不同接种途径对枫叶鸭病毒性肝炎抗体的影响
为探讨不同接种途径对枫叶鸭病毒性肝炎(DVH)抗体的影响,选择非免疫种鸭所产的1日龄体质量均等的360只健康枫叶雏鸭(A组),平均分为4组(饮水组、滴口组、肌注组、对照组);选择免疫种鸭所产的360只雏鸭(B组),也平均分为4组(饮水组、滴口组、肌注组、母源抗体监测组)。在雏鸭5日龄时,除对照组和母源抗体监测组外,对其他组雏鸭进行免疫接种,分别于免疫后5、10、15 d,用ELISA方法检测雏鸭抗体效价,并进行攻毒保护试验。结果显示:A组的肌注组平均抗体效价(0.588±0.018)>饮水组(0.560±0.015)>滴口组(0.541±0.009),雏鸭攻毒保护率依次为91.1%、85.6%、78.9%;B组的肌注组平均抗体效价(0.513±0.021)>饮水组(0.484±0.051)>滴口组(0.466±0.014),攻毒保护率依次为72.2%、66.7%、60.0%,均低于A组。结果表明:无论有无母源抗体干扰,肌内注射的免疫效果最好,其次是滴口,饮水的免疫效果较差;母源抗体可干扰疫苗的免疫效果。因此,需根据使用疫苗种类和实际情况,选择最合适、最有效的免疫途径,同时做好母源抗体监测,选择最佳免疫时间。本研究为鸭场免疫途径的选择提供了参考。Effect of Different Vaccination Methods on the Antibodies against Viral Hepatitis in Maple Duck In order to study the effect of different vaccination methods on the antibodies against viral hepatitis(DVH)in maple ducks,360 one-day-old healthy maple ducklings with equal weight(group A)born by unvaccinated ducks were divided into four groups on average:drinking water,oral drop,intramuscular injection and control group;360 ducklings born by vaccinated ducks(group B)were also equally divided into four groups on average:drinking water,oral drop,intramuscular injection and maternal antibody monitoring. Except the control group and maternal antibody monitoring group,all the ducklings were vaccinated when they were five days old,their antibody titers were detected by ELISA method at 5,10 and 15 days after vaccination,and the virus challenge test was carried out. The results showed that,for group A,the average antibody titers were as follows:the intramuscular injection group(0.588±0.018)>drinking water group(0.560±0.015)>oral drop group(0.541±0.009),and the protection rates were 91.1%,85.6% and 78.9% respectively;for group B,the average antibody titers were as follows:the intramuscular injection group(0.513±0.021)>drinking water group(0.484±0.051)>oral drop group(0.466±0.014),and the protection rates were 72.2%,66.7% and 60% respectively,all of which was lower than that of group A. In conclusion,the intramuscular injection received the best effect no matter whether there was maternal antibody interference or not,followed by oral drop,and the effect of drinking water was the poorest;The vaccine effect might be interfered by maternal antibody. Therefore,it was necessary to select the most appropriate and effective method according to the type of vaccines used and actual status,meanwhile,the maternal antibody should be monitored to select an appropriate vaccination time. This study would provide some reference for selection of vaccination method in duck farms.全文下载链接:https://kns.cnki.net/KCMS/detail/detail.aspx?dbcode=CJFQ&dbname=CJFDAUTO&filename=ZGDW202007021&v=MDY1NDViRzRITkhNcUk5SFpZUjhlWDFMdXhZUzdEaDFUM3FUcldNMUZyQ1VSN3FmWU9kb0Z5emtXcnZCUHlyUGU=
2020-07-17
云南省香格里拉市猪肠气泡病病例的组织病理学观察与病原检测
云南省香格里拉市某藏猪场育肥猪出栏屠宰时,陆续发现部分个体小肠肠系膜浆膜下有大量葡萄串状透明无味气泡,病因不明,但病猪在仔猪阶段有腹泻病史。为调查发病原因和致病机理,以便提出防控措施,采集该养猪场4头患病猪和1头正常猪的抗凝全血、小肠组织和肠系膜淋巴结,进行病毒核酸PCR扩增、细菌分离培养和组织病理显微观察。在患病猪中,未检出与腹泻有关的猪瘟病毒、猪流行性腹泻病毒、传染性胃肠炎病毒和轮状病毒核酸;血琼脂有氧和厌氧培养均只分离到大肠杆菌,并排除常见致病血清型及常见毒力基因;组织病理检查发现,肠黏膜下层有大量空泡,肠腺及其杯状细胞体积明显增大,组织间隙扩大,肠系膜及其淋巴结分别见嗜酸性粒细胞和巨噬细胞增多,病变肠系膜淤血。分析认为,该病病因应考虑养殖区域海拔较高,猪肠内大肠杆菌异常发酵产气的可能,但如何防治需进一步研究。 Histopathological Observation and Pathogen Detection of Porcine Intestine Pneumatosis Cystoides in Shangri-La City of Yunnan ProvinceA large number of grape-shaped transparent odorless bubbles were successively found under serosa of small intestine mesenterium in some fattening pigs in a Tibetan pig farm in Shangri-La City of Yunnan Province when the pigs were sold for slaughtering,with unknown causes,while the sick pigs had suffered from diarrhea at the piglet stage. In order to investigate the cause and pathogenesis so as to put forward prevention and control measures,the anticoagulant whole blood,small intestine tissues and mesenteric lymph nodes were collected from four sick pigs and one normal pig for PCR amplification of viral nucleic acids,bacterial isolation and culture and microscopic observation of histopathology. No any classical swine fever virus,porcine epidemic diarrhea virus,transmissible gastroenteritis virus and rotavirus nucleic acid related to diarrhea were detected;only Escherichia coli was isolated by blood agar aerobic and anaerobic culture,and the common pathogenic serotypes and virulence genes were excluded;the following symptoms were found through pathological examination such as a large number of vacuoles in the intestinal submucosa,the increased volume of intestinal gland and its goblet cells,the widened space among various tissues,the increased eosinophils and macrophages in the mesentery and its lymph nodes,and congestion in the diseased mesentery. It was concluded that the disease was likely to be caused by higher altitude,abnormal fermentation and air produced by E. coli,and it was necessary to continue to study how to prevent and control it. 全文下载链接:https://kns.cnki.net/KCMS/detail/detail.aspx?dbcode=CJFQ&dbname=CJFDAUTO&filename=ZGDW202007018&v=MjQ2OTMzcVRyV00xRnJDVVI3cWZZT2RvRnl6a1Zydk1QeXJQZWJHNEhOSE1xSTlFYklSOGVYMUx1eFlTN0RoMVQ=
2020-07-17
牛白血病的危害及防控策略
牛白血病是牛白血病病毒(bovine leukemia virus,BLV)感染引起的牛最常见的肿瘤性疾病之一,对养牛业和人类健康的潜在威胁较大。本文分别从病原学、流行现状、危害及潜在风险、诊断和防控策略等方面概述了BLV及其引发疾病的研究进展。BLV自首次发现以来,已广泛流行于世界各国,并很容易跨物种传播,且所有感染动物都会终身带毒,引起广泛的免疫功能异常,从而导致产奶量降低、产肉量下降和动物流产。此外,有实验已证实了在人类乳腺癌、肺癌组织和部分人类血液中有BLV的存在。目前尚无有效治疗和预防BLV感染的药物和疫苗,因此准确诊断和清除BLV感染牛是控制牛白血病最有效的措施之一。目前常用的诊断方法包括血清学抗体检测和前病毒DNA检测,主要的防控策略包括净化、通用的牧场管理与生物安全防护以及阻断向人类传播的途径。综上所述,应严格落实上述防控策略,加强诊断方法研究和监测,做到早发现早处理。 Study on the Hazards of Bovine Leukemia Virus and Relevant Control MeasuresAs one of the common neoplastic diseases in cattle caused by bovine leukemia virus(BLV),bovine leukemia has posed a great threat to cattle industry and human health. In this paper,the research progress on BLV and corresponding disease caused therefrom was summarized in terms of etiology,prevalence status,hazards and potential risks,diagnosis,prevention and control measures,etc. BLV has been widely spread all over the world since it was first found,which is prone to cross-species transmission,and is carried by all infected animals all life,resulting in abnormal immunity and causing the reduction of milk and meat production as well as abortion. In addition,it was confirmed by some experiments that BLV appeared in human breast cancer tissue,lung cancer tissue and some of human blood. However,no effective drugs or vaccines were available to prevent and control BLV infection,so the most effective measure was to accurately diagnosis and eliminate all infected cattle. Serological antibody detection and proviral DNA detection were commonly used at present,and the prevention and control measures mainly included disease purification,general management and bio-safety protection of pastures,and blocking all pathways of human infection. In short,it was necessary to implement the above mentioned measures,strengthen the monitoring of BLV,so as to guarantee“early detection and early disposal”.全文下载链接:https://kns.cnki.net/KCMS/detail/detail.aspx?dbcode=CJFQ&dbname=CJFDAUTO&filename=ZGDW202007017&v=MDMwMDNQZWJHNEhOSE1xSTlFWTRSOGVYMUx1eFlTN0RoMVQzcVRyV00xRnJDVVI3cWZZT2RvRnl6a1VMN01QeXI=
2020-07-17
布鲁氏菌病研究进展
布鲁氏菌病的早期诊断及鉴定是该病防控的重要环节,其诊断技术主要包括细菌学诊断技术、血清学诊断技术以及分子生物学诊断技术。布鲁氏菌病防控应坚持预防为主的策略,其免疫主要以弱毒活疫苗为主,随着分子生物学及重组DNA技术的发展,基因工程疫苗成为近年研究热点,但目前尚未有鉴别疫苗免疫和自然感染的鉴别诊断技术及相应的疫苗制品。本文对布鲁氏菌病的病原学、流行病学、诊断技术和疫苗研究进展等方面进行概述,以期为建立灵敏性高、特异性强、高效便捷且易推广的布鲁氏菌病诊断技术和研发更加安全有效的布鲁氏菌病疫苗提供基础。Research Progress on BrucellosisIt is the key to prevent and control brucellosis by early diagnosis and identification that mainly include the technologies of bacteriology,serology and molecular biology. The brucellosis vaccines have been playing an important role due to the strategy of“Prevention First”,and the live attenuated vaccines are commonly used at present. With the development of molecular biology and recombinant DNA technology,genetic engineering vaccine has developed rapidly and become a hotspot in recent years. However,no any differential diagnosis technologies and corresponding vaccine products for distinguishing vaccine immunity from natural infection have been available. In this paper,the etiology,epidemiology,diagnosis technology and brucellosis vaccine development status were summarized,which laid a foundation for the establishment of a popularized diagnosis technology with high sensitivity,specificity and efficiency,as well as for the development of the more effective and safer vaccines.全文下载链接:https://kns.cnki.net/KCMS/detail/detail.aspx?dbcode=CJFQ&dbname=CJFDAUTO&filename=ZGDW202007016&v=MDA2MzExVDNxVHJXTTFGckNVUjdxZllPZG9GeTNoVzczQVB5clBlYkc0SE5ITXFJOUVZb1I4ZVgxTHV4WVM3RGg=
2020-07-17
新型冠状病毒研究进展
自2019年12月新型冠状病毒肺炎(coronavirus disease 2019,COVID-19)暴发以来,世界各国相继出现了大量新型冠状病毒(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)感染病例。SARS-CoV-2对全球公共卫生安全造成了巨大威胁。疫情发生以来,相关科研人员从SARS-CoV-2的病原学特点、流行病学特征、病原学检测、治疗与愈后症状等方面进行了研究。本文对上述几个方面的研究成果进行了汇总。研究发现:SARS-CoV-2以其遗传多样性和重组频繁的特点不断产生变异体,以更大的传播力感染人群;目前已知的2种传染途径为跨物种传播和人际传播,其中飞沫传播和接触传播是人际传播的主要传播途径;感染人群遍布各个年龄段,其中患有慢性疾病的老年人更容易发展成危重病人;临床检测时联合核酸和抗原抗体检测,将有助于提高SARS-CoV-2感染的检出率并排除其他冠状病毒感染的可能性;目前尚未有治疗COVID-19的特效药物,临床上广泛使用广谱抗病毒药物或中药来治疗各种症状;全球各国正在加紧研发SARS-CoV-2疫苗,部分疫苗已进入临床试验阶段。本文有助于全面了解SARS-CoV-2的研究状况,也为其后续研究及防治提供了参考。Research Progress on SARS-CoV-2A large number of cases infected with severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)have appeared in many countries since December 2019 when the outbreak of coronavirus disease 2019(COVID-19)occurred,which has posed huge threat to global public health. SARS-CoV-2 has been studied by relevant researchers based on its etiology,epidemiological characteristics,etiology detection,therapy and prognosis since the outbreak,and the research results were summarized in this paper as follows:variants had been produced by SARS-CoV-2 due to its genetic diversity and frequent recombination,which had infected an increasing number of people with stronger transmission;two pathways including cross-species and interpersonal transmission had been known at present,in which,the main pathways of interpersonal transmission included droplet and contact transmission;people at all ages might be infected,especially the elderly with chronic diseases were more likely to become critical patients;the detection rate might be improved by the combination of detection of nucleic acid and antigen antibody that could eliminate the possibility of other coronavirus infection;no effective drugs for COVID-19 had been available at present,instead,broad-spectrum antiviral drugs or traditional Chinese medicine were widely used in clinical practice;the development of vaccines against SARS-CoV-2 had been sped up by all countries,and some vaccines had been at clinical trial stage. The status of development of SARS-CoV-2 were summarized in this paper,which also provided some references for future study and control of it.全文下载链接:https://kns.cnki.net/KCMS/detail/detail.aspx?dbcode=CJFQ&dbname=CJFDAUTO&filename=ZGDW202007015&v=MjY0MzRxVHJXTTFGckNVUjdxZllPZG9GeTNoVkwzUFB5clBlYkc0SE5ITXFJOUVZWVI4ZVgxTHV4WVM3RGgxVDM=
2020-07-17
上海市某奶牛场牛型结核菌素皮内变态反应疑似结果影响因素分析
为评估奶牛年龄分布、棚舍位置、胎次、泌乳等因素,对牛型结核菌素(PPD)皮内变态反应试验特异性的影响,对上海市某奶牛场2031头奶牛的PPD皮内变态反应试验初筛结果进行分析和回顾性问卷调查,并利用Epi infoTM 7软件,对初筛结果和问卷调查结果进行单因素分析。初筛和问卷调查结果显示:该奶牛场PPD皮内变态反应试验初筛疑似反应率为15.75%,疑似反应主要集中在2岁以下、0~2胎的青年奶牛,其中1.5~2岁青牛奶牛疑似反应率最高;随着年龄、泌乳量和胎次的增加,疑似反应率呈下降趋势。单因素分析结果显示:未配种奶牛(OR=2.02,P<0.01),未怀孕奶牛(OR=1.37,P<0.01),未泌乳奶牛或干奶牛(OR=3.72,P<0.01)是导致奶牛场出现PPD皮内变态反应试验疑似反应的危害性风险因素。结果表明:2岁以下青年奶牛易出现PPD皮内变态反应试验疑似反应,呈现出显著的年龄相关性;是否配种、怀孕以及泌乳量多少均能够影响PPD皮内变态反应试验的特异性。建议奶牛场将犊牛结核病首次检测日龄提前至90日龄以内,并对初筛出现疑似反应的奶牛立即隔离,采用γ-干扰素试验和PPD比较皮内变态反应试验的平行检测策略进行确诊。本研究全面了解了奶牛场初筛疑似反应奶牛的个体特征和风险因素,有助于提高检测方法的敏感性,减少假阴性。 Analysis on the Influence Factors for the Suspected Reaction to Intradermal Allergy Test of Purified Protein Derivative in a Dairy Farm in Shanghai CityIn order to evaluate the age,shed location,parity and milk production of cows and other factors influencing on the specificity of intradermal allergy test of purified protein derivative(PPD),the preliminary test results for 2 031 cows in a diary farm in Shanghai City were analyzed,and a follow-up questionnaire was carried out,single-factor analysis on the results was carried out by use of the software of Epi infoTM 7. It was found that the suspected reaction rate of PPD was 15.75%,mainly in the young cows less than 2 years old and with 0 to 2 parities,especially the young cows at 1.5 to 2 years old;the rate trended to decrease with the increasing age,milk production and parity. It was found that,by the single-factor analysis,the suspected reaction was caused by the hazardous risk factors including unmated cows(OR=2.02,P<0.01),non-pregnant cows(OR=1.37,P<0.01),unlactating or dry cows(OR=3.72,P<0.01). The results showed that the suspected reaction was likely produced in young cows less than 2 years old,which was obviously related to age;the specificity of the reaction could be influenced by whether the cows had been mated or pregnant and the amount of milk production. It was suggested that the first time of tuberculosis detection should be brought forward within 90 days old,the cows with suspected reaction should be quarantined immediately for confirmation by the parallel detection strategy of γ-interferon test and intradermal allergy test of PPD. 全文下载链接:https://kns.cnki.net/KCMS/detail/detail.aspx?dbcode=CJFQ&dbname=CJFDAUTO&filename=ZGDW202007007&v=MDA0NzN5M2hVTHZCUHlyUGViRzRITkhNcUk5Rlk0UjhlWDFMdXhZUzdEaDFUM3FUcldNMUZyQ1VSN3FmWU9kb0Y=
2020-07-17
上海市伪狂犬病病毒相关毒力基因分子变异分析
为了解伪狂犬病病毒(pseudorabies virus,PRV)4种主要毒力基因(gB、gC、gD和gE)的分子特征和变异情况,采用PCR技术,对2010—2015年分离到的28株PRV进行基因扩增和测序。结果显示:2010—2015年分离毒株的gB、gC、gD和gE基因均存在多位点突变,且位于抗原表位区;与2010年分离毒株的gD基因相比,2012年后分离毒株存在6个连续氨基酸插入。基于gB、gC、gD和gE基因的进化树显示:上海市2010年分离的4个毒株与2012年以后分离的毒株虽属于同一大的分支,但与经典毒株亲缘关系近,处于同一小的分支中;2012年分离毒株与2011年后国内变异株亲缘关系近,处于另一分支中。这些毒力基因抗原表位区域氨基酸的突变可能导致其毒力及抗原性发生改变;2012年以后分离毒株均为变异株,并已成为上海市的主要流行毒株。本研究为PRV分子致病机制及分子流行病学研究提供了依据。 Molecular Variation Analysis on Virulence Genes Related toPseudorabies Virus in Shanghai CityIn order to identify the molecular characteristics and variation of major virulence genes(gB,gC,gD and gE)of pseudorabies virus(PRV),28 strains isolated from 2010 to 2015 were amplified and sequenced by PCR assay. The results showed that multi-site mutations appeared in the epitope zones of all the genes;compared to the gD gene isolated in 2010,six consecutive amino acid insertions appeared in the strains isolated since 2012. Based on the genetic evolution trees of gB,gC,gD and gE gene,the four strains isolated in 2010 belonged to the same big branch as the strains isolated since 2012,but they were closely related to the classical strains,which were in the same small branch;the strains isolated in 2012 were closely related to the domestic variant strains isolated since 2011,which belonged to another branch. The virulence and antigenicity of the genes might be changed by the mutations of amino acids in the epitope zones. All the strains isolated since 2012 were variant,and had become prevalent in Shanghai City. Therefore,some references were provided for the study of PRV molecular pathogenesis and molecular epidemiology.全文下载链接:https://kns.cnki.net/KCMS/detail/detail.aspx?dbcode=CJFQ&dbname=CJFDAUTO&filename=ZGDW202007005&v=MTA5NzN5M2hVci9CUHlyUGViRzRITkhNcUk5RllZUjhlWDFMdXhZUzdEaDFUM3FUcldNMUZyQ1VSN3FmWU9kb0Y=
2020-07-17
2018—2019年我国部分地区猪肺炎支原体多位点序列分型
为了解我国猪肺炎支原体种群结构和进化关系,2018—2019年采集山东、广西、河南、江西、云南、天津、湖南等地猪组织样品1 242份,进行猪肺炎支原体荧光定量PCR和巢式PCR检测,并对测序确认的87份阳性样品,采用adk、rpoB、tpiA 3个管家基因作为分型参考进行多位点序列分型(MLST),并对3个管家基因均扩增成功的阳性样品进行p146基因分型,分析我国猪肺炎支原体种群结构,并结合pubmlst数据库公布的151个序列类型(ST)进行eBURST分析。结果显示:有24份阳性样品的3个管家基因全部扩增且测序成功,共分为10个ST,均为国内外首次报道;10个ST分为3个克隆复合体(CC)和4个独特型,其中ST128占41.7%(10/24),样品来自江苏、河南和广西,为本次检测的主要流行型。结果表明,我国猪肺炎支原体分布较广,具有一定的基因型独特性。本研究为我国猪肺炎支原体的流行病学数据库研究提供了新资料,有助于掌握国内猪肺炎支原体基因型分布情况,进一步完善了猪肺炎支原体流行病学数据库。 Multi-locus Sequence Typing of Mycoplasma hyopneumoniae in Some Regions of China during 2018 to 2019 In order to investigate the genetic characteristics and evolutionary relationship of Mycoplasma hyopnumoniae(M. hyopnumoniae)in China,1 242 swine tissues were collected during 2018 to 2019 from Shandong,Guangxi,Henan,Jiangxi,Yunnan,Tianjin and Hunan for detection of M. hyopnumoniae by real-time PCR and nested PCR,and 87 positive samples were confirmed. DNA was extracted from positive samples and classified according to p146 gene and multi-locus sequence typing(MLST)with adoption of 3 housekeeping genes including adk,rpoB and tpiA,then the population structure of M. hyopnumoniae in China was analyzed,and eBURST analysis was carried out in combination of 151 STs published in pubmlst database. The results showed that all the three housekeeping genes of 24 positive samples were successfully amplified and sequenced,which were divided into 10 STs and firstly reported in the world,involving three clone complexes(CCs)and four singletons,in which,ST128 was prevalent and accounted for 41.7%(10/24)and the samples were collected from Jiangsu,Henan and Guangxi. It was concluded that M. hyopnumoniae spread widely with unique genotypes to some extent. Therefore,some new data was provided for the study on epidemiological database of M. hyopnumoniae,which contributed to the identification of its distribution and further enriched relevant database.全文下载链接:https://kns.cnki.net/KCMS/detail/detail.aspx?dbcode=CJFQ&dbname=CJFDAUTO&filename=ZGDW202007004&v=MTQ2NDlOSE1xSTlGWUlSOGVYMUx1eFlTN0RoMVQzcVRyV00xRnJDVVI3cWZZT2RvRnkzZ1ZMektQeXJQZWJHNEg=
2020-07-17
A型流感病毒分型基因芯片在流感病毒各亚型监测中的应用
为验证自行研制的A型流感病毒分型基因芯片方法在实际中的应用效果,了解A型流感病毒各亚型在我国不同地区和不同宿主中的分布情况,对我国20个地区、不同宿主源(鸡、鸭、鹌鹑、鸽子、猪、人等)拭子和组织样品共16 852份进行了检测。利用自行研制的A型流感病毒分型基因芯片方法,对样品进行RNA提取、多重不对称RT-PCR扩增、芯片杂交、洗涤和扫描分析;同时将基因芯片检测阳性的样品,用普通RT-PCR扩增后进行序列测定。结果显示:利用该方法检出阳性样品2 582份,总检出率为15.32%(2 582/16 852),共检测到H1N1、H2N9、H3N2、H4N6、H5N1、H7N1、H7N9、H9N2、H10N5、H16N3等10个亚型,且均与阳性样品测序结果一致。结果表明,该方法可准确检测不同地区、不同宿主中的A型流感病毒不同亚型。同时,该方法能在同一张芯片上准确鉴定A型流感病毒的多种亚型,解决了其他常规方法无法检测已发现和可能发生变异的所有亚型的棘手问题,从而为A型流感诊断和分子流行病学监测提供了一种新技术。Application of Influenza A Virus Genotyping Microarray in Surveillance of Influenza Virus SubtypesIn order to verify the effect of influenza A virus genotyping microarray in practice,and to make clear the distribution of all subtypes of influenza A virus in various hosts in different regions in China,16 852 swabs and tissue samples collected from different hosts(chickens,ducks,quails,dove,pigs and human)in 20 regions were tested,and then RNA extraction,multiple asymmetric RT-PCR amplification,chip hybridization,cleaning and scanning analysis were carried out by the self-developed genotyping microarray. Meanwhile,the positive samples were amplified by conventional RT-PCR followed by sequencing. The results showed that 2 582 positive samples were detected out,with a total detection rate of 15.23%(2 582/16 852),involving 10 subtypes including H1N1,H2N9,H3N2,H4N6,H5N1,H7N1,H7N9,H9N2,H10N5 and H16N3,which was consistent with the result of conventional RT-PCR. It was concluded that different subtypes of influenza A virus could be detected in various hosts in different regions in China by the genotyping microarray that could also accurately identify various subtypes in the same microarray and solve the difficulty in detecting all identified or variant subtypes compared to the conventional methods,so a new technology was developed for confirmation of influenza A virus and molecular epidemiological surveillance.全文下载链接:https://kns.cnki.net/KCMS/detail/detail.aspx?dbcode=CJFQ&dbname=CJFDAUTO&filename=ZGDW202005021&v=MDQ3NDZHNEhOSE1xbzlIWllSOGVYMUx1eFlTN0RoMVQzcVRyV00xRnJDVVI3cWZZZVpzRnl6aFViM0xQeXJQZWI=
2020-06-04
猪弓形虫实时荧光PCR检测方法的建立与应用
为建立一种快速、敏感、特异的猪弓形虫检测方法,根据弓形虫保守基因序列,设计一套特异性引物和FAM荧光素标记的MGB探针;通过对 PCR反应体系和反应条件进行优化筛选,建立了猪弓形虫实时荧光定量PCR检测方法,并对此PCR检测方法进行了特异性、敏感性、重复性试验;利用所建立的方法对60份疑似弓形虫感染的临床样品进行了检测。结果显示:建立的猪弓形虫实时荧光定量PCR检测方法在101~107拷贝/μL模板范围内有很好的线性关系;对弓形虫重组阳性质粒出现阳性扩增信号,但对阴性对照的水和其他7种病原对照未扩增出特异性曲线;最低检测模板浓度为10拷贝/μL;自60份疑似猪弓形虫感染样品中检出32份阳性,并且和克隆测序结果一致。结果表明,本研究建立的猪弓形虫实时荧光定量PCR检测方法可用于猪弓形虫的快速检测,从而为猪弓形虫病的诊断提供了特异、敏感、高通量的方法。Development and Application of a Real-time PCR Method for Detection of Toxoplasma gondii In order to establish a rapid,sensitive and specific method for detection of Toxoplasmas gondii(T. gondi),a pair of specific primers and FAM(fluorescein)-labeled MGB probes were designed based on the conserved gene sequences of T. gondii. Followed by optimizing PCR reaction system and conditions,a real-time fluorescent PCR assay was established,and the specificity,sensitivity and reproducibility test were carried out;then 60 clinical samples suspected of T. gondii infection were tested by the established method. The results showed that the method expressed a good linear relationship when the template was within the range of 101–107 copies/μL. Specificity test showed that positive signal was observed only when amplifying the recombinant plasmids of T. gondii,rather than the water of negative contrast or other 7 pathogens;the minimum concentration of detection template was 10 copies/μL;32 out of 60 suspected samples were detected positive,and the result was consistent with that of cloning and sequencing. As a conclusion,the established method in this study could be used for rapid detection of T. gondii,which provided a specific,sensitive and high-throughput method for the identification of toxoplasmosis.全文下载链接:https://kns.cnki.net/KCMS/detail/detail.aspx?dbcode=CJFQ&dbname=CJFDAUTO&filename=ZGDW202005020&v=MTYwNzhyTVB5clBlYkc0SE5ITXFvOUhaSVI4ZVgxTHV4WVM3RGgxVDNxVHJXTTFGckNVUjdxZlllWnNGeXpoVXI=
2020-06-04
美洲型猪繁殖与呼吸综合征病毒实时荧光RT-PCR定量检测试剂盒测量不确定度研究
为填补测量不确定度在兽医领域病原学检测的空白,建立猪繁殖与呼吸综合征病毒(PRRSV)实时荧光定量RT-PCR检测试剂盒测量不确定度评估模型,对市售的3种美洲型PRRSV实时荧光RT-PCR检测试剂盒进行了测量不确定度评估,评估了移液器、温度、重复性测量因素对试验结果产生的不确定度影响。研究数据表明:移液器与重复性测量对试验结果产生的不确定度影响较大,应予以重视,需规范操作;建立的3种试剂盒测量不确定度模型缩小了试剂盒对可疑样品的判定区域,对样品临界参考值的检测结果判定具有实际意义。通过比对4个厂家的核酸提取试剂,筛选出了提取效率最高的试剂盒。本研究建立的PRRSV实时荧光RT-PCR定量检测试剂盒测量不确定度模型为兽医实验室检测结果不确定度评估及应用奠定了基础。 Study on the Uncertainty of Real-time PCR Kits for Quantitative Detection of PRRSV of American TypeIn order to fill in the blank of the measurement uncertainty in the field of veterinary etiology,an evaluation model of measurement uncertainty for real-time PCR kits for quantitative detection of PRRSV was established,and three kinds of commercial RT-PCR kits(detecting PRRSV strain of American type)were evaluated,including the influence of pipette,temperature and repeated measurement on test results. It was found that the test results were greatly influenced by the pipette and repeated measurement,which should be paid attention to,and the operations should be standardized;the determination area of the kits for suspected samples was reduced,which contributed to the determination of the critical reference value of samples. The kit with highest extraction efficiency was selected through comparing the nucleic acid extraction reagents produced by four manufacturers. In conclusion,a foundation was hereby laid for evaluation and application of measurement uncertainty in veterinary laboratories by the model established in this study. 全文下载链接:https://kns.cnki.net/KCMS/detail/detail.aspx?dbcode=CJFQ&dbname=CJFDAUTO&filename=ZGDW202005018&v=MjI2NzZXcnpOUHlyUGViRzRITkhNcW85RWJJUjhlWDFMdXhZUzdEaDFUM3FUcldNMUZyQ1VSN3FmWWVac0Z5emc=
2020-06-04
基于CRISPR/Cas9技术快速构建PRV gE基因缺失病毒
为快速、高效构建伪狂犬病病毒(pseudorabies virus,PRV)糖蛋白E基因(gE)缺失病毒,基于CRISPR/Cas9基因编辑技术,首先将pSpCas9(BB)-2A-GFP荧光质粒转染至VERO细胞和PK-15细胞,选出转染效率较高的细胞系,同时于http://crispr.mit.edu/网站设计并合成3个高评分的小导向RNA(small guide RNA,sgRNA),通过噬斑形成试验,筛选出高效sgRNA。其次将筛选出的针对PRV gE基因的sgRNA转染于PK-15细胞,然后接种PRV-1病毒,经过5 轮噬斑克隆纯化获得PRV-1-ΔgE。结果显示:VERO细胞比PK-15细胞具有更好的转染效果;gE-sgRNA1和gE-sgRNA2可作为针对gE基因的高效sgRNA;获得了1株PRV gE基因缺失291 bp的病毒,将其命名为PRV-1-ΔgE。研究表明,CRISPR/Cas9基因编辑技术可作为一种高效编辑PRV-1缺失病毒基因的方法,同时也为后续快速应对PRV变异株研究提供了新思路。 Construction of PRV gE-deleted Strain Based on CRISPR/Cas9 TechnologyIn order to rapidly and effectively construct the gE-deleted strain of pseudorabies virus(PRV),the fluorescent plasmid of pSpCas9(BB)-2A-GFP was firstly transfected into VERO cells and PK-15 cells to select the cell lines with high efficiency. Meanwhile,three small guide RNAs(sgRNAs)with high scores were designed and synthesized on http://crispr.mit.edu/ to select high-efficiency sgRNAs through plaque formation experiments. Then the selected sgRNA was transfected into PK-15 cells which subsequently were inoculated with PRV-1,and finally PRV-1-ΔgE was obtained after five rounds of plaque clone purification. The results showed that VERO cells could be well transfected compared to PK-15 cells;gE-sgRNA1 and gE-sgRNA2 could be used as the high-efficiency sgRNAs for gE gene;a strain of gE-deleted virus with a deletion of 291 bp was obtained and named as PRV-1-ΔgE. It was found that CRISPR/Cas9 gene editing technology could be used for effectively editing PRV-1 gene-deleted strain,which also provided new ideas to rapidly response to PRV variants in the future.全文下载链接:https://kns.cnki.net/KCMS/detail/detail.aspx?dbcode=CJFQ&dbname=CJFDAUTO&filename=ZGDW202005017&v=MjE0MjlOSE1xbzlFWTRSOGVYMUx1eFlTN0RoMVQzcVRyV00xRnJDVVI3cWZZZVpzRnl6Z1Y3N09QeXJQZWJHNEg=
2020-06-04
A型塞内卡病毒研究进展
A型塞内卡病毒(Senecavirus A,SVA)是新发现的一种影响养猪业的RNA病毒,现已在美洲的美国、加拿大、巴西、哥伦比亚以及亚洲的中国、泰国、越南等多个国家被发现。2015年SVA首次在我国广东省发现后,已有15个省、自治区和直辖市报道检测到该病毒,表明SVA已在我国不同地域广泛分布。猪感染SVA后的临床症状与口蹄疫、猪水泡病及水泡性口炎等类似,难以鉴别。SVA的实验室诊断多采用病原学和血清学方法。对于该病目前仍无商品化疫苗可用,但已有研究出重组活疫苗候选株和油佐剂灭活候选疫苗的报道。SVA未来的流行态势目前难以预测,因此需密切关注SVA在全球的流行现状,并深入研究SVA的致病及免疫机理,同时尽快研发有效的疫苗和诊断试剂。通过病原学、临床症状、流行状况、诊断方法、疫苗研发等方面的综述,本文为我国SVA防控提供了参考。Research Progress on Senecavirus AAs an emerging RNA virus in pig industry,Senecavirus A(SVA)has been found in many countries,including the United States,Canada,Brazil,Columbia,China,Thailand,Vietnam,etc. In China,the virus has been reported by 15 provinces/autonomous regions/municipalities since 2015 when it was firstly found in Guangdong,indicating a wide prevalence in different regions. It was difficult to identify the clinical symptoms of infected pigs that were similar to those of foot-and-mouth disease,swine vesicular disease and vesicular stomatitis,hence the methods of etiology and biology were widely used by SVA detection laboratories. No any corresponding commercial vaccines were available,but recombinant live vaccine candidate strains and oil-adjuvan inactivated candidate vaccines had been developed and reported. Considering current difficulty in prediction of the development of SVA,it was necessary to pay attention to its global prevalence,and to study its pathogenesis and immune mechanism so as to develop effective vaccines and diagnostic reagents soonest. In this paper,the etiology,clinical symptoms,epidemic status,diagnostic methods,vaccine research were discussed for the purpose of providing some references for the prevention and control of SVA in China. 全文下载链接:https://kns.cnki.net/KCMS/detail/detail.aspx?dbcode=CJFQ&dbname=CJFDAUTO&filename=ZGDW202005014&v=MjQwNDhZZVpzRnl6blc3N0tQeXJQZWJHNEhOSE1xbzlFWUlSOGVYMUx1eFlTN0RoMVQzcVRyV00xRnJDVVI3cWY=
2020-06-04
非洲猪瘟病原学和流行病学研究新进展
全球非洲猪瘟疫情形势十分严峻,2019年以来共29个国家/地区报告了新发或正在流行的疫情,非洲猪瘟的防控备受全世界关注。为全面了解非洲猪瘟病毒(ASFV)特性,概述了近年来对ASFV病原学特性和流行病学特点的一些新认识:ASFV对外界抵抗力较强,但怕干燥、高温、强酸和强碱;除家猪、野猪、软蜱外,苍蝇也可能扮演着储存宿主的角色;污染的饲料、饮用水也是ASFV的重要传染源,其中饮水造成ASFV感染的剂量更低,二者的风险点相差1万倍,防控时要高度重视饮用水;非洲猪瘟传播途径较多,但肉制品、环境、设备或车辆、人员是传播的重要风险因素,管理好其流动是防控的关键。本文对于识别非洲猪瘟的所有潜在传染源和传播途径,以便采取更有效的防控措施具有参考意义。 Research Progress on Etiology and Epidemiology of African Swine FeverThe emerging or prevalent outbreaks of African swine fever(ASF)have been reported in 29 countries/territories since 2019,its prevention and control has been concerned around the world due to the serious threats. In order to make clear the characteristics of African swine fever virus(ASFV),some new knowledge about the etiology and epidemiology found in recent years was summarized,that was,the virus was with strong resistance to outside conditions,except dryness,high temperature,strong acid and strong alkaline;its reservoir hosts might include flies in addition to domestic pigs,wild boars and soft ticks;the virus might spread via polluted feed and drinking water,especially the latter that needed to be highly concerned due to its risk level which was 10 000 times higher than that of polluted feed;in spite of various transmission pathways,it was the key to manage the movements of meat products,equipment or vehicles and personnel as well as environmental changes that were the major risk factors. As a conclusion,the study might contribute to the implementation of effective prevention and control measures through identifying all potential sources and transmission pathways of ASFV.全文下载链接:https://kns.cnki.net/KCMS/detail/detail.aspx?dbcode=CJFQ&dbname=CJFDAUTO&filename=ZGDW202005013&v=MjY2MzBac0Z5em5VTHZOUHlyUGViRzRITkhNcW85RVo0UjhlWDFMdXhZUzdEaDFUM3FUcldNMUZyQ1VSN3FmWWU=
2020-06-04
2014—2018年我国5株基因VII型新城疫病毒的基因组特性
为了解我国基因VII型新城疫病毒的分子生物学特性,对2014—2018年分离的5株基因VII型新城疫病毒代表株进行了基因组测序和生物信息学分析。序列分析显示:5株病毒F蛋白裂解位点为112RRQKR/F117或112RRRKR/F117,具有强毒株的典型特征;病毒基因组长度均为15 192 nt,其F蛋白融合肽、七肽重复区和跨膜区,以及HN蛋白跨膜区、中和抗原表位等功能区有多处氨基酸变异。病毒F基因遗传进化分析显示:2014—2015年分离的2株病毒属于基因VII.1.1亚型;2016—2018年分离的3株病毒属于基因VII.2亚型,暗示此亚型毒株可能已逐步取代基因VII.1.1亚型,成为家禽中的优势流行毒株。本研究进一步掌握了我国基因VII型新城疫病毒的遗传特性,从而为科学防控该病提供了参考。 Genomic Characteristics of Five Strains of Genotype VII Newcastle Disease Viruses Isolated in China from 2014 to 2018In order to recognize the molecular biological characteristics of genotype VII Newcastle disease virus(NDV)in China,five strains of representative genotype VII NDVs isolated from 2014 to 2018 were sequenced and analyzed. It was found that the cleavage site of F protein of the five isolates was 112RRQKR/F117 or 112RRRKR/F117,with the typical characteristics of virulent strain;the complete length of virus genome was 15 192 nt,and several amino acid mutations were identified in the fusion peptide,heptad repeat region and transmembrane domain of F protein,and the transmembrane domain and neutralizing epitope of HN protein. Analysis on genetic evolution of F genes showed that,the two strains isolated from 2014 to 2015 belonged to sub-genotype VII.1.1,and other three isolated from 2016 to 2018 fell into sub-genotype VII.2,which might gradually replace the sub-genotype VII.1.1 and become dominant in poultry. In short,the genetic characteristics of NDV genotype VII were identified,which provided some references for scientific prevention and control of Newcastle disease. 全文下载链接:https://kns.cnki.net/KCMS/detail/detail.aspx?dbcode=CJFQ&dbname=CJFDAUTO&filename=ZGDW202005008&v=MjQ3NjllWnNGeXpuVXJ6QVB5clBlYkc0SE5ITXFvOUZiSVI4ZVgxTHV4WVM3RGgxVDNxVHJXTTFGckNVUjdxZlk=
2020-06-04
我国台湾地区H5亚型禽流感病毒遗传进化分析
2019年以来,我国台湾地区多次暴发H5亚型禽流感疫情,病原包括H5N2、H5N5、H7N7等亚型禽流感病毒。为了解台湾地区流行的H5亚型禽流感病毒的分子流行特点,下载GenBank Influenza Virus Database和GISAID中公布的台湾地区所有H5亚型禽流感病毒HA序列,对近年来公开报道的台湾地区流行的H5亚型禽流感病毒进行遗传进化分析,并与大陆地区流行的H5亚型禽流感病毒进行比较。结果显示:台湾地区同时存在欧亚谱系和美洲谱系两个分支的H5亚型禽流感病毒流行,而大陆地区流行的H5亚型病毒都是欧亚谱系。欧亚谱系中,台湾地区流行的主要是第2.3.4.4a分支病毒,而大陆地区流行的主要是2.3.4.4d分支病毒,两个地区流行的H5病毒谱系并不完全一致。由于台湾地区的禽流感生态系统与大陆地区南方相似,因此台湾地区流行的美洲谱系H5亚型禽流感病毒有通过野鸟传播传入大陆地区的潜在风险,应提高警惕,加强禽流感调查监测,严厉查处非法走私禽类及其产品的活动。本研究为我国H5亚型禽流感防控提供了信息支持。。 Phylogenetic Analysis on H5 Subtype Avian Influenza Virus in Taiwan of ChinaH5 subtype avian influenza(AI)including H5N2,H5N5 and H7N7 had taken place several times in Taiwan of China since 2019. In order to identify the molecular characteristics of H5 subtype AIV prevalent in Taiwan,all HA sequences of the viruses isolated from Taiwan registered in GenBank Influenza Virus Resource and GISAID Database were downloaded to analyze their evolutionary relationships,and these H5 subtype AIV strains were compared with those prevalent in mainland China. The results showed that the H5 subtype AIV prevalent in Taiwan belonged to Eurasian or American lineage,while the strains prevalent in mainland China only fell into Eurasian lineage. For Eurasian lineage,clade 2.3.4.4a virus was mainly prevalent in Taiwan,while clade 2.3.4.4d virus was prevalent in mainland China. As avian influenza ecosystem in Taiwan was similar to that in southern mainland China,the viruses of American lineage prevalent in Taiwan had a potential risk of being introduced into mainland China via wild birds,so it was necessary to improve early warning system,strengthen investigation and surveillance on AIV,investigate and handle with any illegal smuggling of poultry and related products. In conclusion,some data was provided to prevent and control H5 subtype AI in China..全文下载链接:https://kns.cnki.net/KCMS/detail/detail.aspx?dbcode=CJFQ&dbname=CJFDAUTO&filename=ZGDW202005007&v=MTA5MTR6bVZMM09QeXJQZWJHNEhOSE1xbzlGWTRSOGVYMUx1eFlTN0RoMVQzcVRyV00xRnJDVVI3cWZZZVpzRnk=
2020-06-04
2019年我国部分地区屠宰生猪旋毛虫和弓形虫检测
为了解我国部分地区屠宰生猪旋毛虫和弓形虫感染情况,2019年在广东(5个)、山东(4个)、云南(6个)等省份的15个大型、中小型生猪屠宰场,采集猪膈肌样品315份,用PCR方法进行旋毛虫和弓形虫病原学检测;在以上3省15个大型、中小型生猪屠宰场(每省5个),采集血清样品254份,用ELISA方法进行旋毛虫和弓形虫血清学检测。结果显示:经PCR检测,采样地区所有膈肌样品均为旋毛虫阴性,仅在云南省120份膈肌样品中检出1份弓形虫阳性;经ELISA检测,采样地区血清样品的旋毛虫和弓形虫抗体阳性率分别为1.97%和2.36%,不同省份的阳性率分布不一致,中小型屠宰场阳性率均高于大型屠宰场。结果表明,广东、山东、云南等省份屠宰生猪的旋毛虫和弓形虫携带率极低,基本可以保证猪肉的产品安全;部分地区屠宰生猪存在一定的弓形虫感染抗体,尤其是中小型屠宰场,表明此类猪群需要加强饲养环节的弓形虫感染控制。本研究为保障猪肉食品安全及饲养环节的猪旋毛虫和弓形虫感染控制提供了依据和指导。 Detection of Trichinella spiralis and Toxoplasma gondii in Pigs for Slaughtering in Some Regions of China in 2019 In order to make clear the prevalence of Trichinella spiralis(T. spiralis)and Toxoplasma gondii(T. gondii)in pigs for slaughtering in some regions of China,in 2019,315 diaphragmatic samples were collected from 15 large or small and medium slaughterhouses in Yunan(5 farms),Shandong(4 farms)and Guangdong(5 farms)provinces for etiological detection by PCR;and 254 serum samples were collected from the slaughterhouses in the above three provinces(5 slaughterhouses per province)for serological test by ELISA. The results showed that,for PCR detection,all the diaphragmatic samples were negative for T. spiralis,and only one out of 120 samples from Yunnan was positive for T. gondii;for ELISA detection,the positive rates of antibodies against T. spiralis and T. gondii were 1.97% and 2.36%,respectively,with different distribution in different provinces,that was,the positive rate of small and medium slaughterhouses was higher than that of large ones. In short,the carrying rate of T. spiralis and T. gondii in the pigs for slaughtering in the above provinces was extremely low,which could ensure the safety of pork products;infection of T. gondii were presented in some regions in certain degree,especially in small and medium slaughterhouses,indicating that T. gondii should be controlled in the feeding process of such pig population. Some basis and guidance were hereby provided to safeguard the safety of pig products and to control infection of T. spiralis and T. gondii in feeding process.全文下载链接:https://kns.cnki.net/KCMS/detail/detail.aspx?dbcode=CJFQ&dbname=CJFDAUTO&filename=ZGDW202005001&v=MDMxMjhITkhNcW85RlpZUjhlWDFMdXhZUzdEaDFUM3FUcldNMUZyQ1VSN3FmWWVac0Z5am5WTDNKUHlyUGViRzQ=
2020-06-04
志贺氏菌检测和分群实时荧光PCR方法的建立
为对志贺氏菌属细菌进行快速、准确检测和分群鉴定,根据志贺氏菌invC、rfc、wbgZ和rfpB基因,分别设计引物和探针,建立了鉴定志贺氏菌属以及福氏志贺氏菌、宋内志贺氏菌和痢疾志贺菌氏菌实时荧光PCR方法,同时将根据ompA基因设计的引物和探针作为内参照,并通过特异性试验和人工接种试验等对该方法进行验证。结果显示,该方法对 17 株志贺氏菌和 19株非志贺氏菌均出现100%的特异性;用增菌肉汤直接进行PCR检测,发现在消毒奶、冰淇淋、酸奶、奶酪、熟肉和香肠等即食食品中的志贺氏菌检出限为1~3 cfu/25 g(mL),在原奶、生肉和奶粉中的检出限分别为≤8 cfu/25 mL、12 cfu/25 g和≤12 cfu/25 g;分离培养后再对可疑菌落进行实时荧光PCR鉴定,发现所有样品的检出限为1~3 cfu/25 g(mL),与传统培养法检出限一致。结果表明,该实时荧光PCR方法是一个快速、敏感、特异的检测方法,适合于志贺氏菌的常规检测分析。Establishment of a Real-time PCR Assay for Detecting and Classifying ShigellaIn order to detect and classify Shigella bacteria rapidly and accurately,the primers and probes were respectively designed according to the genes of invC,rfc,wbgZ and rfpB,and a real-time PCR assay was established to identify Sh. castellani including Sh. flexneri,Sh. sonnei and Sh. dysenteriae,then it was verified by specific test and artificial inoculation test with reference to the results of using the primers and probes designed according to ompA gene. The results showed that the specificity was 100% when all 17 strains of Shigella and 19 non-shigella strains were amplified by the established assay;through PCR assay with enrichment broth,the detection limits of Shigella in pasteurized milk,ice cream,acidophilus milk,cheese,cooked meat,sausage and other instant foods were 1–3 cfu/25 g(mL),and that in raw milk,raw meat and milk powder were≤8 cfu/25 mL,12 cfu/25g and≤12 cfu/25 g,respectively;the suspicious bacterial colonies were isolated and cultivated for real-time PCR assay,it was found that the detection limit of all samples was 1–3 cfu/25 g(mL),which was consistent with the result by traditional cultivation method. In short,the real-time PCR assay was rapid,sensitive and specific,which was appropriate for routine detection and identification of Shigella.全文下载链接:https://kns.cnki.net/KCMS/detail/detail.aspx?dbcode=CJFQ&dbname=CJFDAUTO&filename=ZGDW202003023&v=MjUxNzhNMUZyQ1VSN3FmWk9kdEZ5am1WN3ZJUHlyUGViRzRITkhNckk5SFo0UjhlWDFMdXhZUzdEaDFUM3FUclc=
2020-03-12
鸡传染性喉气管炎病毒实时荧光PCR检测方法的建立与应用
为探索适合临床高通量鸡传染性喉气管炎检测的方法,针对鸡传染性喉气管炎病毒gB基因序列保守区域,筛选1对特异性引物和1条特异性探针,建立了实时荧光PCR检测方法,并对该方法进行特异性、敏感性评估和临床应用检测。结果显示:筛选出的引物和探针与其他禽类常见病毒无交叉反应,检测下限达到100拷贝/反应;对来自13个场的480份临床样本进行检测,检测出17个阳性样品,与常规PCR检测结果符合率为100%。结果表明,此方法特异性强,灵敏度高、耗时少,可用于鸡传染性喉气管炎的快速检测。Establishment and Application of a Real-time PCR for Detecting Infectious Laryngotracheitis VirusIn order to develop an appropriate method for high-throughput detection of avian infectious laryngotracheitis(AILT),a real-time PCR assay was established with a pair of specific primers and a probe based on the conservative zone of gB gene sequence of AILT,then its specificity and sensitivity were evaluated,and its clinical application was tested. The results showed that the selected primers and probe failed to cross-react with other common poultry viruses,with the detection limit of 100 copies/reaction. 17 out of 480 clinical samples collected from 13 farms were positive. The results completely conformed to the test result by common PCR assay. As a conclusion,AILT could be rapidly detected by the established method with high specificity,high sensitivity and less time-consuming.全文下载链接:https://kns.cnki.net/KCMS/detail/detail.aspx?dbcode=CJFQ&dbname=CJFDAUTO&filename=ZGDW202003021&v=MTM1MDBQeXJQZWJHNEhOSE1ySTlIWllSOGVYMUx1eFlTN0RoMVQzcVRyV00xRnJDVVI3cWZaT2R0RnlqbVU3N04=
2020-03-12
禽源HSP70、HSP40和RPL4基因的克隆和表达
本研究参照GenBank中编码禽源热休克蛋白HSP70、HSP40 和核糖体蛋白RL4等的基因序列,分别设计特异性引物,采用RT-PCR扩增HSP70、HSP40和RPL4基因,经双酶切后克隆到真核表达载体pEF1α-Myc,得到重组质粒pEF1α-Myc-HSP70、pEF1α-Myc-HSP40和pEF1α-Myc-RPL4;将构建好的重组质粒,转染HEK293T细胞后,采用间接免疫荧光和Western-blot技术,对目的蛋白进行验证。结果表明,Myc-HSP70、Myc-HSP40和Myc-RPL4融合蛋白在HEK293T细胞内得到了正确表达。本研究为后续禽源HSP70、HSP40和RPL4基因的功能研究奠定了基础。Cloning and Expression of Avian-origin Genes of HSP70,HSP40 and RPL4In this study,specific primers were respectively designed according to the gene sequences of the heat-shock protein(HSP70 and HSP40)and the ribosomal protein(RPL4)registered in GenBank,then the genes of HSP70,HSP40 and RPL4 were amplified by RT-PCR,and after double enzyme digestion,the qualified genes were cloned into the eukaryotic expression plasmid,pEF1α-Myc,to obtain the recombinant plasmids including pEF1α-Myc-HSP70,pEF1α-Myc-HSP40 and pEF1α-Myc-RPL4;after the recombinant plasmids were transfected into HEK293T cells,the expressed protein was verified by indirect immunofluorescence and Western-blot. The results showed that the recombinant proteins of Myc-HSP70,Myc-HSP40 and Myc-RPL4 were correctly expressed in HEK293T cells. Therefore,the foundation was laid for further study on the genes of HSP70,HSP40 and RPL4.全文下载链接:https://kns.cnki.net/KCMS/detail/detail.aspx?dbcode=CJFQ&dbname=CJFDAUTO&filename=ZGDW202003020&v=MDI5OTRqbFdyN0xQeXJQZWJHNEhOSE1ySTlIWklSOGVYMUx1eFlTN0RoMVQzcVRyV00xRnJDVVI3cWZaT2R0Rnk=
2020-03-12
山东省3家鹦鹉养殖场多瘤病和喙羽病的病原检测与序列分析
2018年山东省某养殖场发生大量幼龄鹦鹉死亡事件,疑似为病毒感染。为探寻病因,开展了该场及省内另外两个鹦鹉养殖场的流行病学调查。利用PCR技术对临床样品中提取的DNA或RNA进行检测,结果发现新城疫与禽流感病毒均呈阴性,而禽多瘤病毒(APV1)与鹦鹉喙羽病病毒(PBFDV)呈现为强阳性,由此推断此3个鹦鹉养殖场存在APV1和PBFDV感染,平均病毒检出率分别为67.9%与71.4%,共感染检出率率为58.0%。对阳性样品进化全基因分析发现:区域内的PBFDV流行毒株同源性高,与该地区早期报道的毒株亲缘关系较为接近;APV1的VP1基因同源性较高,与欧洲分离毒株亲缘关系较为接近,说明我国流行的APV1或来源于进口鹦鹉。本研究警示,需要加强鹦鹉疾病防控,严格鹦鹉进出口检疫,并制定和健全标准的鹦鹉病检疫程序。Pathogen Detection and Sequence Analysis of Aves Polyomavirus 1 and Psittacine Beak & Feather Disease Virus in Three Parrot Farms in Shandong ProvinceIn 2018,a large number of young parrots died in a farm in Shandong Province,which was suspected of being infected with virus. In order to find out relevant causes,an epidemiological investigation was carried out in the infected farm and other two farms in Shandong Province. DNA or RNA extracted from clinical samples was tested by PCR,it was found that Newcastle disease virus(NDV)and avian influenza virus(AIV)were detected negative,while Aves polyomavirus 1(APV1)and psittacine beak & feather disease virus(PBFDV)were detected strongly positive,therefore,a conclusion was given that parrots in the three farms had already been infected with APV1 and PBFDV,with average positive rate of 67.9% and 71.4% respectively,and the average co-infection rate was 58.0%. As per the results of whole-genome analysis on these positive samples,the prevalent strains of PBFDV in the region shared high homology,which was more similar to those reported earlier;the VP1 genes of APV1 were with higher homology,which were more closer to those isolated in Europe,indicating that the APV1 prevalent in China might be introduced by imported parrots. Hence,it was necessary to strengthen the prevention of parrot diseases and increase quarantine indicators related to imported parrots in China.全文下载链接:https://kns.cnki.net/KCMS/detail/detail.aspx?dbcode=CJFQ&dbname=CJFDAUTO&filename=ZGDW202003022&v=MTAxMjJDVVI3cWZaT2R0RnlqbVVidk5QeXJQZWJHNEhOSE1ySTlIWm9SOGVYMUx1eFlTN0RoMVQzcVRyV00xRnI=
2020-03-12
结核病实验室检测技术研究进展
结核病(tuberculosis,TB)是由结核分枝杆菌(Mycobacterium tuberculosis,MTB)引起的一种人兽共患慢性传染病,其早期诊断和检测非常困难。常规的TB实验室检测技术主要是痰涂片和痰培养,这是诊断肺结核的金标准,但均有一定的局限性,故对其优化和改良是目前TB检测研究的一个重要方向,目前主要集中在硬件设备的升级改造上。近年来,TB实验检测诊断技术发展较快。核酸扩增方法成为TB诊断研究的新方向,包括环介导等温扩增(loop-mediated isothermal amplification method,LAMP),结核分枝杆菌/利福平耐药实时荧光定量核酸扩增(Xpert Mycobacte-rium tuberculosis/Rifamfampin,Xpert MTB/RIF),交叉引物恒温扩增(crossing priming amplification,CPA),实时荧光恒温扩增(simultaneoous amplification and testing method,SAT)以及PCR技术等。免疫学检测技术是TB检测的另一个重要发展方向,现已研制出两种快速检测方法T-SPOT.TB和QuantiFERON-Gold test(QFT)。还有一些新技术正在研发中,如基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption/ionization time of flight mass spectrometry,MALDI-TOF-MS)以及代谢标记检测、纳米孔测序等。TB检测手段正由传统的细菌学检查转向免疫、分子诊断等更为精确的方法。然而,不同诊断方法均有局限性,在实际应用中需采用多种检测方法相结合的方式。本文对于全面了解多种TB检测技术的优点和局限性,从而选择合适的检测方法具有帮助意义。Research Progress on Laboratory Technologies for Detection of TuberculosisTuberculosis(TB)is a chronic zoonosis caused by mycobacterium tuberculosis(MTB),which is very difficult to diagnose and detect at its early stage. Fortunately,the technologies of TB laboratory testing and diagnosis has been developing rapidly in recent years. As the main conventional testing technologies,smear and cultivation of sputum are gold standards,but both of the two technologies have some limitations,hence,their optimization and improvement have become the important direction for TB testing,with an emphasis on upgrading of related hardware equipment. In order to solve such limitations,nucleic acid amplification methods have been studied and explored,including Loop-mediated Isothermal Amplification(LAMP),Xpert Mycobacterium tuberculosis/Rifampin(Xpert MTB/RIF),crossing priming amplification(CPA),simultaneous amplification and testing(SAT)and PCR. Immunological assays are also used for TB testing,in which,T-SPOT.TB and QuantiFERON-Gold test(QFT)have been already developed. Other new technologies are also in development,such as Matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF-MS),metabolic marker testing,nanopore sequencing,etc. At present,TB testing methods are turning to immunoassay,molecular diagnosis and other more accurate methods from traditional bacteriological testing. However,all the above methods are limited,therefore different technologies should be used together in practice. In conclusion,the advantages and limitations of various TB testing technologies were summarized,which would help to select appropriate method in practice.全文下载链接:https://kns.cnki.net/KCMS/detail/detail.aspx?dbcode=CJFQ&dbname=CJFDAUTO&filename=ZGDW202003018&v=MDc1MjV4WVM3RGgxVDNxVHJXTTFGckNVUjdxZlpPZHRGeWpsVnJ2QlB5clBlYkc0SE5ITXJJOUViSVI4ZVgxTHU=
2020-03-12
冷冻原肠中细菌基因组DNA提取方法的建立
为建立冷冻原肠中细菌基因组DNA的直接提取法,采用震荡洗脱、金属网过滤和梯度离心相结合的方法,对2份冷冻原肠样品进行前处理;对经前处理后的原肠样品,采用酚/氯仿法提取细菌基因组DNA,然后进行琼脂糖电泳和OD260/OD280比值测定;以细菌基因组DNA为模板,用PCR技术扩增细菌16S rDNA并构建克隆文库,并从2个文库中各随机挑取3个菌落进行16S rDNA 的PCR鉴定和DNA测序。结果显示:样品提取的DNA浓度、纯度较高,OD260/OD280比值介于1.8~2.0;挑取的6个菌落中,1个为阴性,剩余5个为阳性,为肠球菌属(Enterococcus)和魏斯氏菌属(Weissella)的5种细菌。结果表明,本研究提取的原肠细菌基因组DNA质量较好,可用于非培养法研究冷冻原肠中细菌的多样性。本研究解决了因缺乏原肠细菌基因组DNA提取方法,无法进一步应用现代分子生物学方法研究冷冻原肠细菌的多样性、菌群组成结构和动态变化等难题。Establishment of a Method for Extracting Bacterial Genome DNA from Frozen Archenterons In order to establish a direct method for extracting bacterial genome DNA from frozen archenterons,two samples of frozen archenterons were pretreated by combination of shake-elution,filtration via metal mesh and gradient centrifugation technique;then the bacterial genome DNA was extracted by use of phenol-chloroform for agarose gel electrophoresis and measurement of OD260/OD280 ratio;taking the bacterial genome DNA as the template,16S rDNA was amplified by PCR assay,then the clone libraries were constructed. Three bacterial colonies were randomly selected from each of the two libraries for PCR identification and DNA sequencing of 16S rDNA. The results showed that the concentration and purity of DNA extracted from the samples were relatively high,and the OD260/OD280 ratio ranged from 1.8 to 2.0;one out of six selected bacterial colonies was negative,and others were positive,furthermore,the five species of bacteria belonged to Enterococcus and Weissella. In conclusion,the bacterial genome DNA extracted from frozen archenterons was of good quality,which could be used to study the diversity of bacteria in frozen archenterons by culture-independent method. With the method established in this study,it was possible to conduct further study on bacterial diversity,community structure and variation by using modern molecular biology technologies.全文下载链接:https://kns.cnki.net/KCMS/detail/detail.aspx?dbcode=CJFQ&dbname=CJFDAUTO&filename=ZGDW202002020&v=MjY5MzJGeW5oVkw3T1B5clBlYkc0SE5ITXJZOUhaSVI4ZVgxTHV4WVM3RGgxVDNxVHJXTTFGckNVUjdxZlpPZHQ=
2020-03-12
我国牦牛源冠状病毒流行现状分析
为了解我国牦源牛冠状病毒(BCoV)流行现状,通过查阅文献资料方法,汇总分析我国牦牛主产地区的BCoV检测情况。资料显示:2012年以来,我国青海、四川、新疆、西藏等牦牛主产省(自治区)的牦牛群中普遍存在BCoV感染且感染率较高,平均血清抗体阳性率多在80%以上,病原学阳性率多在60%以上。需采取加强饲养管理、控制混合感染、做好隔离净化等措施,控制牦牛群中的BCoV流行。我国的BCoV研究起步较晚,流行病学方面的报道较少,有必要进一步加强该病毒在牦牛中致病机理、流行情况和防治技术等方面的调查研究。本文为掌握我国牦牛中的BCoV流行情况及其防控提供了参考。Analysis on the Prevalence Status of Yak-derived Bovine Coronavirus in ChinaIn order to investigate the prevalence status of yak-derived bovine coronavirus(BCoV) in China,relevant testing results in main production regions of yak were summarized and analyzed by means of reviewing literature. As it was found that,since 2012,BCoV had been widely prevalent in the main provinces(autonomous regions),including Qinghai,Sichuan,Xinjiang and Tibet,with a high infection rate,and the average positive rates of serum antibodies in most regions were above 80%,the positive rates of pathogens were above 60%. In order to control the disease,a series of measures should be carried out,such as,strengthening husbandry management,preventing any mixed infection,and striving to isolate and purify the virus. However,there were few epidemiological reports due to late beginning of BCoV study in China,it was necessary to further strengthen relevant investigation and research on the pathogenic mechanism,prevalence and control technology of the disease. This study would provide some references for identification of BCoV prevalence and its prevention and control in China.全文下载链接:https://kns.cnki.net/KCMS/detail/detail.aspx?dbcode=CJFQ&dbname=CJFDAUTO&filename=ZGDW202003003&v=MzA0NDZZUzdEaDFUM3FUcldNMUZyQ1VSN3FmWk9kdEZ5amtXN3pBUHlyUGViRzRITkhNckk5Rlo0UjhlWDFMdXg=
2020-03-12
新型冠状病毒2019-nCoV与动物冠状病毒进化关系分析
新型冠状病毒(2019-nCoV)感染肺炎疫情自2019年12月在湖北省武汉市发生以来,已在我国和世界多个国家传播,引发全球关注。为阐明2019-nCoV与已知动物冠状病毒的关系,从冠状病毒的宿主分布、遗传进化分析、宿主受体分析等角度,对2019-nCoV的可能来源进行了综合分析。结果显示:2019-nCoV与从云南省中菊头蝠中检测到的冠状病毒分离株RaTG13基因组同源性最高,为96.2%,两者遗传进化关系也较为接近;从我国广东省和广西壮族自治区查获的走私穿山甲中检测到的冠状病毒则与2019-nCoV遗传关系相对稍远,与2019-nCoV基因组同源性分别为90.4%和85.5%;2019-nCoV与已知家畜、家禽,以及犬猫等宠物中检测到的冠状病毒基因组同源性≤54.2%,遗传进化关系较远。结合监测结果,基本排除2019-nCoV来源于已知家畜、家禽以及犬猫等宠物冠状病毒的可能性。Phylogenetic Analysis between the Novel Coronavirus(2019-nCoV)and Animal CoronavirusesSince the outbreak of pneumonia caused by the novel coronavirus(2019-nCoV)in Wuhan City of Hubei Province in December 2019,it has spread in China and many other countries around the world,which is the focus of global attention. In order to clarify the relationship between 2019-nCoV and the known animal coronaviruses,the comprehensive analysis was performed to find the possible source of 2019-nCoV based on the host distribution,genetic phylogenetic analysis,host receptor analysis of coronaviruses in the study. The results showed that the genome homology between 2019-nCoV and the coronavirus RaTG13 detected from Rhinolophus affinis in Yunan Province was the highest(96.2%),and 2019-nCoV was closely related to RaTG13 in genetic phylogenetic analysis. The genetic relationship between 2019-nCoV and the two coronaviruses detected from seized smuggling pangolin in Guangdong and Guangxi were relatively longer compared with RaTG13,and the genome homology of the 2019-nCoV with coronaviruses detected from seized smuggling pangolin in Guangdong and Guangxi was 90.4% and 85.5%,respectively. Genetic distance between 2019-nCoV and coronaviruses obtained from known domestic livestock,poultry,pets including dogs and cats was genetically long,and the genome homology of 2019-nCoV with them was≤54.2%. Combined with the published surveillance results,the possibility of 2019-ncov originating from the known coronaviruses of domestic livestock,poultry,pets including dogs and cats is basically excluded. 全文下载链接:https://kns.cnki.net/KCMS/detail/detail.aspx?dbcode=CJFQ&dbname=CJFDAUTO&filename=ZGDW202003002&v=MjI3MTVUcldNMUZyQ1VSN3FmWk9kdEZ5amtWTC9QUHlyUGViRzRITkhNckk5RlpvUjhlWDFMdXhZUzdEaDFUM3E=
2020-03-12
农业农村部派工作组赴玉树州指导抗灾保畜
依法做好非洲猪瘟防控工作——农业农村部畜牧兽医局负责人就非洲猪瘟防控答记者问
市场监管总局、农业农村部要求猪肉制品生产企业进一步做好非洲猪瘟防控
农业农村部部署2019年畜牧兽医重点工作
中心组召开学习(扩大)会议传达学习“不忘初心、牢记使命”主题教育工作会议精神
韩长赋在全国非洲猪瘟防控工作视频会上强调 确保打好打赢非洲猪瘟防控攻坚战 努力保障生猪产业持续健康发展
2018年我国农产品市场整体供应较为充裕
澳大利亚默多克大学代表团访问中心
农业农村部关于印发《2019年兽药质量监督抽检和风险监测计划》的通知
淋巴细胞脉络丛脑膜炎病毒实时荧光RPA快速检测方法的建立