猪急性腹泻综合征病毒RT-PCR检测方法的建立及应用
为建立一种鉴定猪急性腹泻综合征病毒(SADS-CoV)的RT-PCR方法,根据NCBI登录的SADS-CoV N基因保守区域序列,设计了3对引物(P1、P2和P3),通过对SADS-CoV及其他6种病毒进行检测,筛选出最合适的引物,然后对退火温度、引物浓度、dNTPs浓度、rTaq DNA聚合酶浓度和循环次数等反应条件进行了优化。结果显示,P3引物对SADS-CoV特异性最好,与其他病毒无交叉反应。敏感性试验显示,该方法最低检测限可达9.55×102 copies/μL;重复性试验显示该方法重复性良好。运用建立的RT-PCR方法,对广东省的21份临床样品进行检测,结果检出SADS-CoV阳性样品4份,阳性样品的测序结果与其RT-PCR完全一致。结果表明,本研究成功建立了一种鉴定SADS-CoV的RT-PCR方法,且该方法具有检测快速、成本廉价、特异性强、敏感性高和重复性好的优点,可以用于SADS-CoV的临床诊断。Establishment and Application of a RT-PCR Assay for Detection of Porcine Acute Diarrhea Syndrome CoronavirusIn order to establish a RT-PCR assay for detection of porcine acute diarrhea syndrome virus(SADS-CoV),three pairs of primers(P1,P2 and P3)were designed based on N gene sequence of SADS-CoV registered in NCBI,and the most appropriate primer was selected through the detection of SADS-CoV and other six kinds of viruses,followed by the optimization of reaction conditions including annealing temperature,primer concentration,dNTPs concentration,rTaq DNA polymerase concentration,cycle index,etc. The results showed that P3 primer was with the best specificity against SADS-CoV,and failed to react with other viruses. It was shown that,by sensitivity test,the lowest detection limit was 9.55×102 copies/μL;the established assay was with good repeatability as tested. 21 clinical samples from Guangdong were detected by the RT-PCR assay,4 positive samples were found,which was consistent with the sequencing results. Therefore,a RT-PCR assay was successfully established for detection of SADS-CoV,which was characterized by rapid detection,low cost,strong specificity,high sensitivity and good repeatability,and could be used for identification of SADS-CoV in practice.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202011015&v=Bh8a8ZT44%25mmd2BV3EfgMfB9vvEyWkNTMtTs%25mmd2BzGvsReoG5nuBXoFx9PNzPdOLTK1PFyhD
2020-11-06
环境DNA应用研究进展
近年来,环境DNA(eDNA)应用发展迅速,已涉及食品微生物、生物监测、群落生态学、古环境研究、保护生物学和入侵生态学等多个领域。基于eDNA的研究手段具有非侵入性、易于取样和能够探测隐秘物种等优点,因此eDNA是生态系统广泛直接取样的一种有吸引力的替代品。本文综述了eDNA的定义和发展历史,介绍了DNA条形码选择和设计、样品收集、DNA抽提、DNA扩增测序、DNA条形码数据分析等eDNA研究步骤,描述了eDNA在外来物种入侵、物种检测、生物量估算、水中生态系统监测等领域的应用,总结出eDNA研究中存在的问题,并展望该方法的应用前景。本文为全面了解eDNA的应用提供了参考。 Research Progress on the Application of Environmental DNAIn recent years,environmental DNA(eDNA)has been widely used in various fields,including food microbiology,biological monitoring,community ecology,study on palaeo-environment,conservation biology and invasion ecology,etc. As the study methods of eDNA were advantaged by non-invasiveness,convenience for sampling and detection of cryptic species,eDNA could be considered as an attractive alternative to extensive direct sampling in ecological system. In this paper,its definition and development history were summarized;the study steps were introduced from the aspects of DNA barcode selection and design,sample collection,DNA extraction,DNA amplification and sequencing,and analysis on DNA barcode data;then its application in such fields as alien species invasion,species detection,biomass estimation and aquatic ecosystem monitoring,was described;all problems encountered during the above steps were summarized,and its application prospect was prospected. The review provided some references to fully learn the application of eDNA. 全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202011014&v=Bh8a8ZT44%25mmd2BXhkriUVQ308eGbsO7mbLugIY40POMzvPIr%25mmd2F7pVBhEQB7yCBgcfpV7P
2020-11-06
卤虫作为生物饵料传播虾类病原风险的研究进展
卤虫是虾类育苗的关键性生物饵料,其是否具有传播虾类病原的风险已引起多方关注。本文梳理了卤虫作为生物饵料传播虾类病原的研究进展,并提出了防控建议。卤虫是白斑综合征病毒(white spot syndrome virus,WSSV)的机械携带者,也可能是对虾传染性肌坏死病毒(infectious myonecrosis virus,IMNV)、肝胰腺细小病毒(hepatopancreatic parvovirus,HPV)、罗氏沼虾野田村病毒(Macrobrachium rosenbergii nodavirus,MrNV)和极小病毒(extra small virus,XSV)的传播载体,但是目前的研究由于缺少原位杂交或组织细胞水平的病理学证据,均尚未证实卤虫可被这些病毒直接感染;研究已证明卤虫可被副溶血弧菌(Vibrio parahaemolyticus)、坎贝氏弧菌(V. campbellii)和哈维氏弧菌(V. harveyi)等水产养殖致病菌感染,存在传播急性肝胰腺坏死病(acute hepatopancreatic necrosis disease,AHPND)的风险。此外卤虫可能被虾肝肠胞虫(Enterocytozoon hepatopenaei,EHP)污染而存在传播风险。因此,建议系统开展卤虫的病原学和流行病学研究,通过规范卤虫卵生产管理和孵化操作、探索卤虫生物安保产业发展途径和替代产品等措施,降低卤虫传播相关病原的风险。本文可为正确使用卤虫等生物饵料提供技术支持,为水产养殖绿色发展和生物安保体系建设提供参考。 Research Progress on the Risk of Artemia Acting as a Kind of Live Feed to Spread Shrimp PathogensArtemia is a kind of critical live feed for shrimp breeding,whether it could bring the risk of spreading pathogens has been concerned by the society. In this paper,the research progress on Artemia acting as a kind of live feed to spread pathogens was summarized,and relevant suggestions were put forward. Artemia served as the physical carrier for white spot syndrome virus(WSSV),as the vector carrying infectious myonecrosis virus(IMNV),hepatopancreatic parvovirus(HPV),Macrobrachium rosenbergii nodavirus(MrNV),and extra small virus(XSV). However,current studies have not proved that Artemia can be infected with the above-mentioned viruses,due to luck of evidences of in situ hybridization or other cyto-or histopathology. It was demonstrated that Artemia can be infected with aquaculture pathogenic bacteria,such as vibrio parahaemolyticus,V. campbellii and V. harveyi,resulting in a risk of spreading acute hepatopancreatic necrosis disease(AHPND). Additionally,enterocytozoon hepatopenaei(EHP)may be transimitted through Artemia by contamination. Therefore,it was suggested to systematically carry out the study on the etiology and epidemiology of artemia,and reduce the risk of spreading pathogens through standardizing production management or hatching control of artemia cytes,identifying the biosecurity pathways of artemia and developing alternative products. Technical support could be provided to correctly use artemia and other live feed and some references were provided for construction of a biosecurity system and for healthy development of aquaculture.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202011013&v=Bh8a8ZT44%25mmd2BXAoeiGBd4M8wAjjf78KMC0IGJlzeY1p3prVG5rt1B7dRNsg%25mmd2BjdZ%25mmd2FLC
2020-11-06
2016—2019年我国I类新城疫病原学监测与遗传进化分析
为掌握我国I类新城疫病毒(Newcastle disease virus,NDV)的分布及分子演化特征,2016—2019年,随机从国内23个省(自治区、直辖市)998个采样点,采集家禽口咽/泄殖腔双拭子样本、野鸟粪便样本和环境样本共45 458份,通过病毒分离和RT-PCR鉴定,共分离到I类NDV 558株。从时间分布看,2016—2019年I类NDV分离率分别为0.95%、0.89%、1.88%和1.37%,有增高趋势;从地理分布看,我国16个省(自治区、直辖市)分离到I类NDV,主要集中于华东、西南和西北地区;从场点分布看,活禽批发市场和农贸市场的I类NDV阳性率明显高于养殖场和屠宰场;从宿主分布看,I类NDV主要来源于鸡和鸭,少量来自鹅、鸽和环境样本;从基因型分布看,558株I类NDV属于2个基因亚型,其中1.1.2亚型为近年来我国流行的主要基因亚型。研究表明,我国I类NDV污染面广,宿主范围广泛,且存在2种基因亚型。研究提示,应加强I类NDV的免疫、监测,同时加强饲养管理,提高家禽抵抗力,降低病毒基因变异致毒力增强的风险。本研究为我国科学防控新城疫提供了参考数据。Pathogenic Surveillance and Phylogenetic Analysis on Class I Newcastle Disease Virus during 2016 to 2019 in ChinaIn order to study the distribution and molecular evolution characteristics of class I Newcastle disease virus(NDV)in China,a total of 45 458 samples including avian oropharynx/cloacal swabs,wild bird feces and environmental samples were randomly collected from 998 sampling sites in 23 provinces(autonomous regions and municipalities)from 2016 to 2019,and 558 strains of class I NDV were isolated through virus isolation and RT-PCR. For time distribution,the isolation rates from 2016 to 2019 were 0.95%,0.89%,1.88% and 1.37%,respectively,and tended to increase;for geographical distribution,such NDV strains were isolated from 16 provinces(autonomous regions and municipalities),particularly in East,Southwest and Northwest China;for farms/premises,the positive rate of NDV in live poultry markets and agricultural markets was obviously higher than that in farms and slaughterhouses;for host distribution,the NDV strains were mainly derived from chickens and ducks,and fewer from geese,pigeons and environment;and for genotype distribution,the isolated strains fell into two genotypes,in which,1.1.2 subtype was predominant in China in recent years. It was concluded that the NDV was widely spread with a large range of hosts and two genotypes. Therefore,the vaccination and surveillance against NDV should be strengthened,meanwhile,feeding management should be enhanced so as to improve relevant resistance of poultry and reduce the risk of increased virulence produced by virus genetic variation. In view of the above,some reference data were provided for scientific prevention and control of NDV in China.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202011001&v=Bh8a8ZT44%25mmd2BUut1OAVi1CyFvtFbyONbzZR0JlI3CIO8pIj5EAp%25mmd2B61e0YSoH%25mmd2FA7kcV
2020-11-06
猪繁殖与呼吸综合征病毒GP5蛋白的原核表达及多克隆抗体制备
为体外表达猪繁殖与呼吸综合征病毒(PRRSV)糖基化囊膜蛋白GP5,以及制备其多克隆抗体,以PRRSV PC疫苗株RNA为模板,应用RT-PCR扩增GP5基因,并克隆至原核表达载体pET-32a(+),构建重组表达载体pET-32a-GP5。经过酶切和测序鉴定后,将阳性重组质粒转化到大肠杆菌BL21(DE3)感受态细胞中,加入IPTG低温诱导表达。使用亲和层析法纯化PRRSV GP5蛋白。然后将纯化的蛋白免疫北京大耳白兔制备多克隆抗体,并进行酶联免疫吸附试验(ELISA)和间接免疫荧光分析(IFA)。结果显示,表达的PRRSV GP5原核蛋白以可溶性和包涵体两种形式存在,相对分子质量大约30 kDa;制备的GP5蛋白多克隆抗体ELISA效价可达1:64 000;IFA检测显示,制备的多克隆抗体能够识别PRRSV。重组PRRSV GP5蛋白及其多克隆抗体的成功制备为PRRSV血清学检测方法的建立提供了良好的生物学材料。Prokaryotic Expression of GP5 Protein of PRRSV and Preparation of Its Polyclonal AntibodyIn order to express the glycosylated envelope protein GP5 of porcine reproductive and respiratory syndrome virus(PRRSV)in vitro,and to prepare its polyclonal antibody,GP5 gene was amplified by RT-PCR by taking the RNA of PRRSV PC vaccine strain as a template,then cloned into the prokaryotic expression vector pet-32a(+)to construct the recombinant expression vector of pET-32a-GP5. After enzyme digestion and sequencing analysis,the positive recombinant plasmid was transformed into E. coli BL21(DE3)cells,and induced by IPTG at low temperature. The PRRSV GP5 protein was purified by affinity chromatography,and then was vaccinated into Beijing white rabbit to prepare polyclonal antibody,then enzyme linked immunosorbent assay(ELISA)and indirect immunofluorescence analysis(IFA)were carried out. The results showed that the expressed GP5 prokaryotic protein was available in soluble supernatant and inclusion body,with a molecular weight of approximate 30 kDa;the ELISA titer of the polyclonal antibody against GP5 protein was up to 1:64 000,and it was shown that PRRSV could react with the polyclonal antibody. In conclusion,good biological materials were provided for establishing serological detection methods for PRRSV through successful preparation of recombinant PRRSV GP5 protein and its polyclonal antibody.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202010023&v=Bh8a8ZT44%25mmd2BWcYOYsnvnN1LgJcL%25mmd2B%25mmd2Fr4rETku%25mmd2BJXQKrBmkCRpxQZ4AMvi%25mmd2FkTKYxYSI
2020-10-14
猪瘟病毒C株检测用国家核酸标准物质的研制
为研制均匀、稳定的猪瘟病毒(CSFV)核酸标准物质,选择CSFV C株接种于PK-15细胞并收获病毒液,然后经过病毒灭活、分装、冻干,最终形成核酸标准物质。用微滴式数字PCR对制备的CSFV标准物质进行均匀性和稳定性评估、标准物质合作定值,在评定不确定度后计算得出最终量值结果,并开展临床试用。结果显示,该标准物质均匀,4 ℃条件下可稳定存放4周,25 ℃可稳定存放1周,-20 ℃可稳定存放11个月,量值结果为(5.1±0.7)×105 copies/mg;临床试用显示,所制备的标准物质与临床样本的互通性良好。本研究制备的CSFV C株国家核酸标准物质均一性良好、稳定、量值准确度高,并通过了全国标准物质管理委员会评定,可用于动物及动物产品质量安全检测,从而有效保障猪瘟防治工作的开展。Preparation of National Nucleic Acid Reference Materials of CSFV C Strain for Detection In order to prepare a kind of uniform and stable nucleic acid reference materials of classical swine fever virus(CSFV),CSFV C strain was selected and inoculated into PK-15 cells to obtain viral fluid,followed by the process of inactivation,subpackage and freeze-drying,then the nucleic acid reference materials were produced. The homogeneity and stability of prepared materials were evaluated by droplet digital PCR,and the value of materials was determined by 8 collaborative laboratories,the quantity value was ultimately calculated after evaluation of uncertainty,then a clinical trial was carried out. The results showed that the material was homogeneous and could be stored stably for four weeks at 4 ℃,for one week at 25 ℃,and for 11 months at -20 ℃,the quantity value was(5.1±0.7)×105 copies/mg;it was found that the materials showed good interactivity with clinical samples in clinical trial. In conclusion,the prepared materials were advantaged by good homogeneity,stability and high accuracy,which had been successfully evaluated by National Administrative Committee for CRM's and could be used in the field of quality and safety detection of animals and animal products,so as to effectively guarantee the prevention and control of CSFV.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202010022&v=Bh8a8ZT44%25mmd2BUcz357Gp6m%25mmd2FNVNsXMlCDIj%25mmd2BoMrJ31MCG%25mmd2FRTZxo4dMrPllwgYbd35ap
2020-10-14
PCV4 SYBR Green I实时荧光定量PCR方法的建立及应用
为建立一种灵敏、可快速检测猪圆环病毒4型(PCV4)的SYBR Green I实时荧光定量PCR方法,根据PCV4 Rep基因的保守区域设计引物,建立针对PCV4的SYBR Green I实时荧光定量PCR检测方法,并对该方法的特异性、灵敏性和重复性进行评价。结果显示,该检测方法的Ct值与标准品在5.64×102~5.64×109 copies/μL范围内呈良好的线性关系,R2为0.999,斜率为-3.638,检测下限为5.64×102 copies/μL,且无非特异性扩增。利用该方法对56份临床样品进行检测,发现PCV4核酸阳性率为3.57%,比普通PCR更灵敏可靠。结果表明,本试验建立的SYBR Green I实时荧光定量PCR方法可用于PCV4的快速诊断。Establishment and Application of SYBR Green I Real-time PCR for Detection of PCV4In order to rapidly and sensitively detect porcine circovirus type 4(PCV4),the SYBR Green I real-time quantitative PCR assay was established based on the primers that were designed according to the conserved region of PCV4 Rep gene. Then its specificity,sensitivity and repeatability were evaluated. The results showed that its Ct value remained a good linear relationship with the standard materials within the range of 5.64×109~5.64×102 copies/μL,specifically,R2 was 0.999,the slope was -3.638,and the detection limit was 5.64×102 copies/μL without non-specific amplification. 56 clinical samples were tested by the method,it was found that the nucleic acid positive rate of PCV4 was 3.57%,which was more sensitive and reliable than that of conventional PCR. Therefore,the established SYBR Green I real-time PCR method could achieve the rapid detection of PCV4.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202010021&v=Bh8a8ZT44%25mmd2BX%25mmd2B788oXBniPXK%25mmd2BKSJj9SS83bukL0TwOF3EqaFRYut6xL7od5eXFwsk
2020-10-14
血清型特异性水泡性口炎病毒同步检测和鉴别方法的建立
为准确地鉴别诊断新泽西型水泡性口炎病毒(VSV-NJ)和印第安纳型水泡性口炎病毒(VSV-IND),本研究建立了可以同时检测和鉴别诊断VSV-NJ和VSV-IND的双重荧光RT-PCR方法,并对方法性能进行了测试和初步应用。结果显示,建立的双重荧光RT-PCR特异性为100%,最低检测限1~10个拷贝/μL,扩增效率E值均在99%左右,R2值分别为0.999和0.998。VSV-NJ和VSV-IND单荧光RT-PCR与双重荧光RT-PCR的相关性系数分别为0.999 7和0.999 4,与单荧光RT-PCR相比,该引物和探针的双重体系混合对特异性和敏感性均未产生明显干扰。用已知VSV-NJ及VSV-IND阳性核酸验证,发现符合率为100%,对50份临床样品的检测均为阴性,与单荧光RT-PCR结果一致。本研究建立的双重荧光RT-PCR方法实现了VSV-NJ和VSV-IND的同步检测和鉴别,为水泡性口炎的防控提供了技术储备。Establishment of a Duplex Real-time RT-PCR for Detection and Identification of Serotype-specific VSV StrainsIn order to accurately identify and differentiate the serotype-specific strains of New Jersey type of vesicular stomatitis virus(VSV-NJ)and Indiana type of vesicular stomatitis virus(VSV-IND),a duplex real-time RT-PCR method was established for simultaneous detection of the two serotypes of viruses,and its performance was tested and initial application was carried out. The results showed that the specificity of the established method was 100%,with the lowest detection limit of 1~10 copies/μL,both of the amplification efficiencies(E)were about 99%,and R2 values were 0.999 and 0.998,respectively. The correlation coefficients between singular real-time RT-PCR and duplex real-time RT-PCR for VSV-NJ and VSV-IND were 0.999 7 and 0.999 4,respectively. Compared to singular real-time RT-PCR,both the specificity and sensitivity were not affected by the mixture of the primers and probes. The coincidence rate was 100% as verified by the known positive nucleic acids of VSV-NJ and VSV-IND,and 50 clinical samples were tested to be negative,which was consistent with the results by singular real-time RT-PCR. As a conclusion,the method established in this study could simultaneously detect and differentiate VSV-NJ and VSV-IND,which would provide technical reserves for prevention and control of VSV.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202010020&v=Bh8a8ZT44%25mmd2BVeM1YDq6a8EjAd9sCihEm3ObfSAW0yv0S397NvPYV7ZDJgBAp1of7G
2020-10-14
严重急性呼吸综合征冠状病毒2型4种结构蛋白特性分析
为深入了解严重急性呼吸综合征冠状病毒2型(SARS-CoV-2)S、M、E、N蛋白的分子生物学特征和基本结构特征,选取S、M、E、N蛋白序列进行生物信息学分析和预测,使用DNAstar、SignalP、TMHHM、PSORT Prediction、BUSCA和ABCpred软件,分析并相互验证4种蛋白的结构域特征和S蛋白的潜在B细胞抗原表位,结果预测出了4种结构蛋白的功能结构域,并分析预测出S蛋白潜在的B细胞抗原表位为25~29、75~81、112~116、148~152、773~779 aa。研究结果可为SARS-CoV-2相关的分子生物学和结构生物学研究提供参考,并为疫苗开发等提供理论依据。Characteristics of Four Kinds of Structural Proteins of SARS-CoV-2In order to further identify the molecular biological characteristics and general structural characteristics of S,M,E and N proteins of severe acute respiratory syndrome coronavirus type 2(SARS-CoV-2),the amino acid sequences of the above proteins were selected for bioinformatics analysis and prediction. The structural domain characteristics of the above proteins and the potential B cell epitopes of S protein were analyzed and mutually verified by DNAstar,SignalP,TMHHM,PSORT Prediction,BUSCA and ABCpred softwares. The results showed that the functional domains of the four proteins were predicted,and the potential B cell epitopes of S protein was analyzed and predicted to be 25 to 29,75 to 81,112 to 116,148 to152 and 773 to 779 aa. Based on the results,some references were provided for further study on molecular biology and structural biology related to SARS-CoV-2,and theoretical basis was provided for vaccine development.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202010018&v=Bh8a8ZT44%25mmd2BUnxRinWoOlcFhZMdOudDd1JIPIlGMwpAhOsdGh10NuiNy9Bd18v2rS
2020-10-14
犬冠状病毒研究进展
2020年2月,我国香港报道了新冠肺炎(COVID-19)患者家养的宠物犬感染了新型冠状病毒(SARS-CoV-2),这引起了人们对犬冠状病毒的关注。为有助于人们全面了解犬冠状病毒,做好犬冠状病毒病防治,本文从病原学与流行病学、临床症状以及诊断和防治措施等方面进行了综述。犬冠状病毒在犬群中广泛存在,主要包括α冠状病毒属的犬肠炎冠状病毒和β冠状病毒属的犬呼吸道冠状病毒。犬冠状病毒致病性不强,引起的临床症状通常较轻,但对幼犬或与犬细小病毒、犬瘟热病毒等发生混合感染时,会引起较严重症状,且易发生突变,产生新的变异毒株,具有致病性增强的风险。目前,犬冠状病毒检测方法主要包括病原学检测、血清学检测以及RT-PCR、qRT-PCR、纳米PCR等分子生物学检测。犬冠状病毒感染的防治主要通过疫苗免疫以及科学饲养管理、卫生消毒和对症治疗等综合措施。虽然犬可感染SARS-CoV-2,但尚不清楚感染犬是否可将病毒再传染给其他动物或人类。因此,应加强犬冠状病毒的病原生态学研究和监测预警。Research Progress on Canine CoronavirusIn February 2020,it was reported in Hongkong,China,that a pet dog kept by a patient confirmed with coronavirus disease 2019(COVID-19)was infected with severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),which caused the concern about canine coronavirus. In order to fully understand the study on canine coronavirus,prevent and control the disease,the etiology and epidemiology,clinical symptoms,measures of diagnosis and control were summarized in the paper. The canine coronavirus was widely distributed in dog populations,mainly including canine enteritis coronavirus of Alphacoronavirus and canine respiratory coronavirus of Betacoronavirus. The canine coronavirus was with poor pathogenicity,which could cause slightly clinical symptoms,but would be more serious and likely to mutate to new mutant strains when mixed infection of it with canine parvovirus and canine distemper virus occurred,with the risk of increasing pathogenicity. At present,the detection methods for canine coronavirus were mainly including pathogen detection,serological detection,and molecular biological detection methods such as RT-PCR,qRT-PCR and Nano PCR. Canine coronavirus could be controlled by a series of comprehensive measures including vaccination,scientific feeding and management,disinfection and symptomatic treatment,etc. It was still unclear whether the virus could be spread to other animals or human via the infected dogs,although dogs could be infected with SARS-CoV-2. Therefore,it was necessary to strengthen study on pathogenic ecology as well as monitoring and early warning against the virus.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202010017&v=Bh8a8ZT44%25mmd2BXJIZxJ9AXLnmQLd%25mmd2BGaTsBTZkV2VNkT6%25mmd2By5cE%25mmd2F1YOsAS0uFHMQ4f5Dz
2020-10-14
国内外奶牛疫病预警监测技术发展现状
为了解国内外奶牛疫病预警技术及其应用现状,归纳了奶牛疾病的预警要求,分析了国内外8种疫病预警技术的发展现状及其在奶牛养殖中存在的局限性。一是兽医巡检预警技术:该技术较为成熟,但不能满足大规模养殖牛场迅速、高效预警的需求。二是奶样体细胞数测定技术:可初步诊断隐性乳腺炎,但需要额外细菌培养和检测确诊。三是奶牛群体改良测定技术:以监测生产性能为主,仅可预警奶牛乳腺炎及酮病。四是计步器及发情、反刍项圈预警技术:同时具备发情与反刍监测功能,但仅适用于牛群的发情与反刍监测。五是大数据挖掘技术:在数据库基础上,利用各种模型或算法预测传染病的发生和发展,但预警效率受限。六是智能化体温连续远程监测及预警技术:可预警口蹄疫等重大传染病,但无法完全替代临床诊断,不能监测病原微生物。七是基于AI和群体热成像的奶牛行为分析技术:可以监视记录奶牛行为与温度特征,锁定病畜,但对发病初期无异常行为的牛只无效。八是生物气溶胶激光光谱测量技术:通过检测病原微生物种类和浓度,达到预警疫病的目的,目前主要用于军方监测人类重大疫病。未来的奶牛疫病预警技术需要进一步相互结合、取长补短,充分利用大数据、物联网等新兴技术进行奶牛重大疫病预警诊断。本文为预警监测技术的本地化发展提供了参考。 Development Status of Early Warning and Surveillance Technologies for Cow Diseases In order to identify the development and application of early warning and surveillance technologies for cow diseases in the world,relevant requirements for the technologies were summarized,and the development status of 8 different technologies and their limitations in cow breeding industry were analyzed. Specifically,for the technology of routing inspection and early warning by veterinarian,it was relatively mature,but failed to meet the need for rapid and efficient early warning in large-scale farms;for the technology of somatic cell count(SCC)of milk samples,by which,recessive mastitis could be initially diagnosed,but additional bacterial culture and testing would still be needed;for the dairy herd improvement(DHI)technology,it mainly focused on the monitoring of production performance,and only could be applied to early warning of cow mastitis and ketosis;for the pedometer and collar early warning technology for estrous and ruminant,it was only appropriate for monitoring of estrous and ruminant of the cows;for big data mining technology,the occurrence and development of infectious diseases could be predicted by various models or algorithms based on the database,but its efficiency was limited;for intelligent continuous remote temperature monitoring and early warning technology,major infectious diseases such as foot-and-mouth disease could be warned,but it could not fully replace clinical diagnosis and failed to monitor pathogenic microorganisms;for the cow behavior analysis technology based on AI and population thermal imaging,the behavior and temperature characteristics of cows could be monitored and recorded,sick cows could be targeted,but the technology was invalid for the cows without any abnormal behaviors at the early stage of the disease;and for bio-aerosol laser spectrometric measurement technology,the purpose of early warning could be achieved through the testing of species and concentration of pathogenic microorganism,which was mainly used to monitor major diseases in human by military at present. In conclusion,the early warning technologies for cow diseases should be further combined with each other in the future,learn from each other and make full use of big data,internet of things and other emerging technologies,which would contribute to early warning and diagnosis of major cow diseases. Some references were provided for the localization development of early warning and surveillance technologies. 全文下载链接:https://kns.cnki.net/KCMS/detail/detail.aspx?dbcode=CJFQ&dbname=CJFDAUTO&filename=ZGDW202010016&v=MjUzODc0SE5ITnI0OUVZb1I4ZVgxTHV4WVM3RGgxVDNxVHJXTTFGckNVUjdxZVorZHNGeS9rVTc3SlB5clBlYkc=
2020-10-14
我国部分地区送检样品3种常见猪病血清学和病原学检测
为了解我国部分地区猪瘟、猪繁殖与呼吸综合征和猪伪狂犬病3种常见疫病的流行情况,采用ELISA、聚合酶链式反应(PCR)和反转录PCR(RT-PCR)等方法,对2019年11个省(自治区)172个猪场送检的3 550份猪血清样品进行猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)及伪狂犬病野毒(PRV-gE)抗体检测,对9个省(自治区)72个猪场送检的371份病料进行抗原检测。结果显示:CSFV、PRRSV、PRV-gE抗体阳性率分别为82.75%、84.08%、31.86%,抗原阳性率分别为2.62%、26.15%、1.67%。保育猪群CSFV抗体平均阻断率最低,为44.62%,抗体整齐度差,CV为58.26%,且CSFV抗原主要在哺乳仔猪和保育猪病料中被检出,检出率分别为3.57%、3.00%;除育肥猪群外,其他各阶段猪群的PRRSV抗体S/P平均值为1.0~2.0,抗体整齐度整体较差,且平均抗原阳性率较高(26.15%),其中保育阶段猪群病原检出率最高(37.68%);育肥猪、母猪、公猪的PRV-gE抗体阳性率分别为36.82%、35.31%、15.65%,病原主要从死胎/木乃伊胎和哺乳仔猪病料中被检出,检出率分别为8.70%和2.99%。结果表明,PRRSV对我国猪场不同阶段猪群均有较大威胁;保育猪群CSFV抗体水平不理想,暴发疫情风险较高;公猪、母猪及育肥猪群普遍存在PRV感染,尤其是母猪。提议各猪场采取免疫、监测、净化等综合措施,加强这3类常见猪群疫病的防控。Serological and Pathogenic Detection of Three Common Swine Diseases in Some Regions of ChinaIn order to identify the prevalence status of three common swine diseases including classical swine fever(CSF),porcine reproductive and respiratory syndrome(PRRS)and porcine pseudorabies(PR)in some regions of China,by the methods of ELISA,PCR and RT-PCR,3 550 swine serum samples delivered by 172 farms in 11 provinces(autonomous regions)in 2019 were tested for the antibodies against CSFV,PRRSV and PRV-gE,and 371 lesion samples from 72 farms in 9 provinces(autonomous regions)were detected for virus antigens. The results showed that the antibody positive rates of CSFV,PRRSV and PRV-gE were 82.75%,84.08% and 31.86% respectively,and the antigen positive rates were 2.62%,26.15% and 1.67% respectively. The average blocking rate of CSFV antibody in nursery pigs was 44.62%,which was the lowest with poor antibody uniformity,and the CV value was 58.26%,CSFV was mainly detected from the lesions of suckling piglets and nursery pigs,with the detection rates of 3.57% and 3.00% respectively. Except for fattening pigs,the mean S/P value of PRRSV antibody was 1.0 to 2.0 for other pigs,with poor antibody uniformity,and the average positive rate of PRRSV antigen was higher(26.15%),in which,the detection rate of PRRSV antigen in nursery pigs was the highest(37.68%). The positive rates of PRV-gE antibody in fattening pigs,sows and boars were 36.82%,35.31% and 15.65% respectively,and the virus pathogens were mainly detected out in stillborn/mummy fetuses and lesions of suckling piglets,with the positive rates of 8.70% and 2.99% respectively. It was concluded that PRRSV had brought great threat to pig populations at different growth stages in China;the nursery pigs antibody level of CSFV was poor with high risk of outbreak;boars,sows and fattening pigs had been widely infected with PRV,especially sows. Therefore,a series of comprehensive measures including immunization,monitoring and purification and so on should be applied in all farms,so as to strengthen the prevention and control of these common diseases. 全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202010005&v=Bh8a8ZT44%25mmd2BXxmHa%25mmd2F3pXZzppSm8lNN5tOtvxTeoAZGuhyS9TRPv%25mmd2FYabVKxDG%25mmd2Bf1nv
2020-10-14
云南省边境地区送检牛血清样品蓝舌病病毒抗体检测与血清型鉴定
为了解云南边境地区的蓝舌病流行和分布情况,采用竞争ELISA方法,2019年对云南省边境地区5个州市12个县(市)的1 580份牛血清样品进行蓝舌病病毒抗体检测,同时从检出的阳性血清中,每县(市)随机选取15~55份,采用微量血清中和试验进行血清型鉴定。结果显示:12个边境县(市)均检出阳性血清样品,但有一定的地区差异,其中芒市最高(94.1%),沧源县最低(17.0%);本地牛和境外牛血清样品阳性率差异不大(P>0.05),均在70.0%以上;各县(市)均存在5个以上血清型的交叉感染。结果表明,蓝舌病在云南边境地区广泛存在,且呈多种血清型混合流行态势,需要持续开展血清学调查。本研究为我国西南边境地区蓝舌病防控提供了数据参考。Detection of Bluetongue Virus Antibodies in Bovine Serum Samples from Border Areas of Yunnan Province and Relevant Serotype IdentificationIn order to investigate the prevalence and distribution of bluetongue disease in border areas of Yunnan Province,1 580 cattle serum samples collected from 12 counties(cities)of 5 prefectures(municipalities)in border areas in 2019 were tested for bluetongue virus antibodies. For the positive serum samples,15 to 55 samples were randomly selected from each county(city)to identify serotypes by micro-serum neutralization test. The results showed that positive serum samples were detected out from all the 12 border counties(cities),with certain regional difference,among which,maximum positive rate occurred in Mang City(94.1%)and minimum rate in Cangyuan County(17.0%). The serum positive rate of local cattle was slightly different from that of foreign cattle(P>0.05),both of them were higher than 70.0%. Mixed infection of more than 5 serotypes was present in all counties(cities). It was concluded that bluetongue disease was widely distributed in the border areas,and tended to spread with mixed serotypes,so serological investigation should be continued to carry out. Some data references were provided for the prevention and control of the disease in the border areas in Yunnan Province.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202010004&v=Bh8a8ZT44%25mmd2BUA18IL%25mmd2FvvYlJeua9SVCqZZWmUVoNbW7uYr4JfTu5x3Fjl5GE%25mmd2FC6i6H
2020-10-14
猪瘟病毒和牛病毒性腹泻病毒双重荧光RT-PCR检测方法的建立
为同时检测猪源临床样本中的猪瘟病毒(CSFV)和牛病毒性腹泻病毒(BVDV),基于这两种病毒的5'UTR序列设计特异引物和TaqMan探针,建立了一种检测CSFV和BVDV的双重荧光RT-PCR检测方法,并对该方法的特异性、最低检出限和重复性等进行了评价。结果显示,该方法只对CSFV和BVDV呈现特异性扩增,对猪伪狂犬病病毒、猪繁殖与呼吸综合征病毒、猪传染性胃肠炎病毒、猪流行性腹泻病毒、猪圆环病毒2型不发生交叉反应,对阳性标准对照CSFV-5'UTR-RNA、BVDV-1-5'UTR-RNA和BVDV-2-5'UTR-RNA,最低可分别检出27、36和32拷贝/μL。该方法的组内和组间试验Ct值变异系数介于0.11%~1.20%,具有良好的重现性。对152份猪组织样本用该方法进行CSFV和BVDV核酸检测,结果检出CSFV阳性样本16份,BVDV阳性样本3份,CSFV和BVDV双阳性样本1份,与国标和OIE《陆生动物诊断试验和疫苗》相应的荧光RT-PCR方法阳性符合率为100%。结果表明,本研究建立的双重荧光RT-PCR方法可用于临床样本中的CSFV和BVDV检测,从而为猪瘟防制和净化提供了一种有效的技术手段。Establishment of a Duplex Fluorescent RT-PCR Assay for Detection of Classical Swine Fever Virus and Bovine Viral Diarrhea VirusIn order to simultaneously detect classical swine fever virus(CSFV)and bovine viral diarrhea virus(BVDV)in clinical samples from pigs,the specific primers and TaqMan probes were designed based on 5'UTR sequence of the two kinds of viruses,and a duplex fluorescent RT-PCR was established,the specificity,minimum detection limit and repeatability were evaluated. The results showed that the assay could react specifically with CSFV and BVDV only,but failed to crossly react with other viruses including pseudorabies virus(PRV),porcine reproductive and respiratory syndrome virus(PRRSV),transmissible gastroenteritis virus(TGEV),porcine epidemic diarrhea virus(PEDV)and porcine circovirus-2(PCV-2). The minimum detection limits were 27,36 and 32 copies/μL for the positive standard plasmid control of CSFV-5'UTR-RNA,BVDV-1-5'UTR-RNA and BVDV-2-5'UTR-RNA respectively. The coefficients of variation(CV)of intra-and inter-group ranged 0.11% to 1.20%,showing good reproducibility. Atotal of 152 tissue samples were tested for CSFV and BVDV by the assay,with the results of 16 CSFV positive samples,3 BVDV positive samples and 1 CSFV-BVDV dual positive samples,which were completely consistent with the national standard and the results of corresponding fluorescent RT-PCR assay specified in OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. In conclusion,the assay established in this study could be used for the detection of CSFV and BVDV in clinical samples,which provided an effective technical method for control and purification of the two diseases.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202009022&v=Bh8a8ZT44%25mmd2BXfswhduqZcZQVHMHhHXi9wSi2xg6rwQX6OsZDtjLz8LFamaifdRAHg
2020-09-27
鹦鹉热衣原体荧光环介导等温扩增(LAMP)检测方法的建立
鹦鹉热衣原体(Chlamydia psittaci)是一种人兽共患病原体,能引起自然疫源性衣原体病,目前广泛分布于世界各地。本研究应用环介导等温扩增技术(LAMP),针对鹦鹉热衣原体23S RNA基因序列设计引物,并进行筛选以及敏感性和特异性试验,建立了鹦鹉热衣原体恒温荧光扩增方法。该方法在63 ℃恒温条件下1 h内即可显示结果,操作简单、快速,特异性强,灵敏度可达100拷贝/µL,且与流产衣原体、动物布鲁氏菌、牛结核分枝杆菌、沙门氏菌、大肠杆菌、金黄色葡萄球菌无交叉反应;用国产仪器可直接通过检测荧光信号判读结果,既增强了准确性,也避免了开盖污染产生假阳性;应用建立的LAMP方法对实验室保存的临床DNA样品进行检测,发现检测结果与QPCR相同。该方法的建立弥补了传统检测技术的不足,为实现鹦鹉热衣原体的现场快速诊断提供了技术支持。Establishment of a Loop-mediated Isothermal Amplification Method for Chlamydia psittaciChlamydia psittaci,a kind of zoonotic pathogen,is widely distributed all over the world,by which,chlamydiosis of natural origin might be caused. In this study,the primers were designed based on 23S RNA gene sequence of Chlamydia psittaci,and then screened. After sensitivity and specificity tests,a thermostatic fluorescence amplification method for Chlamydia psittaci was established according to the loop-mediated isothermal amplification method(LAMP). By the established method,test results could be shown within one hour under the constant temperature of 63 ℃ due to its advantages including simple operation,rapidity,strong specificity,sensitivity of 100 copies/µL and no cross-reaction with Chlamydia abortus,Brucella animalis,Mycobacterium bovis,Salmonella,Escherichia coli and Staphylococcus aureus. The results could be interpreted directly by fluorescence signals with domestic instruments,which not only enhanced the accuracy,but also avoided false positive due to pollution brought by cap opening. The clinical DNA samples stored in laboratories were detected by the LAMP method,it was found that the results were same as that by QPCR. It was concluded that traditional methods were remedied by the established method,which provided technical support for rapid field detection of Chlamydia psittaci.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202009021&v=Bh8a8ZT44%25mmd2BXxGnjxzXiJU3p%25mmd2FivzuIEWb0bnO6OLe8PeO%25mmd2Bmd6CaSRqBjEGndhokrs
2020-09-27
牛结节性皮肤病诊断方法研究进展
牛结节性皮肤病(lumpy skin disease,LSD)是由牛结节性皮肤病病毒(lumpy skin disease virus,LSDV)引起的急性、亚急性传染病。我国于2019 年8 月在新疆伊犁地区首次确诊该病。快速准确检测是防控该病的关键。目前,应用常规PCR、实时荧光定量PCR、高分辨率熔解曲线分析、环介导等温扩增等分子检测方法,已实现了LSDV与其同属不同种病毒的通用检测和鉴别检测以及LSDV野毒株与疫苗株的区分;应用病毒中和试验、酶联免疫吸附(ELISA)和蛋白印迹试验(WB)等免疫学方法,可进行羊痘病毒属的特异性抗原与抗体检测,而对于LSDV特异性抗体检测方法尚有待研究。LSD作为一种新传入我国的外来动物疫病,有必要运用快速准确的分子或免疫学检测方法加强监测。本文概述了LSD诊断方法研究进展,为我国LSD诊断技术研发提供参考。 Research Progress on Diagnostic Methods of Lumpy Skin DiseaseLumpy skin disease(LSD),an acute and subacute infectious disease,is caused by lumpy skin disease virus(LSDV). In August 2019,Chinese first case was confirmed in Yili Prefecture,Xinjiang. To prevent and control the disease is subject to rapid and accurate detection. Presently,the goal of generic detection of LSDV and its congeneric virus of different species as well as differential diagnosis of wild strains and vaccine strains have been achieved by a series of molecular detection methods,such as conventional PCR,real-time fluorescent quantitative PCR,high-resolution melting(HRM)curve analysis,loop mediated isothermal amplification(LAMP);the specific antigen and antibody of capripoxvirus could be detected by the immunological methods,such as virus neutralization test(VNT),enzyme-linked immunosorbent assay(ELISA),Western Blot(WB),etc. However,the detection methods for specific antibodies against LSDV still need further studies. As a new exotic animal disease,LSD should be further monitored by rapid and accurate molecular or immunological methods. In conclusion,relevant research progress was summarized for the purpose of providing some references for the development of LSD diagnosis technology in China. 全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202009018&v=Bh8a8ZT44%25mmd2BWJazKXHZJ2lAttfvyxPfcF6AGOiS28OYFQzAbEmEhkOi8ryjg0p66%25mmd2B
2020-09-27
副猪嗜血杆菌的实验室检测技术
冠状病毒在自然界中普遍存在,可感染多种哺乳动物和鸟类,给畜牧业生产安全和副猪嗜血杆菌病发病率和死亡率较高,给养猪业造成了严重损失。为及时确诊该病,本文从细菌分离鉴定、血清学检测和分子生物学检测3个方面,对副猪嗜血杆菌实验室诊断方法的研究进展及其优缺点进行了综述。细菌分离鉴定特异性较强,但操作复杂、用时长、所需仪器多,无法满足快速检测需求;酶联免疫吸附试验(ELISA)、间接血凝试验(IHA)和补体结合试验(CF)3种血清学检测方法操作简便、快速,但特异性和敏感性不高,主要适用于早期诊断和筛查。近年来,分子生物技术快速发展,已建立了PCR、荧光定量PCR、数字PCR和环介导等温扩增(LAMP)等多种分子生物学检测方法。这些分子生物学检测方法敏感、快速、特异和稳定,其中PCR、荧光定量PCR已广泛应用于实践中,而数字PCR和LAMP检测技术尚需进一步优化。多种特异性好、灵敏度高、重复性好、检测快速的实验室检测方法的建立和改进,为副猪嗜血杆菌病检测和流行病学调查提供了有效的技术支撑。 Laboratory Testing Technologies for Haemophilus parasuisHaemophilus parasuis has brought huge losses to pig industry due to its high morbidity and mortality. In order to confirm the disease timely,the research progress on relevant laboratory testing technologies as well as their advantages and disadvantages were summarized from the aspects of isolation and identification of bacteria,serological detection and molecular biological detection. Specifically,the method of isolation and identification of bacteria was with strong specificity,but was complex and time-consuming for operation,which would require for more instruments and fail to meet the needs of rapid detection;the serological methods including enzyme linked immunosorbent assay(ELISA),indirect hemagglutination assay(IHA)and complement fixation test(CF)were simple and rapid,mainly appropriate for early diagnosis and screening,but their specificity and sensitivity were relatively low. In recent years,with rapid development of molecular biotechnology,a variety of molecular biological methods including PCR,fluorescence quantitative PCR,digital PCR and loop mediated isothermal amplification(LAMP)had been established,which were sensitive,rapid,specific and stable,especially,PCR and fluorescent quantitative PCR had been widely used in practice. But digital PCR and LAMP still needed to be further optimized. In conclusion,an effective technical support was provided for detection of the disease and epidemiological investigation through establishing and improving a variety of laboratory testing methods with the advantages of good specificity and repeatability,high sensitivity and rapid detection speed. 全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202009017&v=Bh8a8ZT44%25mmd2BW9paznIkMtVgYb7DdBga1vvYUGvtvTHRfdlrLjxzvQYt%25mmd2B6li8ZxOQy
2020-09-27
我国不同地区猪源致泻性大肠杆菌的多重PCR鉴定及2b-RAD序列分析
为了解我国不同地区屠宰生猪肉品中致泻性大肠杆菌污染情况及系统进化关系,降低由其带来的公共卫生危害,将近几年分离自华北、华东、华中及西南地区猪屠体表面的283株大肠杆菌进行了致泻性大肠杆菌多重PCR鉴定及2b-RAD测序分型。多重PCR鉴定结果显示,共检出36株致泻性大肠杆菌,检出率为12.7%,其中西南地区检出率(26.92%)高于华北(13.33%)、华东(10.57%)和华中(10.81%)地区;5种致泻类型中,产肠毒素大肠杆菌(ETEC)、肠致泻性大肠杆菌(EPEC)、肠聚集性大肠杆菌(EAEC)、肠出血性大肠杆菌(EHEC)和肠侵袭性大肠杆菌(EIEC)的占比分别为33.33%、30.56%、25.00%、5.56%和5.56%。将致泻性大肠杆菌进行2b-RAD测序,种群聚类分析显示,这36株致泻菌可分为8个亚群;系统发生树分析显示,亲缘关系较近菌株的致泻类型基本一致,分离时间也比较接近,而且来自同一地区菌株的亲缘关系相对较近。可见,我国屠宰生猪肉品中污染的致泻性大肠杆菌以ETEC、EPEC和EAEC类型为主,其流行分布有一定的地域差异,且其亲缘关系呈一定的时空特异性。本研究为精准防控生猪屠宰环节致泻性大肠杆菌污染,保障肉品卫生安全提供了科学数据和技术支撑。 Multiplex PCR Identification and 2b-RAD Sequence Analysis of Porcine Diarrheagenic Escherichia coli in Different Regions in ChinaIn order to make clear the status of contamination and phylogenetic relationship of diarrheagenic Escherichia coli(E.coli)in raw pork products in different regions of China and to reduce the public health hazards that might occur therefrom,diarrheagenic E.coli strains were identified by multiplex PCR from 283 E.coli strains isolated from the surface of pig carcasses in North China,East China,Central China and Southwest China in recent years,then 2b-RAD sequencing and gene typing were carried out. It was found that,by multiplex PCR,36 strains of diarrheagenic E.coli were detected out,with the detection rate of 12.7%,among which,the detection rate in Southwest China(26.92%)was significantly higher than those in North China(13.33%),East China(10.57%)and Central China(10.81%);for the five types of diarrhea,enterotoxigenic E.coli(ETEC),enteropathogenic E.coli(EPEC),enteroaggregative E.coli(EAEC),enterohemorrhagic E.coli(EHEC)and enteroinvasive E.coli(EIEC)accounted for 33.33%,30.56%,25.00%,5.56% and 5.56%,respectively. After 2b-RAD sequencing for species clustering analysis,it was found that the 36 strains could be classified into 8 subgroups;it was shown that,by phylogenetic tree analysis,the strains with close genetic relationship belonged to similar diarrheagenic types,and their isolation time was also close,in particular,the strains from the same region were with closely genetic relationship. It was concluded that the diarrheagenic E.coli contaminated in raw pork products mainly included three types of ETEC,EPEC and EAEC,the diarrheagenic E.coli strains were distributed with regional diversity,and their genetic relationships were with certain spatial and temporal specificity. In conclusion,some scientific data and technical support were provided to prevent and control the containment with diarrheagenic E.coli during the process of pig slaughtering and to safeguard hygiene and safety of pork products.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202009010&v=Bh8a8ZT44%25mmd2BUeRq5k%25mmd2BkfhEPYt2%25mmd2FmPx9quStu%25mmd2F7HhxzlR4Jr8qSYJnW%25mmd2B7QKhWBaKBd
2020-09-27
基于实时荧光PCR法定量检测食品中鸡源性成分
肉类食品真伪鉴定是目前食品安全检测领域的重点研究内容之一。为准确定量检测食品中的鸡源性成分,根据现行检验检疫标准(SN/T 2727—2010)合成引物及探针,并拓展了特异性、灵敏度、检出限和定量分析等试验。结果显示:特异性试验中,仅鸡成分检测出现特异性扩增,其他动物组织均未出现特异性扩增;梯度稀释鸡源性DNA模板原液进行灵敏度评价,发现引物和探针灵敏度较高,可以检测到100 fg级的鸡组分模板DNA,且建立的标准曲线具有良好的线性关系,R2达0.99以上,定量准确;加工制品中的验证试验结果显示,合成的引物及探针具有良好的适用性和定量检测能力。以上研究结果均说明,此引物和探针适用于市场食品中鸡源性成分的定性、定量检测。本研究为今后TaqMan实时荧光PCR法不同动物源性引物和探针的自主研发奠定了基础。Quantitative Detection of Chicken-derived Components inFood Based on Real-time PCRAt present,the identification of meat products is emphatically studied in the field of food safety. In order to establish a quantitative detection method for chicken-derived components in food,primers and probes were synthesized according to current standard of inspection and quarantine(SN/T 2727—2010),and specificity test,sensitivity test,detection limit and quantitative analysis were expanded. The results showed that the specific amplification was found only in chicken-derived components instead of other animal tissues;gradient dilution of chicken-derived DNA template were used to evaluate the sensitivity,and it was found that the sensitivity of primers and probe was high,which could detect the DNA template at 100 fg level,and a good linear relationship was observed in the standard curve,R2 could reach above 0.99 with an accurate quantitative detection. For the processed products,the synthesized primers and probes were with good applicability and quantitative detection capacity. It was concluded that the primers and probes were suitable for qualitative and quantitative detection of chicken-derived components in food markets. A foundation was laid for the independent development of different animal-derived primers and probes of TaqMan real-time PCR in the future.全文下载链接:https://kns.cnki.net/KCMS/detail/detail.aspx?dbcode=CJFQ&dbname=CJFDAUTO&filename=ZGDW202008023&v=MDU5MDFYMUx1eFlTN0RoMVQzcVRyV00xRnJDVVI3cWZiK2RtRnlqa1dyN0JQeXJQZWJHNEhOSE1wNDlIWjRSOGU=
2020-08-20
猪血凝性脑脊髓炎病毒实时荧光RT-PCR检测方法的建立与应用
为建立一种快速、准确、常规的猪血凝性脑脊髓炎病毒(porcine hemagglutinating encephalomyelitis virus,PHEV)检测方法,根据PHEV N蛋白基因序列,设计1对引物和1条探针,建立了PHEV实时荧光RT-PCR检测方法。该方法特异性好,与猪繁殖与呼吸综合征病毒(PRRSV)、猪伪狂犬病病毒(PRV)、猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)、猪瘟病毒(CSFV)和猪圆环病毒2型(PCV2)均无交叉反应;用阳性质粒Puc57-PHEVn评估其敏感性,发现检测下限达10-6 ng/μL(224拷贝/μL)。采集233份发病猪组织样品进行临床检测,发现该方法与巢式RT-PCR的符合率达98.3%,准确筛查出巢式RT-PCR并测序阳性的样品14份,且检出率高于巢式RT-PCR。结果表明,本方法敏感、特异,可用于PHEV临床样品的检测。Establishment and Application of a Real-time RT-PCR Assay for Detection of Porcine Hemagglutinating Encephalomyelitis VirusIn order to establish a rapid,accurate and routine method for detection of porcine hemagglutinating encephalomyelitis virus(PHEV),a pair of primers and a probe were designed based on the N gene sequence of PHEV,and a real-time RT-PCR assay was developed,which was with good specificity,and failed to crossly react with porcine reproductive and respiratory syndrome virus(PRRSV),porcine pseudorabies virus(PRV),porcine epidemic diarrhea virus(PEDV),transmissible gastroenteritis virus(TGEV),classical swine fever virus(CSFV)and porcine circovirus type 2(PCV2). The sensitivity of the method was evaluated based on positive plasmid Puc57-PHEVn,and it was found that the limit detection was 10-6 ng/μL(224 copies/μL). A total of 233 swine tissues were tested by the method,and it was found that 14 positive samples were detected out,and the coincidence rate with the nested RT-PCR was 98.3%,but the detection rate was higher than that by nested RT-PCR. It was concluded that the method could be used to detect PHEV clinical samples benefited from its high sensitivity and specificity..全文下载链接:https://kns.cnki.net/KCMS/detail/detail.aspx?dbcode=CJFQ&dbname=CJFDAUTO&filename=ZGDW202008022&v=MzAwOTVUcldNMUZyQ1VSN3FmYitkbUZ5amtWNzdLUHlyUGViRzRITkhNcDQ5SFpvUjhlWDFMdXhZUzdEaDFUM3E=
2020-08-20
布鲁氏菌S2疫苗阴道途径免疫羊的安全性初步研究
布鲁氏菌病(简称布病)是一种由布鲁氏菌引起的严重危害人和动物健康的人兽共患病。疫苗免疫是畜间布病流行率较高地区控制该病的重要手段。辽宁省建昌县经过多年实践,创新出一套免疫保护效果较好的阴道途径免疫S2疫苗的新型家畜免疫技术。为了解该技术对家畜的安全性,本研究采用该技术免疫了3只山羊,在免疫1个月后检测其抗体滴度、体内含菌量,观察脾脏和肝脏的病理变化。结果显示,3只免疫山羊均抗体阳转,所有采集的脏器均未分离到布鲁氏菌,且脾脏和肝脏未出现布病相关病理变化。初步研究表明,通过阴道途径对羊进行S2疫苗免疫是安全的。Preliminary Study on the Safety of Brucella Vaccine S2 Strain on Goats Immunized via Vagina Brucellosis is a kind of zoonosis caused by Brucella,which seriously endangers human and animal health,and is mainly controlled through vaccination in areas where animal brucellosis is prevalent. After practices for several years,a new immune technology(immunization via vagina)was developed in Jianchang County of Liaoning Province,and good immune effects have been achieved. In order to make clear the safety of the technology on animals,3 goats were vaccinated,one month later,their antibody titer and bacterial content were detected and the pathological changes of their spleens and livers were observed. The results showed that the antibodies in the three goats turned to be positive,no Brucella was isolated from all the organs collected,and no pathological changes related to brucellosis were found in spleens and livers. These results preliminarily indicated that it was safe to immunize goat with Brucella vaccine S2 strain via the vagina.全文下载链接:https://kns.cnki.net/KCMS/detail/detail.aspx?dbcode=CJFQ&dbname=CJFDAUTO&filename=ZGDW202008019&v=MjU3NjBlWDFMdXhZUzdEaDFUM3FUcldNMUZyQ1VSN3FmYitkbUZ5amtVN3ZJUHlyUGViRzRITkhNcDQ5RWJZUjg=
2020-08-20
化学发光免疫分析技术在动物疫病检测中的应用
化学发光免疫分析(chemiluminesecence immunoassay,CLIA)是用于疫病检测的一种免疫检测技术,是基于免疫反应原理,结合化学发光检测技术而建立的。与放射免疫分析、酶免疫分析等标记免疫技术相比,具有无放射性污染、灵敏度高和特异性强的特点,已被广泛应用于疫病流行病学调查、诊断、药物分析以及微生物检测等方面。本文简要介绍了CLIA技术的基本原理及发展,从细菌、病毒和寄生虫检测方面,综述了近年来该技术在动物疫病检测中的应用,提出该技术正向高特异性、高灵敏度、高通量、快速和自动化检测的方向发展,这为今后CLIA在动物疫病检测中的应用研究提供参考。Application of Chemiluminescence Immunoassay in Detection of Animal Diseases As a kind of immunological technology for detection of animal diseases,chemiluminescence immunoassay(CLIA)is developed in combination with chemiluminescence detection technology based on the principle of immune response,which is characterized by high sensitivity and good specificity with no radioactive pollution compared to the immunolabelling technology such as radioimmunoassay(RIA)and enzyme immunoassay(EIA),and has been widely used for epidemiological investigation and diagnosis of animal diseases as well as drug analysis and microbial detection. In this paper,the basic principle and development of CLIA technology were introduced,and its application in animal disease detection in recent years was summarized from the aspects of bacteria,virus and parasite detection. It was shown that chemiluminescence immunoassay was turning to be a rapid and automatic detection technology with high specificity,high sensitivity and high throughput. This study would provide some reference for application of CLIA in animal disease detection in the future. 全文下载链接:https://kns.cnki.net/KCMS/detail/detail.aspx?dbcode=CJFQ&dbname=CJFDAUTO&filename=ZGDW202008018&v=MDMwNDkrZG1GeW5oV3JyT1B5clBlYkc0SE5ITXA0OUViSVI4ZVgxTHV4WVM3RGgxVDNxVHJXTTFGckNVUjdxZmI=
2020-08-20
水生动物冠状病毒研究进展
冠状病毒在自然界中普遍存在,可感染多种哺乳动物和鸟类,给畜牧业生产安全和公共卫生安全带来严重威胁。自北京新发地批发市场从切割进口三文鱼案板上检测到人新型冠状病毒(SARS-CoV-2)核酸以来,三文鱼以及水生动物冠状病毒与SARS-CoV-2的关系成为公众关注的焦点。本文详细描述了白鲸、宽吻海豚、三文鱼等水生动物感染冠状病毒后的临床症状,以及病毒基因组结构特征,并对水生动物冠状病毒与SARS-CoV-2进行了遗传演化分析,结果显示目前已知的水生动物冠状病毒属于γ冠状病毒属和Alphaletovirus属,而SARS-CoV-2属于β冠状病毒属,二者基因组和主要基因(1ab、S、E、M、N)核苷酸同源性仅为43.7%~54.1%,可初步排除SARS-CoV-2来源于已知水生动物冠状病毒的可能性。本文通过系统阐述水生动物冠状病毒感染状况,旨在进一步提高公众对水生动物冠状病毒的认知,为科学防控冠状病毒提供理论依据。 Research Progress on Coronaviruses in Aquatic AnimalsCoronavirus is widely prevalent in nature and may infect a variety of mammals and birds,which poses a serious threat to the safety of animal husbandry and public health. The relationship between coronaviruses in aquatic animals including salmon and severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has been concerned by the public since the nucleic acid of SARS-CoV-2 was detected on the cutting board for imported salmon in Beijing Xinfadi wholesale market. In this paper,the clinical symptoms of infected aquatic animals such as belugas,bottlenose dolphins and salmon,as well as the structural features of viral genome were described in detail,the genetic evolution of coronaviruses in aquatic animals and SARS-CoV-2 were analyzed. The results showed that the known coronaviruses in aquatic animals belonged to Gammacoronavirus and Alphaletovirus,while SARS-CoV-2 fell into Betacoronavirus,and the nucleotide homology of their genomes and major genes(1ab,S,E,M and N)was 43.7% to 54.1%,which could exclude any possibility of SARS-CoV-2 originating from the known coronaviruses. The infection status of coronaviruses in aquatic animals was systematically described in this paper,hoping to further improve public awareness and provide theoretical basis for scientific prevention and control of coronavirus. 全文下载链接:https://kns.cnki.net/KCMS/detail/detail.aspx?dbcode=CJFQ&dbname=CJFDAUTO&filename=ZGDW202008017&v=MzI2NzkxRnJDVVI3cWZiK2RtRnluaFZML0tQeXJQZWJHNEhOSE1wNDlFWTRSOGVYMUx1eFlTN0RoMVQzcVRyV00=
2020-08-20
云南省蓝舌病病毒“历史毒株”的遗传特征
20世纪90年代,本实验室从云南省哨兵动物采集的血液中分离到7种血清型蓝舌病病毒(bluetongue virus,BTV)。但这些毒株的遗传特征至今仍不清楚,因而阻碍了我国的BTV演化历史分析。为掌握云南省早期流行BTV毒株的遗传特征,对1995—1997年云南省分离的25株BTV毒株的基因组节段2、3、7和10(Seg-2、-3、-7、-10)进行一步法RT-PCR扩增、测序以及序列分析。Seg-2序列分析显示,1995—1997年云南省分离到的7种不同血清型(BTV-1、-2、-3、-4、-12、-15、-16)BTV毒株,分属A、B、G、H、I和J等6种基因型;BTV-12型毒株的Seg-2为西方地域型,其余毒株的Seg-2则属于东方地域型;Seg-3和Seg-10序列分析显示,25株BTV毒株均为东方地域型;Seg-7序列分析显示,1株BTV-2型、2株BTV-12型和1株BTV-16型毒株属于西方地域亚型,并与南非和荷兰BTV毒株的Seg-7具有最近的亲缘关系,其余毒株则均为东方地域型。上述结果表明,云南省存在多种血清型BTV的流行,而Seg-2和Seg-7基因重配毒株的发现,提示国外的西方地域型毒株已侵入云南省,并在传播过程中与我国毒株发生了基因重配。本研究为我国BTV的演化分析与溯源研究提供了数据参考。 Genetic Characteristics of“Historical Strains”of Bluetongue Virus Isolated from Yunnan Province In the 1990s,7 serotypes of bluetongue virus(BTV)were isolated from blood samples collected from sentinel animals in Yunnan Province by our laboratory. However,the genetic characteristics of these strains were still not clear,which hindered the study on the evolutionary history of BTV in China. In order to make clear the genetic characteristics of BTV strains spread early in Yunnan,one-step RT-PCR amplification,sequencing and sequence analysis were carried out for gene segments 2,3,7 and 10(Seg-2,-3,-7 and-10)of 25 BTV strains isolated in the province from 1995 to 1997. It was found that,by Seg-2 sequence analysis,Seven different BTV serotype strains(BTV-1,-2,-3,-4,-12,-15 and -16)were isolated,which fell into 6 nucleotypes of A,B,G,H,I and J,respectively;the Seg-2 of BTV-12 fell into Western topotype,but other strains belonged to Eastern topotype;it was showed that,by Seg-3 and Seg-10 sequence analysis,25 strains of BTV belonged to Eastern topotype;and by Seg-7 sequence analysis,one strain of BTV-2,two strains of BTV-12 and one strain of BTV-16 were clustered into Western topotype,and shared the closest genetic relationship with Seg-7 isolated from South Africa and Netherlands,and the other strains belonged to Eastern topotype. It was concluded that multiple serotypes of BTV were prevalent in Yunnan Province,and foreign Western topotype strains had been introduced into the province and reassorted with Chinese strains in the process of transmission as Seg-2 and Seg-7 gene reassortative isolates were detected. Some data was provided for evolution analysis and traceability study on BTV in China.全文下载链接:https://kns.cnki.net/KCMS/detail/detail.aspx?dbcode=CJFQ&dbname=CJFDAUTO&filename=ZGDW202008007&v=MTYzNDg3RGgxVDNxVHJXTTFGckNVUjdxZmIrZG1GeW5oVnI3TlB5clBlYkc0SE5ITXA0OUZZNFI4ZVgxTHV4WVM=
2020-08-20
马泰勒虫RAP-1基因的原核表达及蛋白性质分析
棒状体相关蛋白1(rhoptry-associated protein 1,RAP-1)是由棒状体分泌的与侵袭宿主细胞相关的蛋白。本研究以顶复门原虫RAP-1基因为靶标,用最大似然法构建了巴贝斯属、疟原虫属、泰勒虫属的RAP-1基因进化树;应用软件预测分析RAP-1蛋白的理化性质、亲疏水性、跨膜区以及二、三级结构,并表达纯化了马泰勒虫(Theileria equi)RAP-1重组蛋白。结果显示:巴贝斯属不同虫株相聚类,测定序列与马泰勒虫聚为一支,又与恶性疟原虫相聚类,表明亲缘关系较近。马泰勒虫RAP-1蛋白理论等电点为9.85,半衰期为30 h,总平均亲水性(GRAVY)为-0.368,无跨膜区;RAP-1蛋白二级结构中,α-螺旋、β-折叠、β-转角和无规卷曲占比分别为38.5%、15.5%、27.8%、18.1%;构建的三级结构立体展现了RAP-1蛋白形态。成功构建了原核表达载体pET-32a-RAP-1;该重组蛋白主要以包涵体形式存在,蛋白大小为65 kDa。RAP-1蛋白可通过原核表达系统高效表达,纯化后的产物能被马泰勒虫标准阳性血清识别,具有良好的反应原性。本研究为深入了解马泰勒虫入侵宿主的机制机理以及RAP-1蛋白的功能研究奠定了基础。Prokaryotic Expression of RAP-1 Gene of Theileriaequi andCharacteristic Analysis on the Encoded ProteinRhoptry-associated protein 1(RAP-1)is a protein related to invading the host cells and secreted by the rhoptry. Targeting at the RAP-1 gene of Protozoan protozoa,the gene evolution trees of Babesia,Plasmodium and Theileria were constructed by the maximum likelihood method. The physical and chemical properties,hydrophobicity,transmembrane zone,secondary and tertiary structures of RAP-1 protein were predicted by software,and the recombinant RAP-1 protein of Theileria equi was expressed and purified. The results showed that the genus of Babesia was clustered together,its sequence and that of Theileria equi belonged to the same branch and clustered with the genus of Plasmodium falciparum,indicating that their genus was closely related. The theoretical isoelectric point of RAP-1 protein was 9.85,the half-life was 30 h,the total average hydrophilicity was -0.368,and there was no transmembrane zone. The secondary structure α-helices,β-sheets,β-turns and random coils of RAP-1 protein accounted for 38.5%,15.5%,27.8% and 18.1%,respectively. The form of RAP-1 protein was shown by the three-level structure,and the prokaryotic expression vector pET-32a-RAP-1 was successfully constructed,the recombinant protein mainly existed in the form of inclusion body with the size of 65 kDa. In conclusion,the RAP-1 protein could be highly expressed by prokaryotic expression system,and the purified products could be identified by the standard positive serum of Theileriaequi due to its good reactogenicity. Therefore,a foundation was provided for further studying the mechanism of Theileriaequi invading into hosts and the functions of RAP-1 protein.全文下载链接:https://kns.cnki.net/KCMS/detail/detail.aspx?dbcode=CJFQ&dbname=CJFDAUTO&filename=ZGDW202007022&v=MTU3NDdTN0RoMVQzcVRyV00xRnJDVVI3cWZZT2RvRnl6bFU3dlBQeXJQZWJHNEhOSE1xSTlIWm9SOGVYMUx1eFk=
2020-07-17