达和多克隆抗体的成功制备,为下一步多杀性巴氏杆菌诊断方法的建立和疫苗研发奠定了基础。Prokaryotic Expression of PlpE Protein of Bovine Pasteurella multocida and Preparation of Polyclonal AntibodiesIn order to express the PlpE gene of bovine Pasteurella multocida in vitro and prepare corresponding polyclonal antibodies,a pair of specific primers were designed according to the nucleic acid sequence of the gene,then pET-32a-PlpE,the recombinant plasmid,was constructed through PCR amplification,after plasmid extraction and identification by double enzyme digestion,the recombinant plasmid was transformed into BL21(DE3)cells and the bacteria was induced by IPTG and the induced protein was identified by SDS-PAGE and Western Blot;Beijing White Rabbits were vaccinated with purified PlpE protein to prepare polyclonal antibodies,which were analyzed by indirect ELISA and Western Blot. The results showed that the amplified PlpE gene was 1 050 bp,and the induced PlpE protein was about 56 kDa. It was shown that,by indirect ELISA,the titer of the polyclonal antibodies could be up to 1:512 000;and by Western Blot,the antibody could react well with the positive serum. It was concluded that a foundation was laid for the establishment of the method for identifying Pasteurella multocida and development of relevant vaccines in the future.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202101019&v=2vpJqQNi66FspGXrYEbDEJvrXEmDYO9pI6yc9rrux3Q6YI6dTxxTKTZwNyU3xVug
2021-01-14
微流控芯片技术在微生物检测中的应用
微流控芯片是一项以芯片为基础,结合化学、物理学、生物学等多学科形成的技术平台,能够在一个芯片上实现样品制备、反应、分离、检测等流程,具有高通量、高效率和易操作的特点。随着材料科学和纳米技术的发展,该项技术得到迅速发展并应用于化学分析、微生物检测等,尤其是在微生物检测和分析方面发挥着重要作用,并逐步产生了商品化的微流控芯片。微流控芯片因具有小型化、节约试剂、速度快、集成化程度高等优点,适用于处理突发公共卫生事件,但目前在动物疫病检测中的应用还很少见。本文对微流控芯片的技术原理、结构及其在微生物检测中的应用进行综述,以期为动物疫病的高通量检测提供借鉴。Application of Microfluidic Chip Technology in Microbial DetectionMicrofluidic chip is a kind of technology platform based on a chip,which combines with several disciplines,such as chemistry,physics,biology and others,and could complete the processes of preparation,reaction,separation and detection of samples on a chip due to its characteristics of high throughput,high efficiency and easy operation. With the development of material science and nanotechnology,the technology has been rapidly developed and applied to chemical analysis,microbial detection and so on,in which,it has been playing an important role in microbial detection and analysis,and gradually leads to the commercial microfluidic chip that is advantaged with miniaturization,reagent saving,fast and high integration,and appropriate for dealing with public health emergencies,but is still seldom used for animal disease detection at present. In the paper,the technical principles and structure of microfluidic chip as well as its application in microbial detection were reviewed,with a view to providing some references for high-throughput detection of animal diseases.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202101018&v=2vpJqQNi66FIJLK2OSEL8Yi4RRdCUFoM1KEDGCyKgnlUlreyqw0sri%25mmd2Bh1Vrcaqxh
2021-01-14
猪繁殖与呼吸综合征检测技术与疫苗研发进展
猪繁殖与呼吸综合征(PRRS)给世界养猪生产造成了巨大经济损失。对PRRS的精准检测以及疫苗的科学使用是临床PRRS防控的重中之重。为此,本文综述了PRRS检测技术和疫苗研发进展,以期为临床PRRS综合防控提供依据。目前临床上检测PRRS的主要技术包括血清抗体ELISA检测以及各种类型的RT-PCR检测技术。ELISA检测以评价疫苗免疫效果或野毒感染情况为主要目的,RT-PCR检测以临床诊断及调查野毒株类型为主要目的。在疫苗方面,目前市场上主要有活疫苗、灭活苗、亚单位疫苗、DNA疫苗以及载体疫苗等不同类型的单苗以及联苗。其中:活疫苗保护效果较好,但交叉保护不强;灭活苗安全性较高,但免疫效力较差;亚单位疫苗可用于妊娠晚期;DNA疫苗可增强活疫苗的保护效果;载体疫苗虽具有部分保护力,但未进行实际应用。目前依旧没有完全符合国际新一代标准的PRRS疫苗上市。因此,通过科学检测以及将生物安全和合理的疫苗接种结合起来,进行田间毒株的检测和基因分析,进而选择相应基因型毒株疫苗进行免疫,对控制农场以及地区的PRRS具有非常重要的作用。Research Progress of the Diagnostic Techniques and Vaccines for PRRSPorcine reproductive and respiratory syndrome(PRRS)has brought huge economic losses to pig production in the world,which should be prevented and controlled with the top priority of accurate detection and scientific application of vaccines. At present,the main techniques to diagnose PRRS included serum antibody ELISA assay and various RT-PCR assays. The former aims to evaluate the immune effect of vaccines or wild virus infection,while the latter focuses on clinical diagnosis and investigation of wild virus strains. For vaccines,all kinds of single and combined vaccines are available in markets,including live vaccines,inactivated vaccines,subunit vaccines,DNA vaccines,vector vaccines,etc.,specifically,live vaccines are with good effect but poor cross protective immunity;inactivated vaccines are with high safety but poor immune effect;subunit vaccines can be used in late pregnancy;DNA vaccines can increase the protection effect of live vaccines;and vector vaccines have not been applied in practice although they are with partial protective immunity. No any vaccines against PRRS that fully meet the new international standards have been available till now. Therefore,it will be very crucial to control PRRS in farms or regions through scientific diagnosis and combination of bio-safety and reasonable vaccination to detect field strains and analyze their genes,and then to vaccinate with corresponding vaccines.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202101016&v=2vpJqQNi66ErxHSyLOD3MXoXltAc3QuZhVZLG5VbgKZIUFf2BdT1YOqRKWETMBD8
2021-01-14
基于网络药理学分析黄芪防控猪繁殖与呼吸综合征的物质基础及靶点信息
为揭示黄芪防控猪繁殖与呼吸综合征(PRRS)的物质基础及靶点信息,采用网络药理学方法,从中药系统药理学数据库与分析平台(TCMSP)筛选黄芪的潜在活性成分、作用靶点,同时从比较毒理基因组学数据库(CTD)收集PRRS相关宿主基因,最后构建“活性成分-关键靶点”网络、蛋白互作网络,并进行GO功能和KEGG通路富集分析。结果显示,从黄芪筛选出的17个潜在活性成分中,有14个潜在活性成分共计65个基因靶点与PRRS相关宿主基因交叉,其中槲皮素、山奈酚、刺芒柄花素、异鼠李素等潜在活性成分可能对治疗PRRS有重要作用,TNF、IL8、TP53、IL6、IL10、IL1B、JUN等45个蛋白靶点间存在相互作用。此外,黄芪治疗PRRS的作用机制可能涉及细胞外间隙和胞浆等组分,并可能与炎症反应、细胞凋亡、T细胞受体信号通路等生物过程相关。本研究为临床应用黄芪及其提取物治疗PRRS奠定了理论基础。Analysis on the Material Basis and Drug Targets of Astragalus Membranaceus in Prevention and Control of PRRS Based on Network PharmacologyIn order to reveal the information concerning the material basis and drug targets of astragalus membranaceus acting on porcine reproductive and respiratory syndrome(PRRS),the potential active components and drug targets related to astragalus membranaceus were screened through traditional chinese medicine systems pharmacology database and analysis platform(TCMSP)by the network pharmacology. Meanwhile,the host genes related to PRRS were gathered from comparative toxicogenomics database(CTD)to construct the“active components-key targets”network and protein-protein interaction(PPI)network,and to analyze the GO functions and KEGG pathway enrichment. The results showed that 14 out of 17 potential active components including 65 gene targets crossed with the host genes related to PRRS,especially,quercetin,kaempferol,formononetin,isorhamnetin and other potential active components played an important role in fighting against PRRS,and 45 targets could interact with each other,including TNF,IL8,TP53,IL6,IL10,IL1B and JUN. In addition,the mechanism of astragalus membranaceus in fighting against PRRS might involve extracellular space and cytoplasmic components,and might be related to biological processes such as inflammatory response,apoptosis,and T cell receptor signaling pathway. Therefore,a theoretical foundation was laid for clinical application of astragalus membranaceus and its extract in fighting against PRRS. 全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202101015&v=2vpJqQNi66FXF9R5APrxlbEWzpYeoKIo%25mmd2FrCDWaNAQ5qxqiEskBfDFtKQggeuabBJ
2021-01-14
鸭坦布苏病毒山东流行株的分离鉴定及其基因组序列分析
为了解山东省鸭坦布苏病毒(duck tembusu virus,DTMUV)的流行与变异情况,对2018年下半年至2019年上半年泰安、济宁和淄博等地送检的临床病鸭样品进行了DTMUV病原学检测,并对阳性病料进行病毒分离鉴定及分离株的全基因组序列分析。结果显示:从102份样品中,共检测到8份DTMUV阳性,阳性率检出率为7.8%,从阳性病料中分离到3株DTMUV分离株;3个分离株基因全长均为10 993 bp,彼此间的氨基酸同源性为99.2%~99.3%,与24个参考毒株的同源性为95.8%~99.9%,均属于中国株I型;与参考毒株相比,分离株的核衣壳蛋白C、囊膜蛋白E和非结构蛋白NS1的同源性相对较低,分别为86.1%~99.2%,95.0%~99.4%和92.3%~99.7%,同时存在数量不等的氨基酸突变位点,但关键的蛋白结构和潜在的糖基化位点无差异。结果表明,DTMUV在山东部分地区仍有流行,但流行毒株基因结构及致病特性与经典毒株相比无明显变化,但应持续关注和深入研究。Isolation and Identification of Shandong Strain of Duck Tembusu VirusIn order to identify the prevalence status and variation of duck Tembusu virus(DTMUV)in Shandong Province,the clinical samples delivered from Tai'an,Jining and Zibo during the second half of 2018 to the first half of 2019 were detected for DTMUV,and virus isolation and identification were carried out towards the positive samples,then the whole genome sequences of the isolates were analyzed. The results showed that 8 out of 102 samples were positive,with the detection rate of 7.8%,from which,3 strains of DTMUV were isolated;the full lengths of all the three strains were all 10 993 bp,the amino acid homology of they ranged from 99.2% to 99.3%,and the homology between the isolates and 24 reference strains ranged from 95.8% to 99.9%,all the isolates fell into Chinese strain I;compared to the reference strains,the homologies of nucleocapsid protein C,envelope protein E and non-structural protein NS1 of the isolates were lower,which were 86.1% to 99.2%,95% to 99.4% and 92.3% to 99.7%,respectively,and with different number of amino acid mutation sites,but major protein structures and potential glycosylation sites had no difference. It was concluded that DTMUV was still prevalent in some regions in Shandong Province,which should be further concerned and studied,although its genetic structure and pathogenicity remained unchanged compared to the classical strains.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202101008&v=2vpJqQNi66HAmotFUcqF5c8IG0hCBj%25mmd2FjC6JPalOXfdjJ1WIX49lsO7qv25k1hfwp
2021-01-14
安徽省6个地市规模鸡场新城疫流行率估计及风险因素分析
为了解安徽省规模鸡场新城疫病毒感染情况及其引发该病的风险因素,结合2019年秋季集中监测采样工作,对6个地市存栏3 000~10 000只的规模鸡场开展横断面研究。采用两阶段随机抽样,在6个地市范围内,随机抽取153个规模鸡场,采集5 369份咽喉/泄殖腔拭子采用荧光PCR进行病原学检测,同时进行问卷调查,分析相关风险因素。结果显示,6个地市153个规模鸡场的场表观流行率和场真实流行率分别为11.1%和11.7%。单因素分析显示:“1个月只清理1次粪便”和“场内共用器具、车辆”是潜在风险因素,有统计学意义(P<0.05);车辆和人员进出场消毒、净道和污道分开、空舍消毒以及消毒前清洗场内可以降低感染风险。Logistic回归分析显示,“净道、污道不分开”和“车辆、人员进出不消毒”是风险因素。结果表明,安徽省部分鸡场仍存在新城疫病毒感染,应加强鸡场生物安全管理,合理规划养殖场布局,场区入口要建造消毒池,场内净道和污道要分开,车辆和人员进出场时要严格消毒,场内要制定详细的消毒计划,切实改善养殖场环境。Estimation of Newcastle Disease Prevalence in Scale Farms in 6 Cities of Anhui Province and Analysis on Relevant Risk FactorsIn order to identify the infection status of Newcastle disease virus(NDV)and risk factors leading to the disease in Anhui Province,an cross-sectional study was carried out in the scale farms with an inventory of 3 000 to 10 000 chickens in six cities in combination with centralized surveillance and sampling in the fall of 2019. A total of 5 369 throat/cloacal swabs were randomly collected from 153 farms by two-stage sampling method. Meanwhile,a questionnaire investigation was conducted to analyze relevant risk factors. The results showed that the apparent prevalence and true prevalence were 11.1% and 11.7% respectively at the farm level. It was shown that,by monofactor analysis,the potential risk factors included“only cleaning feces once a month,sharing equipment and vehicles within the farms”,which was of statistical significance(P<0.05);and the risk of infection could be reduced by disinfecting vehicles and personnel when they were entering or leaving farms,separating clean channels from the dirty ones,disinfecting empty pens and cleaning the premises prior to disinfecting. It was shown by Logistic regression analysis that,the risk factors included“failure to separate clean and dirty channels and to disinfect vehicles and personnel in or out of farms”. As a conclusion,NDV was still prevalent in some farms in Anhui Province,and some suggestions were put forward,such as strengthening bio-security management in the farms,reasonably designing their layout,building a disinfection pool at the entrance,separating clean channels from the dirty ones,strictly disinfecting vehicles and personnel when they were entering or leaving farms,and developing detailed disinfection plan to practically improve the environment of the farms.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202101006&v=2vpJqQNi66HaOQK62RPPfwn8x6iknDBctOCeF2DdBBqt7ZnZcqNDxFfJKn3aOdPd
2021-01-14
羊驼免疫库的制备及抗孔雀石绿纳米抗体的筛选
孔雀石绿(malachite green,MG)是一种易溶于水和乙醇等的基础染料,可在多种行业应用,并曾用于水生动物多种疾病如真菌病、寄生虫病、细菌性疾病等的治疗。但因其具有致癌等毒副作用,目前已被禁用。然而市场上仍有在水产品违法使用MG的现象,因此需要加强对该药品残留的检测。本研究选择羊驼免疫库筛选抗MG纳米抗体,通过噬菌体ELISA筛选高亲和力和特异性的纳米抗体。通过辅助噬菌体M13的救援,使得纳米抗体基因(VHH)与噬菌体外壳蛋白基因融合表达,从而使得VHH蛋白展示在噬菌体表面,通过有限稀释筛选和多次洗脱的办法,获得含有目的基因的单个噬菌克隆;将重组质粒PMES4-VHH转化到表达细胞中,进行体外表达。最后采用NI-NTA柱进行His标签纯化,得到抗MG纳米抗体,并进行SDS-PAGE电泳鉴定。结果显示:噬菌体文库成功构建,库容为3.2×108 cfu/mL;通过ELISA筛选,得到16个具有抑制效果的噬菌体克隆,经对噬菌体基因测序并翻译,得到2种氨基酸序列;将重组质粒转化至表达菌株表达、纯化,最后得到大小约为15 kDa的抗MG纳米抗体,从而为MG的下一步检测奠定了基础。Preparation of Alpaca Immune Repertoire and Selection of Nano-antibody against Malachite GreenMalachite green(MG)is a kind of primary dye that is soluble in water and ethanol,and has been applied in many industries,especially to fight against many diseases of aquatic animals,such as fungal diseases,parasitic diseases and bacterial diseases. But it has been banned due to its adverse reaction such as carcinogenicity. However,it is necessary to strengthen the detection of drug residues from MG that is still illegally used in aquatic products in markets. In this study,nano-antibody with high affinity and specificity against MG was selected from alpaca immune repertoire through the phage by ELISA,then was used to support the rescue of phage M13,so that nano-antibody gene(VHH)could be fused and expressed with the coat protein gene of the phage,and VHH protein could be shown on the surface of the phage to obtain the single phagocytic colonies containing the targeting gene by means of limited dilution screening and multiple elution;the recombinant plasmid PMES4-VHH were transformed into expression cells and expressed in vitro,then His-tag purification was carried out by the NI-NTA column to obtain the nano-antibody against MG that was identified by SDS-PAGE electrophoresis. It was concluded that the immune repertoire was successfully constructed with a capacity of 3.2×108 cfu/mL;16 colonies with inhibitory effect were screened by ELISA,two kinds of amino acid sequences were obtained by sequencing and translating the phage genes;PMES4-VHH was transformed into the expression strain for expression and purification,and the nano-antibody of about 15 kDa against MG was produced,which paved the way to future detection of MG.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTODAY&filename=ZGDW202012025&v=Bh8a8ZT44%25mmd2BXk0ZJ7KNYHWtHzXPUmzT5DElVbdXML%25mmd2FMLk8l%25mmd2FMwEnYZUQ9XabHs0ZU
2020-12-03
应用猪繁殖与呼吸综合征核酸标准物质比较5种核酸提取试剂盒
为比较不同核酸提取试剂盒对猪繁殖与呼吸综合征病毒(PRRSV)核酸的提取效率,使用国家标准物质“猪繁殖与呼吸综合征病毒美洲经典株核酸标准物质”作为模拟样品,分别使用试剂盒A、B、C(磁珠法)以及试剂盒D、E(柱式法)等5种核酸提取试剂盒进行核酸提取,然后通过荧光RT-PCR和数字RT-PCR方法评估提取效率,并分析不同基质对提取效率的影响。结果显示:5种试剂盒均能有效提取PRRSV RNA,其中磁珠法的提取效率高于柱式法;试剂盒B和C对PRRSV RNA的提取效率较高;基质会对提取效率造成影响,精液和抗凝血对试剂盒提取效率的影响较大。综上,试剂盒B、C均可适用于猪繁殖与呼吸综合征临床样品的核酸提取。实际操作中,应根据实验室条件、样品数量、样品类型和时间要求等因素,选择应用合适的提取试剂盒。Comparison of Five Kinds of Nucleic Acid Extraction Kits Based on Nucleic Acid Reference Materials of PRRSVIn order to compare the efficiency of various nucleic acid extraction kits for RNA of porcine reproductive and respiratory syndrome virus(PRRSV),national reference material“PRRSV American classic strain nucleic acid reference material”was respectively extracted by five kinds of kits(three magnetic bead kits including A,B and C and two column kits including D and E),and their efficiency were evaluated by fluorescent RT-PCR and digital RT-PCR. Then,the effect of different substrates on the efficiency was analyzed. The results showed that all the kits could effectively extract RNA of PRRSV,in which,the efficiency of magnetic bead kits was higher than that of column kits;the efficiency of B and C was higher;the efficiency could be affected by substrates,especially semen and anticoagulant. In conclusion,both B and C were appropriate for extraction of nucleic acids from clinical samples of PRRSV. Therefore,appropriate kits should be used subject to laboratory conditions,number of samples,types of samples,urgency of cases and other factors.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTODAY&filename=ZGDW202012023&v=Bh8a8ZT44%25mmd2BVdUZJJ4fPLG4mzwxK%25mmd2FyyV5Gx5EoETBAWZubmLyLyQ6p%25mmd2B%25mmd2BCT0seR3R9
2020-12-03
A型塞内卡病毒VP1结构蛋白在昆虫杆状病毒中的表达与鉴定
目前在昆虫杆状病毒表达系统(baculovirus expression vector system,BEVS)中表达A型塞内卡病毒(Senecavirus A,SVA)结构蛋白VP1尚未见报道。本研究将VP1基因通过限制性酶切位点插入杆状病毒载体pFastBac I,然后将鉴定正确的重组载体转化至DH10Bac感受态细胞,经蓝白斑筛选,对鉴定正确的菌落提取质粒。将质粒转染Sf9昆虫细胞,待有明显细胞病变后收获重组杆状病毒rBac-I-VP1,并进行病毒滴度测定。随后通过蛋白免疫印记(Western Blot)和间接免疫荧光(IFA)鉴定VP1蛋白表达情况。结果显示,重组杆状病毒rBac-I-VP1滴度为6.8×105 pfu/mL;Western Blot可检测到相对分子质量约27 kDa的表达产物,且其能够被SVA兔阳性多克隆血清识别;IFA试验显示,VP1蛋白能够在Sf9细胞内表达。结果表明,本研究利用BEVS表达的SVA VP1蛋白具有良好的免疫反应性,为其后期功能研究及SVA诊断试剂研制提供了技术基础。Expression and Identification of Senecavirus A Structural Protein VP1 in Insect Baculovirus The expression of senecavirus A(SVA)structural protein VP1 in baculovirus expression vector system(BEVS)has been not reported till now. In the study,VP1 gene was inserted into baculovirus vector of pFastBac I through the restriction enzyme site,the identified recombinant vector was transformed into DH10Bac,and the correct colonies were screened by blue and white spots to extract the plasmids that were transfected into Sf9 insect cells,and the recombinant baculovirus rBac-I-VP1 was obtained after obvious cytopathic changes. The expression of VP1 protein was identified by Western Blot and indirect immunofluorescence(IFA). The results showed that the titer of recombinant baculovirus rBac-I-VP1 was 6.8×105 pfu/mL;the expression products with relative molecular weight of about 27 kDa could be detected by Western Blot,which could be recognized by SVA rabbit positive serum;it was shown by IFA that,VP1 protein could be expressed in Sf9 cells. It was concluded that the SVA VP1 protein expressed by the BEVS was with good immunoreactivity,which provided a basis for future study on related functions and the development of diagnostic reagents.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTODAY&filename=ZGDW202012022&v=Bh8a8ZT44%25mmd2BVJojsL36nb%25mmd2BCImZ5SxS65hljHwT7w8vihqJVTRfQYhRa61GRaSRLVI
2020-12-03
塞内卡病毒CH/ZZ/2016株全基因组测序与分析
为了解塞内卡病毒分离株SVA/CH/ZZ/2016全基因组序列,设计4对相互重叠的特异性引物扩增基因片段,将扩增产物分别克隆至pCE2TA/Blunt-Zero载体并进行测序,拼接校正后获得SVA/CH/ZZ/2016株全基因组。结果显示,该毒株基因组全长7 292 bp,包括5'UTR(670 bp)、ORF(6 546 bp)以及3'UTR(76 bp)。选择国内外其他9株参考毒株序列,对编码区12个基因的核苷酸及编码氨基酸进行比对。结果显示,核苷酸同源性最高的是3B基因,最低的是VP1基因,其余基因的核苷酸序列同源性均在85.2%~100%之间。VP1基因遗传进化分析显示,SVA/CH/ZZ/2016株与美国分离株USAIL_Purdue_43_2016和USAIN_Purdue_3698_2016株亲缘关系最近,属同一进化分支,与原始毒株SVV-001株亲缘关系最远。对SVA/CH/ZZ/2016株和原始毒株SVV-001 VP1蛋白的氨基酸序列进行比对,发现共有10处氨基酸差异。本研究通过对SVA/CH/ZZ/2016株全基因组测序及分析,为进一步开展SVA分子生物学研究及流行病学调查提供了基础数据。Sequencing and Analysis on Whole Genome of Senecavirus CH/ZZ/2016 Strain In order to identify the whole genome sequence of senecavirus SVA/CH/ZZ/2016 strain,four pairs of overlapping specific primers were designed to amplify gene fragments. The amplified products were cloned into pCE2TA/Blunt-Zero vector for sequencing. The whole genome of SVA/CH/ZZ/2016 strain was obtained after splicing and correction. The results showed that the total genome length of the strain was 7 292 bp,including 5'UTR(670 bp),ORF(6 546 bp)and 3'UTR(76 bp). Other 9 reference strains both at home and abroad were selected and compared with the nucleotide and amino acid sequences of 12 genes in coding region. It was found that the nucleotide homology was highest in 3B gene,minimum in VP1 gene,and remained 85.2% to 100% in other genes. By phylogenetic analysis,it was shown that SVA/CH/ZZ/2016 strain had the closest genetic relationship with American isolates USAIL_Purdue_43_2016 and USAIN_Purdue_3698_2016,shared the same evolutionary branch,and was with largest evolutionary distance with the original strain SVV-001,a total of 10 amino acid differences were found. Therefore,the basic data was provided for further study on SVA molecular biology and epidemiological investigation.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTODAY&filename=ZGDW202012021&v=Bh8a8ZT44%25mmd2BWe%25mmd2Fu6mMUO8Io9ewTzdK8XE6rL14BCn5ZgS5VrStjQTvAjvZuRVCkn3
2020-12-03
QuEChERS法在兽药残留检测中的优化与应用
兽药的不合理使用可导致动物产品及环境中兽药残留超标,从而对公共卫生安全构成威胁。QuEChERS法是一类快速、简便、经济、高效、耐用和安全的样品前处理方法,常用于植物产品农药检测。通过持续优化,目前该方法开始在动物产品兽药残留检测中得到应用。本文综述了QuEChERS法的特点,探讨了该方法在畜禽产品或样品兽药残留检测中的优化与应用。与植物样品相比,动物相关样品基质复杂,脂肪含量较高,因此需要对QuEChERS法进行优化。常用的优化方式包括提取液优化、脱水盐优化和吸附剂优化。目前QuEChERS法在畜禽肌肉、内脏、鸡蛋、牛奶及相关产品以及尿液、粪便等环境样品兽药残留萃取中得到应用。随着与其他提取净化方法的连用以及自动化提取装置的不断更新,未来QuEChERS法会得到持续优化与发展,其提取效率和净化效果将进一步得到提高,并将在动物产品残留检测中发挥重要作用。Optimization and Application of QuEChERS in Detection of Veterinary Drug Residues The abuse of veterinary drugs would lead to excessive veterinary drug residues in animal products and environment,posing a threat to public health. The QuEChERS,which is quick,easy,cheap,effective,rugged and safe,is a pretreatment method for samples and usually used to detect pesticide in plant products. At present,the method with continuous optimization has been applied in the detection of veterinary drug residues in animal samples. In the paper,the characteristics of the method were summarized,its optimization and application in the detection of veterinary drug residues in livestock and poultry products or samples were discussed. Animal samples were with complex matrix and higher fat content compared to plant samples,so the method should be optimized by common means including the optimizations of extraction solution,dehydration salt and adsorbent. At present,the QuEChERS has been applied in the extraction of veterinary drug residues in animal muscle,viscera,eggs,milk and related products,as well as urine,feces and other environmental samples. With continuous use of other extraction and update of automatic extraction units,the QuEChERS would be continuously optimized and developed in the future,and its extraction efficiency and purification effect would be further improved to play an important role in detection of veterinary drug residues in animal products.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTODAY&filename=ZGDW202012020&v=Bh8a8ZT44%25mmd2BW2NuVVy8plweIK9k3ca%25mmd2B%25mmd2FPefxCuLCIL0oso4h04TfcwyV0b%25mmd2BfKoX13
2020-12-03
我国动物疫病风险评估研究热点和趋势——基于CiteSpace的可视化分析
为探究我国动物疫病传播风险评估的研究热点及趋势,以2000—2019年“中国知网”(CNKI)数据库收录的348篇动物疫病风险评估相关文献为研究对象,利用CiteSpace软件对文献进行时空分析和内容分析。时空分析显示,这20年间动物疫病风险评估文献数量呈现从少到多再减少的动态过程,各大研究机构合作较少。内容分析显示,研究热点主要涉及“风险评估”“风险分析”和“动物疫病防控”等方面。提示研究机构应加大动物疫病风险评估研究力度,加强各大研究机构间的合作,推进动物疫病风险评估的研究、分析、监管,完善预警机制,进一步提升我国动物疫病防控水平。本研究可为深化动物疫病风险评估研究、实践和预警提供参考。Research Hotspots and Trends of Animal Disease Risk Assessment in China According to Visual Analysis Based on CitespaceIn order to explore the research hotspots and trends of animal disease risk assessment in China,348 articles concerning such risk assessment registered in the database of CNKI during 2000 to 2019 were retrieved and analyzed by CiteSpace software based on their time and space as well as contents. It was shown that,by the temporal and spatial analysis,the number of literature on risk assessment went through a dynamic process from emerging to increasing then to decreasing in the past 20 years,and fewer major institutes cooperated with each others. The contents of the literature were analyzed,it was found that“risk assessment”,“risk analysis”and“animal disease prevention and control”were the research hotspots. Therefore,institutes should strengthen their inputs in animal disease risk assessment,and increase cooperation with each others,so as to push forward the study,analysis and supervision concerning risk assessment,improve early warning system,and to further upgrade the control level of animal diseases. Some references were provided to further the study and practice of risk assessment and early warning.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTODAY&filename=ZGDW202012019&v=Bh8a8ZT44%25mmd2BUmenw8z1%25mmd2BeFZoLuHtJ0BefrEsn2GG6E6t0WHpgBiQ2tAHubGLfwaZt
2020-12-03
我国非洲猪瘟相关技术专利发展现状
为了解我国非洲猪瘟(ASF)相关技术专利研发重点,利用中国专利文摘数据库(CNABS)等专利数据库,结合“人工阅读去噪”筛选出295件ASF相关技术专利,并运用Excel软件对2006年1月至2020年7月专利的申请量、技术领域、申请人、研究核心等进行了分析。结果显示:我国专利总体申请量呈现增长趋势,并从2018年开始出现爆发性增长;ASF技术专利以快速、高效、特异地鉴定和监测ASF病毒以及有效、安全地预防和治疗ASF为研究的重点和热点;专利申请人涉及面广,涵盖企业、科研院所、高校和个人,其中企业申请总量最高,其次是科研院所和高校;研究核心各有侧重,科研院所和高校侧重于基础技术的探索和研发,而企业则偏重于技术和产业化的有效结合。随着我国ASF疫情防控重要性和紧迫性的日趋增加以及研究手段的日益更新,可以预期科研院所、高校以及企业等各申请人在该领域中的研究将会更加活跃。本文为我国ASF防控及其技术研发等提供了参考。The Development of Technical Patents Related to African Swine Fever in China In order to identify the research priority of technical patents related to Africa swine fever(ASF),295 relevant patents were selected by use of patent databases including China Patent Abstracts Database(CNABS)and in combination with“manual reading denoising”,and the number of application,technical field,applicant and research core of patents from January 2006 to July 2020 were analyzed by the Excel software. The results showed that the total number of application was increasing and had boomed since 2018;the technical patents focused on rapid,effective and specific identification and detection of African swine fever virus(ASFV)and safe prevention and control of ASF;the applicants might come from enterprises,institutes,colleges or be an individual,most of them were from enterprises,followed by institutes and colleges;for the research core,institutes and colleges focused on the development of basic technology,and enterprises mainly developed the effective combination of technology and industrialization. With the increasing importance and urgency of ASF control as well as the update of research methods in China,it could be expected that the research by the applicants would be more active in the field. Some references were provided for prevention and control of ASF as well as the development of relevant technologies in China.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTODAY&filename=ZGDW202012018&v=Bh8a8ZT44%25mmd2BWtVTc8gWyxxX6VayE8NViQNoQb6N4Khk0FzSxwn7eXtxiG3PSxiVOt
2020-12-03
非洲猪瘟新型疫苗研究进展
非洲猪瘟(African swine fever,ASF)已存在约一个世纪,给世界养猪业带来巨大损失和威胁。为此国内外科学家一直致力于ASF疫苗研究,从早期的灭活疫苗、弱毒活疫苗到目前的新型疫苗,包括基因缺失减毒活疫苗、亚单位疫苗、病毒活载体疫苗、DNA疫苗、单周期病毒疫苗等。但直到现在,世界上仍未制造出切实有效并投入生产的ASF疫苗。研究发现:灭活疫苗不具有保护性或仅具有部分保护性;亚单位疫苗具有部分保护性;基因缺失减毒活疫苗被证实具有同源保护性,但其安全问题仍未得到解决;病毒活载体疫苗虽会产生特异性抗体,但仍需要进一步研究;DNA疫苗虽然能够诱导产生体液免疫和细胞免疫,但不能提供保护。目前,一种新型的单周期病毒疫苗正被研究,也为ASF新型疫苗研制提供了新选择。随着基因工程技术与分子生物学技术的不断发展以及对ASF病毒基因组研究的不断深入,ASF疫苗有望研制成功。Research Progress of Novel Vaccines against African Swine Fever VirusAfrican swine fever virus(ASFV)has remained for about a century,which has brought huge economic losses and threats to the world. Therefore,the study on vaccines against ASFV has been continued by scientists both at home and abroad,from the early inactivated vaccines,attenuated live vaccines to current new vaccines,including gene deletion attenuated live vaccines,subunit vaccines,live vector vaccines,DNA vaccines,single-cycle virus vaccines,etc. But up to now,no effective vaccine had been developed or produced. It was studied that inactivated vaccines were not protective or partially protective;subunit vaccines were partially protective;gene deletion live attenuated vaccines had been proven to be homologous-protective,but their safety had not been solved;although live vector vaccines could produce specific antibodies,further research was still needed;DNA vaccines could induce humoral or cellular immunity,but failed to provide protection. At present,a kind of novel single-cycle virus vaccine was being studied,which also provided a new option for the development of novel vaccines against ASFV. With the continuous development of genetic engineering technology and molecular biology technology as well as the in-depth study on ASFV genome,vaccines against ASF were expected to be successfully developed.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTODAY&filename=ZGDW202012017&v=Bh8a8ZT44%25mmd2BXHiNGRu1LlBDMWBctkn9umG3qE8vPysHtqUmRpYD0vEXOEXJv76oGP
2020-12-03
转基因动物及其产品检测技术研究进展
为探索可靠、灵敏的转基因动物及其产品检测技术,从核酸检测和蛋白检测两个方面,综述了转基因动物及其产品不同检测技术的原理、特点及应用,提出了每种检测技术的不足及今后转基因检测技术的发展趋势和研究方向。目前,已经研发的核酸检测技术主要包括多种PCR方法、环介导等温扩增技术、Southern blot技术和染色体原位杂交技术等,蛋白检测方法主要包括酶联免疫吸附检测技术、免疫层析试纸条、Western blot技术、免疫组织化学和免疫荧光技术等。三代测序技术是近年来提出的一种新型基因检测技术,适用于对未知序列转基因动物的分析。随着转基因技术的发展,转基因动物及其产品越来越多,高通量、高灵敏度和低成本的检测技术将成为未来转基因动物及其产品检测的研究重点。 Research Progress on the Techniques for Testing Transgenic Animals and Their ProductsIn order to explore a kind of reliable and sensitive technique for testing transgenic animals and their products,the principle,characteristics and application of relevant techniques were summarized from the aspects of nucleic acid and protein detection,then the disadvantages of each technique as well as relevant development trend and direction to study were put forward. At present,the established techniques for detecting nucleic acids mainly included PCR,ring mediated isothermal amplification,Southern blot and chromosome in situ hybridization,and those for protein included enzyme-linked immunosorbent assay(ELISA),immunochromatographic strip,Western blot,immunohistochemistry and immunofluorescence assay. The third-generation sequencing technique had been used to detect gene sequences in recent years,which is appropriate to analyze transgenic animals with unknown sequence. With the development of transgenic techniques,the quantity of transgenic animals and their products was increasing,so the techniques with high throughput,high sensitivity and low cost will become a priority in the future. 全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTODAY&filename=ZGDW202012016&v=Bh8a8ZT44%25mmd2BVv3mciFa8kv4rOzUDjKYUNajRapQZ5zhlukwGnapMOdiSX0e2zkBbw
2020-12-03
口蹄疫通过云南中缅边境活牛走私传入我国的定量风险评估
受国内市场需求和价格等影响,在与缅甸、老挝接壤的云南边境地区,活牛走私现象屡禁不止,成为口蹄疫传入我国的主要风险途径。为分析云南中缅边境活牛走私传入口蹄疫的可能性,收集云南边境地区活牛非法调入途径、路线和数量等信息,利用“情景树”法建立传入风险随机模型进行仿真分析。结果显示:从云南中缅边境走私调入1头牛,口蹄疫传入我国的概率为0.81%(95%CI,0.43%~1.47%),且每年约有14 017头(95%CI,7 520~25 660头)口蹄疫感染牛自中缅边境走私进入云南省;口蹄疫通过中缅边境活牛走私传入我国的风险取决于缅甸北部活牛市场内的口蹄疫流行率。结果表明,云南中缅边境活牛走私导致口蹄疫传入我国的可能性不容忽视。建议对缅甸活牛以及过境缅甸活牛入境前进行口蹄疫疫苗免疫,并在云南当地有效监管下屠宰;允许从东南亚国家进口符合我国进口检验标准的冷冻去骨肉。本研究首次对口蹄疫通过中缅边境活牛走私传入我国的风险进行了定量评估,为我国口蹄疫防控提供了重要信息和数据。Quantitative Risk Assessment on FMD Introduced into China via Smuggled Live Cattle across China-Myanmar Border in Yunnan ProvinceAs being affected by the need and price in domestic markets,activities of smuggling live cattle have continued in Yunnan frontier areas bordering Myanmar and Laos in spite of repeated prohibition,which have posed an important risk pathway for foot and mouth disease(FMD)to be introduced into China. In order to analyze the probability of FMD introduced into China via smuggled live cattle across China-Myanmar border in Yunnan Province,collect the information concerning the pathways,routes and quantity of illegally imported live cattle,a stochastic model of entry risk was established and analyzed by the“scene tree”. The results showed that when one cattle was smuggled via the border,the probability to introduce FMD into China was 0.81%(95%CI,0.43%–1.47%),what was worse,about 14 017 cattle(95%CI,720–25 660 cattle)infected with FMD were smuggled into Yunnan Province every year;and such risk depended on the prevalence in cattle markets in northern Myanmar. It was concluded that the probability to introduce FMD into China through smuggling live cattle from Myanmar should not be ignored. In view of the above,the live cattle that were from or transit via Myanmar should be vaccinated against FMD prior to entry,and slaughtered under effective supervision by local authorities;and frozen boneless meat that conformed to China's import inspection standards should be allowed to be imported from Southeast Asian countries. In the study,the risk to introduce FMD into China via China-Myanmar border was quantitatively evaluated for the first time,which provided important information and data for the prevention and control of FMD in China.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTODAY&filename=ZGDW202012002&v=Bh8a8ZT44%25mmd2BUG3I6hkBCxo4jms8VPlghmnIEoyLK0k1b1hIhtiJ70eTzdWrwJ8QAs
2020-12-03
毛皮动物新冠病毒监测简报
2019新型冠状病毒病(COVID-19)给公共卫生安全带来巨大危害,但目前引起该病的新型冠状病毒(SARS-CoV-2)来源还未有定论。现有证据显示,SARS-CoV-2与分离自蝙蝠和穿山甲的 SARS-CoV-2病毒株同源性相近,表明蝙蝠和穿山甲可能作为SARS-CoV-2的中间宿主存在。目前,丹麦、荷兰和美国等国家已有人工饲养水貂及饲养场工作人员感染SARS-CoV-2的报道。试验研究显示,SARS-CoV-2可在雪貂上呼吸道复制,且雪貂对SARS-CoV-2高度易感,表明雪貂可作为一种SARS-CoV-2感染和传播的动物模型。2020年2—7月,本课题组对山东、吉林、辽宁、黑龙江等毛皮动物主要养殖省份的水貂样品进行SARS-CoV-2病原及抗体监测。利用WHO推荐的real-time RT-PCR检测方法,对328份水貂内脏组织及咽拭子样品进行检测,结果所有样品均为SARS-CoV-2阴性;采用北京万泰生物药业有限公司提供的SARS-CoV-2抗体检测ELISA试剂盒,对35份水貂血清样品进行抗体检测,结果样品中抗SARS-CoV-2抗体均为阴性。由于国内养殖多为密集型庭院养殖,养殖人员与水貂密切接触,且国外已有养殖场水貂大规模感染SARS-CoV-2的先例,为保障毛皮动物行业健康发展,此次监测重点对我国北方地区毛皮动物养殖场进行采样,结果并未发现毛皮动物携带SARS-CoV-2和针对SARS-CoV-2的抗体。这可能与目前我国人群的低SARS-CoV-2感染率和未有养殖场工人感染有关。本实验室已建立了检测毛皮动物及宠物的SARS-CoV-2抗原及抗体检测方法,在目前全球COVID-19疫情的严峻形势下,未来仍需对毛皮动物及宠物进行长期的SARS-CoV-2流行病学监测。Overview on the Surveillance of SARS-CoV-2 in Fur Animals The coronavirus disease 2019(COVID-19)has brought great harm to public health safety,but the source of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)causing COVID-19 has not been concluded. It was shown that,by available evidences,the virus was with similar homology with the SARS-CoV-2 isolated from bats and pangolins,which indicated that bats and pangolins might act as the intermediate hosts for the virus. It was reported that captive minks and farm workers had been infected with SARS-CoV-2 in Denmark,Netherlands and the United States. According to related study,SARS-CoV-2 could replicate on the upper respiratory tract of ferrets who were highly susceptible to the virus,which indicated that ferrets could be used as an animal model of infection and transmission of the virus.In February to July of 2020,the mink samples collected from Shandong,Jilin,Liaoning,Heilongjiang and other provinces where most fur animals were kept were monitored for pathogen and antibody of SARS-CoV-2,328 visceral tissue samples and throat swabs were detected by the real-time RT-PCR assay recommended by WHO,and all the samples turned out to be negative;meanwhile,35 serum samples were detected by use of the ELISA kit provided by Beijing Wantai Biological Pharmacy,and all the samples were negative for the antibodies against SARS-CoV-2. The operators had been in close contact with minks due to intensive courtyard farming,especially,there had been cases of minks infected with SARS-CoV-2 on a large scale in foreign countries. In order to safeguard healthy development of fur animal industry,the farms were monitored and sampled as a priority. No SARS-CoV-2 or relevant antibody were found in any fur animals,which might benefit from low infection rate in populations and the absence of infection in farm workers in China. The method for detecting pathogen and antibody of SARS-CoV-2 in fur animals and pets was established by our laboratory,and epidemiological surveillance for SARS-CoV-2 in fur animals and pets should be continued for long term in the future in response to current severe situation of COVID-19 across the world.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTODAY&filename=ZGDW202012001&v=Bh8a8ZT44%25mmd2BV6mndOdWwC3lCFYz%25mmd2F9tG%25mmd2B0Yu7LUoQYiF8eJU%25mmd2FuGJY16mnuTkcHAh0e
2020-12-03
重组禽流感病毒(H5+H7)三价灭活疫苗免疫鸡群的临床效果评价
为评价重组禽流感病毒(H5+H7)三价灭活疫苗(H5N1 Re-11株+Re-12株,H7N9 H7 Re-2株)免疫鸡群的临床应用效果及当前禽流感免疫程序的合理性,对河南省使用重组禽流感病毒(H5+H7)三价灭活疫苗免疫的940个存栏500只以上的养鸡场开展临床应用效果调查,并抽取328个场开展免疫抗体检测。结果显示:免疫后1周内出现不良反应的养鸡场190个,占20.21%(190/940);发病的养鸡场140个,占14.89%(140/940);出现死亡的养鸡场51个,占5.43%(51/940)。免疫后1周内出现不良反应的鸡只占0.11%,发病的占0.09%,死亡的占0.04%。血清样品的血凝(HA)和血凝抑制(HI)试验结果显示:H5亚型Re-11株场群合格率为97.56%,免疫抗体阳性率为94.20%;H5亚型Re-12株场群合格率为96.34%,免疫抗体阳性率为92.70%;H7亚型Re-2株场群合格率为98.17%,免疫抗体阳性率为94.67%。结果表明:重组禽流感病毒(H5+H7)三价灭活疫苗的鸡群免疫效果较好,免疫后的临床不良反应率极低。但在免疫过程中,应尽量消除影响疫苗临床效果的不利因素,定期开展免疫效果监测,及时调整免疫程序。本研究为全国高致病性禽流感疫苗的应用提供了参考。Evaluation on the Clinical Application Effect of Recombinant Avian Influenza Virus(H5+H7)Trivalent Inactivated Vaccines in Chickens In order to evaluate the clinical application effect of recombinant avian influenza virus(H5+H7)trivalent inactivated vaccines(H5N1 Re-11+Re-12 strain and H7N9 H7 Re-2 strain)in chickens and the rationality of current vaccination program,940 farms with more than 500 chickens were investigated in Henan Province,in which,328 farms were selected for detection of immune antibody. It was found that,within one week after vaccination,some adverse reactions revealed in 190 farms,accounting for 20.21%(190/940);avian influenza outbreaks in 140 farms were detected,accounting for 14.89%(140/940);and dead cases were found in 51 farms,accounting for 5.43%(51/940). The proportions of chickens with adverse reactions,being infected and dead were 0.11%,0.09% and 0.04%,respectively. It was found that,by hemagglutination(HA)and hemagglutination inhibition(HI)test for serum samples,the farm/flock qualified rates of H5 Re-11,H5 Re-12 and H7 Re-2 strain were 97.56%,96.34% and 98.17%,respectively,and their positive rates of immune antibodies were 94.20%,92.70% and 94.67%,respectively. It was concluded that the recombinant avian influenza virus(H5+H7)trivalent inactivated vaccine could receive good effect in chickens,with lower level of adverse reaction. However,any adverse factors therefrom should be eliminated to the greatest extent during the process of vaccination,which should be monitored regularly,and the vaccination program should also be timely adjusted. Some references were provided for the application of vaccines against highly pathogenic avian influenza virus in China.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202011022&v=Bh8a8ZT44%25mmd2BViTsUIpnMUhMdfl8JdNVN4nOcJGcWypRTOzA9syj9XPAWc4KBTGMQp
2020-11-06
高致病性猪繁殖与呼吸综合征病毒自然感染与人工感染下的组织病理学观察
为进一步研究高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV)的致病机理,对HP-PRRSV自然感染和人工感染下的病理组织学变化进行比较观察。在组织病理学变化方面,自然感染病例病变性质与人工感染基本相同,但在病变范围以及严重程度方面存在明显差异:自然感染病例的胃肠道病变尤为显著,而人工感染病例却较少引发消化道病变;自然感染病例的炎症波及全身淋巴结,而人工感染病例以肺门淋巴结、下颌淋巴结、肠系膜淋巴结病变最为明显,其他部位淋巴结未见明显病变。自然感染病例中,常见多种病原的混合感染,提示HP-PRRSV可能作为多种疾病的基础病因。自然感染和人工感染病例在肺脏上均表现为严重的间质性肺炎,说明肺脏部位的病理变化对单纯HP-PRRSV感染的诊断具有指证意义。本研究为HP-PRRSV感染的诊断及其致病机理研究提供了理论依据。Histopathological Observation on Natural and Artificial Infection with HP-PRRSVIn order to further study the pathogenesis of highly pathogenic porcine reproductive and respiratory syndrome virus(HP-PRRSV),the histopathological changes of natural and artificial infection with HP-PRRSV were compared and observed. For the nature of lesions,the natural and artificial infection cases were basically similar,but obviously different in the scope and severity degree of the lesions;the gastrointestinal lesions were particularly significant in the natural infection cases,and fewer gastrointestinal lesions were observed in the artificial ones;all the lymph nodes were involved in the inflammation of the natural infection cases,but the lesions of hilar lymph nodes,mandibular lymph nodes and mesenteric lymph nodes appeared in the artificial infection cases,and other lymph nodes were normal. Mixed infection with multiple diseases were common in natural infection cases,indicating that HP-PRRSV might be the general cause for several diseases. Severe interstitial pneumonia was shown in the lungs of both natural and artificial infection cases,which indicated that the pathological changes of the lungs were indicative for the diagnosis of single infection with HP-PRRSV. As a conclusion,a theoretical basis was provided for the diagnosis of HP-PRRSV infection and for the study on its pathogenesis.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202011021&v=Bh8a8ZT44%25mmd2BUVIUjuVi2E9SXrkHJjA%25mmd2F8oezzoQP1ttRyl97yDc5jZ6uBPwCc0mNOi
2020-11-06
重组PCV3 Cap蛋白在酵母细胞中的表达与鉴定
猪圆环病毒3型(PCV3)是影响我国养猪业的重要病原之一,目前尚无商品化疫苗用于防控该病。Cap蛋白是PCV3主要的抗原蛋白。为构建高水平表达可溶性PCV3 Cap蛋白的工程菌株,对Cap蛋白NLS序列进行定点突变,构建重组表达载体pPICZαA-PCV3,经转染、酵母细胞表达以及纯化,最终获得可溶性PCV3 Cap蛋白。随后对纯化蛋白进行Western Blot验证及电镜观察。结果显示:获得的PCV Cap蛋白大小约27 kDa;电镜下观察可见均一的病毒样颗粒(VLP),颗粒直径约18 nm。PCV3 Cap蛋白在酵母细胞中的成功表达为进一步开发Cap蛋白亚单位疫苗奠定了基础。Expression and Identification of Recombinant PCV3 Cap Protein in Yeast CellsPorcine circovirus type 3(PCV3)is one of the most serious pathogens that would affect pig industry in China,but no commercial vaccine has been available till now. In order to establish an engineered strain that could express soluble PCV3 Cap protein at a high level,considering that Cap protein was the main antigenic protein of PCV3. The nuclear localization sequence(NLS)of Cap protein was specifically mutated to establish the recombinant plasmid pPICZαA-PCV3,after transfection of the plasmid as well as expression and purification of recombinant protein,the soluble PCV3 Cap protein was obtained. Then the purified protein was verified by Western Blot and observed under electron microscope. It was concluded that the PCV Cap protein obtained was about 27 kDa,and uniform virus-like particles(VLP)with the diameter of 18 nm were observed under electron microscope. The successful expression of PCV3 Cap protein in yeast cells laid a foundation for further development of Cap protein subunit vaccines.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202011019&v=Bh8a8ZT44%25mmd2BW%25mmd2B2F5ndRK1P%25mmd2FQzKJxnpMmCaZMw9aPlw6lKWcwzPB1vWxk9I2BRd7GJ
2020-11-06
同源血浆及血清中蓝舌病病毒抗体ELISA检测结果比较
为简化动物血液样品中蓝舌病病毒(BTV)检测及研究方法,采用2种酶联免疫吸附试验(ELISA),对采自云南省德宏州芒市一家肉牛养殖场的30对牛同源血浆及血清样品进行BTV总抗体及VP7抗体检测。BTV总抗体ELISA检测结果显示,同源血浆与血清之间的阳/阴性结果一致率为100%,ΔPI在±5%以内的样品占87%;VP7抗体ELISA检测结果显示,同源血浆与血清之间的阳/阴性结果一致率为93%,ΔOD在±0.05范围内的样品占87%。结果表明:利用ELISA检测动物血样中BTV抗体时,血浆样品可替代血清样品;血浆样品的溶血程度不影响检测结果,但粗制血浆中的非溶解性杂质可能影响检测结果,需要使用离心除杂后的血浆样品。Comparison of the Test Results of Antibody against Bluetongue Virus in Homologous Plasma and Serum by ELISAIn order to simplify the test and research method of bluetongue virus(BTV)in animal blood samples,30 pairs of bovine homologous plasma and serum samples collected from a beef cattle farm in Mang City,Dehong Prefecture,Yunnan Province were tested for antibodies against BTV and VP7 protein. The results showed that,for the detection of total antibodies of BTV,the consistency rate of the positive/negative results of homologous plasma and serum was 100%,and samples with ΔPI less than ±5% accounted for 87%;as tested by VP7 Ab ELISA kit,the consistency rate of the positive/negative results of homologous plasma and serum was 93%,and samples with ΔOD less than ±5% accounted for 87%. Therefore,serum samples might be replaced by plasma samples when the antibody against BTV in animal blood was tested by ELISA,and the test results could not be affected by the hemolysis level of plasma samples rather than the insoluble impurities in crude plasma which should be removed by centrifugation before being used.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202011018&v=Bh8a8ZT44%25mmd2BX2Lfl3uaiMzu%25mmd2Bl%25mmd2B1qun3qkzoQgjVsMP8i3HGEkCPfr%25mmd2FsdtRDXv8w98
2020-11-06
单重PCR鉴别犬4种布鲁氏菌检测方法的建立
为建立适合犬布鲁氏菌病临床检测的单重PCR方法,通过对比粗糙型(犬种)与光滑型(羊种、牛种和猪种)布鲁氏菌基因组DNA的序列差异,设计1对引物并优化反应条件,建立了可初步鉴别犬4种布鲁氏菌的PCR方法,然后对该方法的特异性和灵敏度进行评价,并对20份犬布鲁氏菌血清学阳性的血液样品进行临床检测。结果显示,利用建立的PCR反应体系对牛种、羊种和猪种布鲁氏菌均能扩增出717 bp的目的条带,对犬种布鲁氏菌能扩增出366 bp的目的条带;最低可检测到犬种、羊种、猪种和牛种布鲁氏菌基因组DNA浓度分别为5.07×10-2、6.20×10-2、7.80×10-3和5.50×10-2 ng/μL;20份血液样品中共检测到3份犬种布鲁氏菌阳性样品,与使用GB/T 18646—2018引物的PCR检测结果一致。结果表明,本试验建立的单重PCR方法简捷、特异、敏感,适合基层兽医实验室对犬布鲁氏菌病的检测与鉴定。Development of a Single PCR Assay for Detection of Four Kinds of Brucella in DogsIn order to establish a single PCR assay appropriate for detection of brucella in dogs,one pair of primers were designed through comparison of genomic DNA sequences between rough phenotype(B. canis)and smooth phenotype(B. melitensis,B. abortus and B. suis)strains,after optimization of reaction conditions,a PCR assay was established for preliminary detection of the four kinds of brucella,then its specificity and sensitivity were evaluated,and 20 Brucella-seropositive blood samples from dogs were tested clinically. The results showed that,for B. melitensis,B. abortus and B. suis,the fragment with the length of 717 bp could be amplified by the established method,and for B. canis,the fragment with the length of 366 bp could be amplified;the lowest genomic DNA concentrations of B. canis,B. melitensis,B. suis and B. abortus were 5.07×10-2,6.20×10-2,7.80×10-3 and 5.50×10-2 ng/μL,respectively;three positive samples for B. canis were detected out in 20 blood samples,which was consistent with the PCR results of using GB/T 18646—2018 primers. As a conclusion,the established assay was appropriate for detection of brucella in dogs in field veterinary laboratories due to its advantages of simplicity,specificity and sensitivity.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202011016&v=Bh8a8ZT44%25mmd2BV4D1fobHDy%25mmd2BF74F2eWRbGd1dXZ%25mmd2FFC%25mmd2FuzaTjuc0l4HBh%25mmd2BouBJZqFqPU
2020-11-06