单分子免疫阵列技术在传染病诊断中的应用
单分子免疫阵列(single molecule array,Simoa)技术,又称“数字ELISA”,是近年来新开发的一种数字免疫分析方法,将单分子计数用于蛋白质生物标记物的检测。该方法可测量各种不同基质(血清、血浆、脑脊液、尿液、细胞提取物等)中的蛋白质,检测限可达飞摩尔(fg/mL)数量级,灵敏度较传统ELISA提高约1 000倍。目前,该技术在一些重大传染病,如新冠肺炎、结核病、艾滋病、朊病毒病诊断中的优势逐渐凸显。Simoa技术可帮助监测新冠病毒感染进程,有望实现早期高灵敏度诊断,以区分新冠肺炎轻症和重症患者;有望帮助诊断活动性结核病,并可以监测结核病治疗效果;可更准确检测人免疫缺陷病毒(HIV)免疫相关蛋白含量变化,提高早期诊断的灵敏性;可在羊瘙痒病临床症状出现前检测活体动物血清,提高痒病早期检测能力,从而有望用于人类克雅氏症诊断。未来,通过开发相关病种的检测试剂盒,有望利用Simoa技术提高疫病的综合诊断能力,还可进行疫苗免疫效果评价,为疫病防控提供依据。Application of Single Molecule Array in Diagnosis of Infectious DiseasesSingle molecule array(Simoa),also known as“digital ELISA”,was a new digital immunoassay technology developed in recent years,which was generally used to detect protein biomarkers via single molecule counting. The technology could be applied to measure proteins in various specimens,such as serum,plasma,cerebrospinal fluid,urine,cell extracts,etc.,with a limit of detection as low as femtomolar(fg/mL),and an approximately 1000-fold sensitivity compared to traditional ELISA. Currently,the technology was promisingly applied in diagnosis of some major infectious diseases including COVID-19,tuberculosis,AIDS and prion virus. As the process of COVID-19 infection could be monitored with the help of the technology,it was expected that,by the technology,such infection could be early diagnosed with high sensitivity so as to distinguish patients with severe or mild symptoms,so was the active tuberculosis,and relevant therapeutic effect could be monitored;any changes of relevant immunogenic protein content related to human immunodeficiency virus(HIV)could be accurately detected to improve the sensitivity of early diagnosis;serums of live animals could be detected prior to any clinical symptoms of scrapie to improve early detection capacity and then it was expected to be used in the diagnosis of human Creutzfeldt Jakob disease in the future. It was also expected that,through development of kits for corresponding diseases,the technology could be used to improve comprehensive diagnosis capacity,and evaluate the effect of vaccination to provide a basis for disease prevention and control.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202107017&v=2vpJqQNi66HYIULKef58WwZVJVLDZg6qPdhbwq1MqCrryBSve7KdHzDvQhalVaqt
2021-07-26
新型免疫佐剂碳纳米管的研究进展
碳纳米管具有多种良好的生物学特性,是近几年科研工作者研究的热点纳米材料之一,可增强机体免疫应答,提高特异性抗体水平,在免疫佐剂研发领域展现了良好的应用前景。碳纳米管作为免疫佐剂的优势在于其有极高的比表面积,可以非特异性吸附多种药物、基因、蛋白质等物质;可进入免疫细胞,且不破坏细胞膜结构,从而为抗原运输和递送提供了有利条件。目前关于碳纳米管作用机体免疫应答的研究取得了一定进展,但其增强机体免疫应答的确切作用机制尚未揭示,今后需对碳纳米管最佳的功能化修饰方式、最适的免疫剂量及免疫毒性三方面进行深入研究。本文总结分析了碳纳米管作为佐剂的优势及其应用研究情况,以及存在的潜在问题及下一步研究重点,以期为碳纳米管免疫佐剂的研发提供新思路。Research Progress on the Carbon Nanotube as a Novel Immune AdjuvantWith excellent biological properties,carbon nanotube(CNT)has become one of the key nano materials studied by science researchers in recent years,which could improve immune response and increase the level of specific antibodies,with a good application prospect in the development of immune adjuvant. With extremely high specific surface area,CNT,as an immune adjuvant,could non-specifically absorb various drugs,genes,proteins and other substances,and enter immune cells without damaging any structure of cell membrane for the purpose of providing favorable conditions to transport and deliver antigens. At present,the studies on the effect of CNT on immune response have taken a step forward,but the exact mechanism for enhancing immune response has not been identified. Therefore,the optimal functionalization,immune dose and immunotoxicity of carbon nanotubes should be further studied in the future. In the paper,the advantages and application of CNT as an adjuvant,as well as possible problems and future research focus were analyzed with a view to providing some new ideas for next step.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202107016&v=2vpJqQNi66F%25mmd2BSI2TTQzug1SfI0uAyhOwe2b3xgJ8u7TxG1doR7XzGeDmyRHrp5Vo
2021-07-26
雪山草鸡感染传染性法氏囊病病毒新型变异株的鉴定
传染性法氏囊病病毒(infectious bursal disease virus,IBDV)新型变异株正在我国广泛蔓延,给商品肉鸡群和蛋鸡群带来了新的威胁。本研究对一个疑似发生非典型传染性法氏囊病的地方品种雪山草鸡群进行了RT-PCR检测,并对分离的3株毒株进行了基因测序及序列分析。结果显示,引起该鸡群发病的病原是IBDV新型变异株,属于A2dB1基因型;这3个毒株的VP2高变区编码蛋白上含有IBDV变异株的特征性氨基酸,也具有IBDV新型变异株的独特氨基酸。结果提示,我国地方品种鸡群中也有IBDV新型变异株的流行,现有的部分商品化IBDV疫苗已不能有效预防IBDV新型变异株的流行。Identification of Novel Variant Strain of Infectious Bursal Disease Virus in Xueshan ChickenNovel variant strains of infectious bursal disease virus(IBDV)have been spreading widely in China,posing a new threat to commercial broilers and layers. In the study,a local variety of Xueshan chicken with suspected atypical IBD was detected by RT-PCR,gene sequencing and sequence analysis were then conducted for the three isolates. The results showed that the disease was caused by novel variant strain of IBDV that belongs to A2dB1 genotype;in addition to the characteristic amino acids of variant strain,the unique amino acids of novel variant IBDV were also contained in the hypervariable region of VP2 encoding proteins of the three isolates. It was indicated that the novel variant IBDV could not be effectively controlled by some current commercial vaccines,as it has been spreading in local chicken in China.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202105022&v=2vpJqQNi66EG9nURkPtQqnsBL45kjWFuSxjcE%25mmd2BxMFYTC%25mmd2FNabgexGz2EDjIZ%25mmd2BVgWY
2021-05-17
非洲猪瘟病毒抗体间接ELISA检测方法的建立与应用
为建立一种检测非洲猪瘟病毒(ASFV)抗体的间接ELISA(iELISA)方法,对构建的ASFV P30基因表达工程菌诱导表达后,将获取的重组ASFV P30蛋白进行纯化和Western-blot检测,然后以纯化的重组蛋白为抗原,建立了ASFV抗体iELISA检测方法,并进行了特异性、灵敏性、重复性试验;同时与基于ASFV P30-2His6蛋白(N、C末端各融合1个His6标签)的iELISA方法进行兽医临床样本比较试验。结果显示:重组ASFV P30蛋白和重组ASFV P30-2His6蛋白在Western-blot检测中,均能与猪ASFV阳性血清产生特异性杂交带;基于重组ASFV P30蛋白iELISA的最佳反应条件为,抗原蛋白包被质量浓度20 μg/mL、血清样品稀释度1:1 000、酶标二抗稀释度1:40 000、血清样品检测OD450阳性结果临界值0.22。该方法仅对ASFV阳性血清呈特异性反应,1:3 200稀释的阳性血清仍可检出,批内试验和批间试验变异系数均小于10%,可以消除His标签所造成的假阳性反应。本研究建立的ASFV P30 iELISA检测方法为ASFV抗体检测提供了一种有效手段。Establishment and Application of the iELISA for Detection of Antibodies against ASFVIn order to establish an indirect enzyme-linked immunosorbent assay(iELISA)for detecting antibodies against African swine fever virus(ASFV),the gene expression engineering strain of recombinant ASFV P30 was induced and expressed,followed by purification and Western-blot detection of the recombinant proteins,then the iELISA method was established by taking the purified recombinant proteins as antigens,its specificity,sensitivity and repeatability were subsequently evaluated. Meanwhile,it was compared with the iELISA based on ASFV P30-2His6 protein(one His6 tag was fused respectively at N and C terminals)for veterinary clinical samples. The results showed that,by Western-blot detection,both the recombinant proteins of ASFV P30 and ASFV P30-2His6 could produce specific hybrid band with ASFV positive serum;the optimal reaction conditions of iELISA based on the recombinant ASFV P30 proteins were 20 μg/mL mass concentration of antigen protein coating,1:1 000 dilution of serum samples,1:40 000 dilution of goat anti-swine IgG conjugate,and 0.22 critical value of positive serum samples OD450. The established method was specific only to ASFV positive serum,1:3 200 diluted positive serum could still be detected,and the coefficients of variation(CV)of intra assay and inter assay were both less than 10%,which could eliminate any false positive reaction caused by His tag. Therefore,an effective tool was provided for detecting antibodies against ASFV by the established ASFV P30 iELISA in the study.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202105021&v=2vpJqQNi66FSzfXYeD5Lvm3vSgcyl2HyKfSv6OXd%25mmd2BKgpat1OHHXYyFn4oslWpcnF
2021-05-17
五种重要犬病毒微流控芯片检测方法的建立及应用
狂犬病毒、伪狂犬病毒、犬瘟热病毒、犬细小病毒和犬冠状病毒是引起犬科动物重要传染病的5种病毒,严重威胁着犬科动物的健康,是出入境犬科动物重点筛查的致病因子。为提升口岸进出境犬科动物检疫工作效率,建立了上述5种病毒微流控芯片检测方法。在建立5种病毒环等温扩增检测技术(loop mediated isothermal amplification,LAMP)的基础上,将这些LAMP检测体系固化在同一块塑料芯片上,制备了同步高通量快速检测微流控芯片,并将其应用于临床检测。结果显示:微流控芯片检测方法具有良好的特异性,含有目的基因的阳性样本仅在芯片上相应病毒反应槽出现显著扩增,而其他病毒反应槽未出现扩增,彼此无交叉反应;微流控芯片的敏感性与LAMP方法一致;通过检测不同时期收集的感染犬瘟热病毒、犬细小病毒和犬冠状病毒临床样本,证实建立的微流控芯片检测方法具有良好的稳定性和可靠性。与现有核酸检测方法相比,微流控芯片可以在受试核酸上样40 min内完成上述5种犬病毒的快速筛查,从而满足进出境犬科动物快速检疫的需求。Establishment and Application of the Microfluidic Chip Assay for Five Kinds of Major Canine Viruses Rabies virus,pseudorabies virus,canine distemper virus,canine parvovirus and canine coronavirus are five kinds of viruses that cause important infectious diseases of canine,which seriously threaten the health of canine,and are the key pathogenic factors to be screened and examined by the exit-entry ports. In order to improve the efficiency of port entry-exit quarantine for canine,a microfluidic chip assay for the above five viruses was established. Five loop-mediated isothermal amplification(LAMP)assays were specifically established and then solidified on the same plastic chip to prepare a microfluidic chip with high-throughput and rapid detection capacity for clinical detection. The results showed that the established assay had a good specificity where positive samples with targeted genes were obviously amplified only in the reaction tank in corresponding viruses instead of other viruses,and no cross reaction with each other was found;the sensitivity of microfluidic chip was consistent with that of LAMP;the established assay was with good stability and reliability as verified through detecting clinical samples infected with canine distemper virus and canine parvovirus and collected in different periods. Compared to existing nucleic acid detection methods,the microfluidic chip could be used to complete rapid screening of the above five viruses within 40 minutes after the loading of nucleic acids,which could satisfy the needs of rapid quarantine for entry-exit canine animals. 全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202105020&v=2vpJqQNi66EdimcaTwAcYhuaBu2kchUvfLZovQccWmMOE7GMIQI0zM9H4K4ihNQs
2021-05-17
禽白血病病毒抗原高敏荧光微球快速定量检测方法的建立
为建立快速定量检测家禽样本中禽白血病病毒抗原的方法,对禽白血病病毒p27蛋白单克隆抗体细胞株2E5和3D5进行复苏、鉴定,通过反应条件优化,建立了禽白血病病毒抗原高敏荧光微球快速检测方法。该方法能够快速定量检测家禽样本中禽白血病病毒,敏感性高、特异性强,对其他家禽病原无交叉反应且具有良好的检测重复性。对采自全国的240份临床家禽样品进行检测,同时用美国IDEXX公司生产的ELISA抗原试剂盒开展比较,结果发现两种方法符合率达93.98%,其中阳性样品符合率为90.83%,阴性样品符合率为95.83%;组内与组间变异系数分别低于10%和15%。本研究建立的禽白血病病毒p27抗原高敏荧光微球快速检测方法,特异性高、敏感性强,操作简单、快速,具有较高的应用推广价值。Establishment of the Highly Sensitive and Rapid Fluorescence Quantitative Detection for Avian Leukemia VirusIn order to establish a rapid and quantitative method for detection of avian leukosis virus(ALV)in poultry samples,2E5 and 3D5,the monoclonal antibody cell strains of ALV p27 protein were revived and identified,a highly sensitive and rapid fluorescence quantitative detection method,with high sensitivity and specificity and good test repeatability and no cross reaction to other pathogens,was established through optimization of reaction conditions,by which,ALV could be rapidly and quantitatively detected from poultry samples. 240 clinical samples collected across China were detected and compared with ELISA kit produced by IDEXX. It was found that the coincidence rate of the two methods was 93.98%,in which the coincidence rate of positive samples was 90.83%,and that of negative ones was 95.83%. The coefficients of variation in intra group and inter group were lower than 10% and 15%,respectively. In conclusion,the established method was worthy of being applied and expanded as supported by its high specificity and sensitivity,easy and rapid operation. 全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202105019&v=2vpJqQNi66GeYlL7h4X%25mmd2FfETw9jF5iXTqBJjpI7l9VOef8tuE2WJ08X8bEbqL%25mmd2FnyF
2021-05-17
鸡肝破裂血水综合征病因初探
近几年,在鸡群中出现一种以肝破裂血水为主要特征的疾病,简称为肝破裂血水综合征,给养殖单位造成较大经济损失。为探究鸡肝破裂血水综合征发生原因,采集11省区市9种品系鸡的458份临床肝破裂血水样本,开展病原分离以及分子生物学检测、病理学诊断、内毒素检测和动物试验,并结合流行病学调查进行病因探讨。病原分离结果显示:检测到大肠杆菌14份,占比2.8%;沙门氏菌7份,占比1.5%;未检测到弯曲杆菌以及未分离到相关病毒。PCR、RT-PCR结果显示:检测到禽腺病毒(FAdV)3份,占比0.66%;禽戊型肝炎病毒(HEV)1份,占比0.22%;未检测到鸡传染性贫血病毒(CIAV)、马立克病毒(MDV)、禽白血病病毒(ALV)。Random PCR检测未发现RNA和DNA病毒;内毒素检测发现15份样本超标,占比3.3%。动物试验结果显示,腹腔攻毒不能复制出临床肝破裂血水表征。选取11省区市99份有代表性的肝脏样本,固定后病理学诊断发现:无疑似髓样白血病/白血病病理变化;疑似细菌造成的坏死性肝炎4份,占比4.04%;疑似禽网状内皮细胞增生病并发血管瘤病理变化1份,占比2.02%;淀粉样变94份,占比93.94%。流行病学调查发现:该病在全国各地均有发病,发病鸡品种和日龄广泛,无明显季节性;同一鸡场不同品种,在饲料、免疫和管理都一致的情况下,有的品种肝破裂血水,有的品种正常。以上研究表明,近几年鸡群中出现的肝破裂血水综合征病例与常见的可引起鸡肝破裂出血的因素无明显相关性,初步分析与鸡体本身存在遗传自身免疫性缺陷因素有关,机体反应过度,出现细胞因子风暴综合征导致鸡肝脏淀粉样变出血,从而出现肝脏破裂血水的临床症状。Exploration on the Causes of Chicken Liver Hemorrhagic SyndromeIn recent years,an animal disease mainly characterized by liver hemorrhage(liver hemorrhagic syndrome)appeared in chicken,resulting in huge economic loss to poultry industry. In order to identify relevant causes,458 clinical lesion samples were collected from 9 chicken lines in 11 provinces/districts/cities for pathogen isolation,molecular biological detection,pathological diagnosis,endotoxin test and experiment on animals,the causes for the disease were discussed based on epidemiological investigation results. It was shown that,by pathogen isolation,14 samples of Escherichia coli and 7 samples of Salmonella were detected out,accounting for 2.8% and 1.5%,respectively,no Campylobacter or other virus was detected or isolated. It was concluded that,by PCR and RT-PCR,3 samples of fowl adenovirus(fadv)and one sample of avian hepatitis E virus(HEV)were detected out,accounting for 0.66% and 0.22%,respectively,no chicken infectious anemia virus(CIAV),Marek's disease virus(MDV)and avian leukosis virus(ALV)were detected. No RNA or DNA virus was found by Random PCR. 15 samples exceeded the standard for endotoxin,accounting for 3.3%. It was found that,by experiment on animals,the symptoms of clinical liver hemorrhage could not be reproduced by intraperitoneal challenge. 99 representative liver samples were selected from 11 provinces/districts/cities,followed by being fixed for pathological diagnosis,no suspected myeloid leukemia/leukemia pathological changes were found,4 cases of necrotic hepatitis caused by suspected bacteria,one suspected avian reticuloendotheliosis complicated with hemangioma and 94 cases of amyloidosis were observed,accounting for 4.04%,2.02% and 93.94%,respectively. By epidemiological investigation,it was found that the disease occurred all over the country,covering a wide range of varieties and age,without obvious seasonality;for different varieties in the same farm,some were with liver hemorrhage but others were normal under consistent conditions of feed,vaccination and management. In short,the cases of liver hemorrhagic syndrome found in recent years were not related to the common factors leading to liver hemorrhagic,which was connected with the genetic autoimmune defects in chicken as preliminarily analyzed,specifically,the organism reacted overly,followed by cytokine storm,resulting in amyloidosis bleeding in chicken liver,and thereby clinical symptoms of liver hemorrhage.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202105018&v=2vpJqQNi66Gk%25mmd2FmOb8Nn1bPYoKcWscH1oT57w4oQOpUdlnylBTbdSc3QPJgPCq9eb
2021-05-17
无乳链球菌对奶牛乳腺成纤维细胞金属蛋白酶及uPA系统表达的影响
为研究无乳链球菌(Streptococcus agalactiae,S. agalactiae)作用奶牛乳腺成纤维细胞(BMFBs)后对MMPs/TIMPs与uPA系统(uPA/PAI-1)表达的影响,以进一步阐明MMPs/TIMPs与uPA系统在调控细胞外基质(ECM)代谢的作用,将107 cfu/mL热灭活S. agalactiae菌液作用于BMFBs,在作用后不同时间点(6、12、24、48 h)通过RT-qPCR和Western Blot检测MMP-2、MMP-9、TIMP-1、TIMP-2、uPA、PAI-1 mRNA转录水平和相应蛋白表达水平,并用明胶酶谱法检测MMP-2、MMP-9的酶活性。结果显示:热灭活S. agalactiae作用后,MMP-9、TIMP-2、uPA、PAI-1 mRNA转录水平均有不同程度下降趋势,但MMP-2 mRNA水平上调,TIMP-1 mRNA水平呈先上升后下降趋势;明胶酶谱法检测MMP-2的酶活性较MMP-9明显,二者均在作用6 h、48 h活性最强;MMP-2和MMP-9蛋白表达水平与对照组相比主要呈先升高后下降趋势,但在作用后期MMP-2蛋白表达又出现了回升趋势;TIMP-1蛋白水平与对照组相比主要呈先下降后上升趋势,在作用前期与MMP-9蛋白表达相反,TIMP-2蛋白表达与对照组相比呈上升趋势;uPA和PAI-1蛋白水平与对照组相比均呈先降低后升高再降低趋势,均在24 h达到峰值,随后呈下降趋势。研究表明,热灭活S. agalactiae菌液可诱导BMFBs中MMPs/TIMPs表达,影响uPA系统参与调控MMPs的表达及活性。The Effect of Inactivated S. agalactiae on Metalloproteinase and uPA System Expression in BMFBsIn order to study the effect of Streptococcus agalactiae(S. agalactiae)on bovine mammary fibroblasts(BMFBs)and subsequently on the expression of MMPs/TIMPs and uPA system(uPA/PAI-1),and to further clarify the role of MMPs/TIMPs and uPA system in regulating extracellular matrix(ECM)metabolism,107cfu/mL heat-inactivated S. agalactiae was acted on BMFBs,the levels of MMP-2,MMP-9,TIMP-1,TIMP-2,uPA,PAI-1 mRNA transcription and corresponding protein expression were detected by RT-qPCR and Western Blot at different time points(6,12,24 and 48 h),then the enzymatic activities of MMP-2 and MMP-9 were detected by gelatin zymography. The results showed that after the reaction of heat-inactivated S. agalactiae,the level of transcription of MMP-9,TIMP-2,uPA and PAI-1 mRNA trended to reduce to different extent,except that of MMP-2 mRNA,and that of TIMP-1 mRNA increased firstly and then decreased;the enzymatic activity of MMP-2 was more obvious than that of MMP-9 as detected by gelatin zymography,both of which were strongest in 6 and 8 h after reaction;as compared with the control group,the level of protein expression of MMP-2 and MMP-9 trended to increase prior to decrease,but the protein expression of MMP-2 rose again at later stage;as compared to the control group,the level of protein of TIMP-1 showed a trend of decreasing prior to rising,which was contrary to that of MMP-9 at early stage,and TIMP-2 trended to increase;the levels of protein of uPA and PAI-1 both decreased first then increased,and decreased finally,reaching the peak at 24 h after reaction. In conclusion,the expression of MMPs/TIMPs in BMFBs could be induced by heat-inactivated S. agalactiae,which could influence uPA system in regulating the expression and activity of MMPs.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202103017&v=2vpJqQNi66HD98xaKRkUCe2%25mmd2FvY%25mmd2BdoLqNpkBm9OA0N3AHjQbEicL7krHGw89vj4Hl
2021-05-17
新疆野生北山羊小反刍兽疫病毒分子特征
为了解新疆野生北山羊小反刍兽疫病毒(peste des petits ruminants virus,PPRV)的分子特征,根据GenBank公布的PPRV基因组序列设计并合成引物,采用RT-PCR方法和基因测序技术获得病毒全基因序列,应用分子生物学分析软件,对分离的PPRV毒株进行序列分析。结果显示,本次分离的PPRV毒株(China/XJAKS/2017)属于基因IV型,基因组全长15 954 nt,编码6种结构蛋白和2种非结构蛋白;在系统进化上,与国内新疆分离株China/XJBZ/2015、China/XJYL/2013同源率分别高达99.0%、99.6%,与国外的巴基斯坦和塔吉克斯坦分离株亲缘关系最近。结果表明,PPRV已经在家养动物与野生动物之间传播。结果提示,PPRV传入野生动物群为我国消除PPR带来极大困难,需加强我国边疆地区PPR免疫隔离带建设,并对野生动物PPRV分子流行病学特点进行追踪监测。 Molecular Characteristics of PPRV Isolated from Ibex in Xinjiang In order to identify the molecular characteristics of peste des petits ruminants virus(PPRV)in ibex in Xinjiang,primers were designed and synthesized according to the genome sequence of PPRV published in GenBank,the whole gene sequence of the virus was obtained by RT-PCR and gene sequencing,then the isolated PPRV strain was sequenced and analyzed by molecular biology analysis software. The results showed that the isolated PPRV strain(China/XJAKS/2017)fell into genotype IV,with a total genome length of 15 954 nt,encoding 6 structural proteins and 2 non-structural proteins;for phylogenetic evolution,its homology rates with Xinjiang isolates including China/XJBZ/2015 and China/XJYL/2013 were 99.0% and 99.6%,respectively,and it was closest relative to Pakistan and Tajikistan isolates. It was concluded that PPRV had been spreading among domestic and wild animals,especially wild animals,which brought difficulties to eradicate it in China,so it was necessary to strengthen the construction of isolation zone with vaccination against FMDV in border regions in China,and to track and monitor the molecular epidemiological characteristics of PPRV in wild animals.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202105008&v=2vpJqQNi66FB38KGubt17K3%25mmd2BS%25mmd2BOWJtAMbK55OsDUUtzg45nPuZOLL3I5PoszpeXv
2021-05-17
我国西部地区农牧民包虫病防控认知情况及其影响因素
为掌握新疆、青海、甘肃、四川和西藏等西部5省区农牧民包虫病防控相关知识、态度和行为(“知信行”),分析影响“知信行”水平的关键因素,对1 213名农牧民进行问卷调查,用百分比描述“知信行”的每个条目,采用t检验或方差分析,比较不同人群的“知信行”水平差异。结果显示:西部5省区农牧民对包虫病防控相关知识的知晓率为57.0%,其中感染途径知晓率最低,为30.8%;55.6%的农牧民对包虫病防控持有积极态度,68.3%的农牧民对包虫病防控有较好的行为习惯。当地农牧民获取包虫病防治知识的途径多来自医生和传统宣传资料,分别占60.7%和48.2%,未来还希望通过电视、广播和网络等大众媒体获取。青海省农牧民以及文化程度初中及以上、民族为汉族的农牧民具有较高的认知水平。结果表明,西部5省区农牧民对包虫病的认知水平较低,获取相关信息途径较为单一。建议加大宣传力度,突出包虫病源头防控;利用当前主流新媒体,丰富宣传方式,使宣传内容、载体更加契合西部少数民族地区的生活和文化习惯,切实推进农牧民防疫意识和自我防护意识的提升。Awareness of Herdsmen for the Prevention and Control of Echinococcosis and Potential Risk Factors in Western ChinaIn order to investigate relevant knowledge,attitude and practice(KAP)of herdsmen towards the prevention and control of echinococcosis in Western China including Xinjiang,Qinghai,Gansu,Sichuan and Tibet,and to analyze the key factors for KAP level,a questionnaire investigation was conducted for 1 213 herdsmen,each item of KAP was described by percentage,and the KAP levels among different populations were compared by t test or ANOVA. The results showed that the average awareness rate of herdsmen for the knowledge of prevention and control of echinococcosis was 57.0%,and that for infection route was 30.8%,which was the lowest;55.6% of herdsmen were active for the prevention of echinococcosis,and 68.3% performed better behavior habits. Local herdsmen could access to the above knowledge mostly from doctors and traditional publicity materials,accounting for 60.7% and 48.2%,respectively,mass media such as television,radio and Internet were also expected in the future. The herdsmen with education background of junior middle school or above,and from Han ethnic group in Qinghai Province were at high awareness level. It was concluded that the herdsmen knew little about echinococcosis due to single pathway to access to relevant information. It was suggested to strengthen awareness activities with a priority of prevention and control from source,enrich publicity methods by use of current new media to make the contents and carriers conform to the living and cultural habits in the ethnic minority regions,and to effectively improve herdsmen's awareness for prevention and control of the disease and self-protection.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202105001&v=2vpJqQNi66EDmTu0UXlUm6Q%25mmd2BIvqB1ZwkysgLv0jd%25mmd2B2gd8fdMS9VGzNWDhm8218%25mmd2B3
2021-05-17
肠衣传统和新型腌制技术研究及应用进展
我国是世界肠衣出口大国。为使国内腌制肠衣企业全面了解国际肠衣腌制工艺更新动态,及时跟踪相关机构的科研方向及研究进展,本文综述了传统氯化钠(NaCl)腌制肠衣技术和新型磷酸盐补充干盐(P-salt)腌制肠衣技术的研究背景、最新进展及其应用范围,介绍了国外更新肠衣加工工艺的操作模式。NaCl腌制技术一直是肠衣防腐杀菌和常温保存的主要方法,目前国际贸易中绝大多数半成品和成品肠衣采用该技术处理。NaCl腌制技术的杀菌能力与腌制肠衣中的盐浓度即水活性、腌制温度和pH等多种因素联系紧密。P-salt腌制技术能显著提高肠衣的卫生状况和机械特性,既可显著提高肠衣灌注内容物时的润滑度,又能提高肠衣携带病毒的杀灭效果。一些相关研究成果已成为当前制定肠衣腌制处理工艺的重要依据。目前国内外的肠衣腌制技术已由单一的NaCl腌制技术,逐渐进入到传统NaCl腌制和新型P-salt腌制共存的局面,并大有向后者倾斜的趋势。本文为国内肠衣腌制技术的研究和储备提供了参考。Research and Application Progress of Traditional and New Salting Techniques for Casing for SausagesAs China has been a major exporter of casing for sausages in the world,the research background,latest progress and application scope of the salting techniques by traditional sodium chloride(NaCl)and new phosphate supplement dry salt(P-salt)were summarized,and the operation mode of latest casing processing techniques in other countries was introduced in the paper,for the purpose of learning update trend of international salting techniques,and tracing the research direction and progress in relevant organizations. NaCl salting technology has always been the main method for antisepsis,sterilization and room temperature preservation of casings,at present,most semi-finished and finished casings in international trade are treated with this technology. The bactericidal capacity of the NaCl salting technology is closely related to salt concentration(water activity,curing temperature,pH value and other factors)in salted casings. P-salt salting technique can obviously improve sanitary conditions and mechanical properties of casings,including both the lubricity when contents are filled into casings and the eradication effect when viruses are carried by casings. Relevant research results have been considered as the important basis for development of salting technique for casings. Currently,the salting technology of casings at home and abroad has gradually entered into the coexistence of traditional NaCl and new p-salt salting technology,and there is a great trend to the latter. Some references were thereby provided for the research and reserve of casing salting technique in China.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202105015&v=2vpJqQNi66EGhD5qw18P4wrvCIB9HA3PPuI98VprWEkP2jNEHuAwXrt2M%25mmd2FhcJYgx
2021-05-17
液相色谱-串联质谱法测定猪尿液中地西泮及其7种代谢物残留
为建立快速测定猪尿中地西泮及其7种代谢物残留的超高效液相色谱-串联质谱(UPLC-MS/MS)确证方法,本研究优化了样品净化的阳离子交换固相萃取柱(PCX柱)及其淋洗洗脱条件。猪尿液酸化后直接经PCX柱净化,依次用水、60%甲醇水溶液淋洗,最后用5%氨化甲醇洗脱;选用BEH C18色谱柱分离,UPLC-MS/MS进行检测,以基质匹配标准曲线定量。结果显示:8种药物在0.3~20.0 μg/L范围内具有良好的线性关系(R2≥0.995),检出限和定量限分别为0.1 μg/L和0.3 μg/L;各药物在3个添加浓度下回收率为73.6%~95.3%,日内、日间相对标准偏差分别为2.9%~18.6%(n=6)和2.2%~12.6%(n=3)。结果表明,本方法操作简单、快速,灵敏度高、特异性好。Determination of Diazepam and Its Metabolite Residuesin Swine Urine by UPLC-MS/MSIn order to establish an ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)to determine diazepam and its seven metabolite residues(temazepam,nimetazepam,nordiazepam,oxazepam,nitrazepam,7-aminonitrazepam and 7-aminonimetazepam)in swine urine,a cation exchange solid phase extraction column(PCX column)and its conditions of elution were optimized. Swine urine was directly purified by PCX column after being acidized,washed with water and 60% methanol water solution in turn,and then eluted with 5% ammoniated methanol;then the pre-treated urine sample was separated by BEH C18 column,detected by UPLC-MS/MS,and quantified by matrix-matched external standard curves. The results showed that a good linear relationship(R2≥0.995)among the eight drugs was obtained within 0.3 to 20 μg/L,the limits of detection and quantification were 0.1 and 0.3 μg/L respectively;the mean recoveries varied from 73.6% to 95.3% at the three concentrations,the relative standard deviations of intra and inter day were 2.9% to 18.6%(n=6)and 2.2% to 12.6%(n=3),respectively. It was concluded that the proposed method was simple and rapid with high sensitivity and specificity.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202103022&v=2vpJqQNi66FHwRwrKctQIdf4sHDyT80dN7Vep7TY5yE2n5TglxoDELE5pR1HP%25mmd2FaL
2021-03-16
兽用疫苗中BVDV、PCV2和PPV污染三重荧光定量PCR检测方法的建立
为建立可以快速同时检测兽用疫苗中牛病毒性腹泻病毒(BVDV)、猪圆环病毒2型(PCV2)和猪细小病毒(PPV)3种病原的方法,通过研究比对BVDV 5'-UTR、PCV2 Rep以及PPV NS1基因序列,分别设计合成了3对引物和3条荧光探针,在优化反应条件和反应程序后,建立了一种可同时检测BVDV、PCV2以及PPV的三重荧光定量PCR方法,并对其敏感性、特异性进行了评估。结果显示,建立的三重荧光定量PCR方法灵敏度高,对3种病原核酸的最低检测限均为10 copies/µL;特异性强,与其他相关病原(猪瘟病毒、猪繁殖与呼吸综合征病毒、伪狂犬病病毒、非洲猪瘟病毒)均无交叉反应。结果表明,本试验建立的三重荧光定量PCR适于疫苗中外源病毒的检测,可用于疫苗等生物制品质量把控。Establishment of a Triple Fluorescent Quantitative PCR for Detecting BVDV,PCV-2 and PPV in Veterinary VaccinesIn order to establish a rapid method for simultaneously detecting bovine viral diarrhea virus(BVDV),porcine circovirus type 2(PCV2)and porcine parvovirus(PPV)in veterinary vaccines,three pairs of primers and three fluorescent probes were designed and synthesized through comparing the sequences of BVDV 5'-UTR,PCV2 Rep and PPA NS1 genes,followed by the optimization of reaction conditions and procedures,a triple fluorescent quantitative PCR was established for simultaneously detecting BVDV,PCV2 and PPV,then its sensitivity and specificity were evaluated. The results showed that the established method was with high sensitivity and specificity,its minimum detection limits for the three pathogen nucleic acids were 10 copies/μL,and failed to react with other relevant pathogens,including classical swine fever virus(CSFV),porcine reproductive and respiratory syndrome virus(PRRSV),pseudorabies virus(PRV),African swine fever virus(ASFV). It was concluded that the method was applicable for detection of exogenous virus in vaccines,and could be used to control the quality of vaccines and other biological products. 全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202103021&v=2vpJqQNi66F74BtupbXSqOF6E0q2970nQnOlj%25mmd2F98%25mmd2FrBzx%25mmd2BbBHjr31kasB71O7ZIJ
2021-03-16
BVDV、BRV和BCoV TaqMan三重RT-qPCR检测方法的建立
为建立牛病毒性腹泻病毒(BVDV)、牛轮状病毒(BRV)和牛冠状病毒(BCoV)的快速检测方法,根据GenBank中登录的BVDV 5'-UTR、BRV NSP5和BCoV N基因序列设计特异性引物和探针,通过优化反应体系和条件,建立了同时检测上述3种病毒的TaqMan三重RT-qPCR方法。该方法仅对BVDV 5'-UTR、BRV NSP5和BCoV N基因扩增呈阳性,而对牛传染性鼻气管炎病毒、牛副流感病毒、魏氏梭菌(A型、B型和D型)、多杀性巴氏杆菌(A型和B型)等犊牛腹泻相关病原扩增均呈阴性;最低检出限为10拷贝/μL;组内和组间变异系数均小于2%。利用本研究建立的TaqMan三重RT-qPCR方法对29份临床样品进行检测,获得BVDV、BRV、BCoV的4种混合感染型,其中BVDV与BCoV的混合感染率最高(27.6%);与已有单重RT-qPCR方法比较,发现两种方法检测BVDV、BRV和BCoV的符合率分别为100%、96.5%、100%。结果表明,本研究建立的TaqMan三重RT-qPCR方法具有特异性强、敏感性高、重复性好、可行性高等优点,可为今后BVDV、BRV和BCoV共感染引起的牛腹泻性疾病的鉴别诊断和流行病学调查提供新技术手段。Establishment of TaqMan Triple RT-qPCR for Detecting BVDV,BRV and BCoVIn order to establish a rapid method for detecting bovine viral diarrhea virus(BVDV),bovine rotavirus(BRV)and bovine coronavirus(BCoV),the specific primers and probes were designed according to the sequences of 5'-UTR,BRV NSP5 and BCoV N genes registered in GenBank,followed by the optimization of reaction systems and conditions,then the TaqMan triple RT-qPCR was established to simultaneously detect the above three viruses. By which,positive results only occurred in the amplification of BVDV 5'-UTR,BRV NSP5 and BCoV N genes,but negative for infectious bovine rhinotracheitis virus(IBRV),bovine parainfluenza virus(BPIV),Clostridium welchii(type A,B and D),Pasteurella multocida(type A and B)and other pathogens related to calf diarrhea;the minimum detection limit was 10 copies/μL;and the variation coefficients of intra-and inter-group were less than 2%. 29 clinical samples were detected by the established method,the four types of co-infection of BVDV,BRV and BCoV were obtained,in which,the co-infection rate of BVDV and BCoV was highest(up to 27.6%). The coincidence rates of current single RT-qPCR and the established method for BVDV,BRV and BCoV were 100%,96.5% and 100%,respectively. As a conclusion,the established method could be applied in differential diagnosis and epidemiological investigation against bovine diarrhoeal diseases caused by the co-infection with BVDV,BRV and BCoV in the future due to its advantages of good specificity,sensitivity,repeatability and feasibility. 全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202103020&v=2vpJqQNi66FgWN7t02lf8JAPYtfwV3kJao7hAGUuiVll0QCZG9Q10FUq5hMJeVk8
2021-03-16
PCR结合斑点杂交技术检测禽多瘤病毒方法的建立及应用
鹦鹉幼雏病是由禽类多瘤病毒(APV)引起的多种鹦鹉雏鸟死亡的急性病毒性传染病,严重危害鹦鹉养殖业的健康发展。为提高分子生物学方法检测APV的敏感性和特异性,对APV基因片段进行克隆和序列分析,设计合成1对特异性引物,以VP1基因为模板,经PCR扩增获得731 bp的核苷酸DNA,并用DIG标记,制备用于检测APV的特异性核酸探针;对制备的探针进行灵敏度检测,同时与普通PCR进行敏感性比较;使用制备的探针,对经分离鉴定和制备保存的其他7种禽病毒核酸进行特异性检测;用该核酸探针,对疑似感染APV的鹦鹉病料进行斑点杂交检测,并对鉴定为阳性的APV进行全基因组扩增和序列分析。结果显示:该探针可检测到2 pg量的APV特异性核酸片段;仅APV-VP1阳性核酸显色,呈现阳性反应,而阴性核酸和其他7种禽病毒核酸均不显色,呈阴性反应。结果表明:建立的核酸斑点杂交检测方法具有较高的灵敏度和特异性,可用于临床初步诊断。本方法的建立为我国开展APV分子流行病学调查及其感染的临床诊断提供了技术支撑。Establishment and Application of PCR Combined with Dot Blot Hybridization for Detecting Avian PolyomavirusBudgerigar fledgling disease(BFD)is an acute viral infectious disease caused by avian polyomavirus(APV),which could lead to death of various parrot nestlings and seriously endanger the healthy development of parrot industry. In order to improve the sensitivity and specificity of molecular biological methods to detect APV,the APV gene fragment was cloned and sequenced. After designing and synthesizing a pair of specific primers as well as carrying out PCR amplification,gene fragment with the nucleotide length of 731 bp was obtained by taking VP1 gene as a template,and then the fragment was marked with DIG to prepare a specific probe for detecting APV;the sensitivity of the probe was detected and compared with general PCR;other 7 kinds of isolated and prepared virus nucleic acids were used to evaluate the specificity of the probe;the lesions of parrots suspected to be infected with APV were detected by dot blot hybridization using the probe,and the whole genome sequences of the positive APV were amplified and analyzed. The results showed that the probe could detect 2 pg of APV specific nucleic acid fragment;and only the positive nucleic acids of APV-VP1 showed positive reaction,while the negative one and other 7 kinds of virus nucleic acids all showed negative reaction. In conclusion,the established method could be used for clinical preliminary diagnosis due to its high sensitivity and specificity. Therefore,molecular epidemiological investigation and clinical diagnosis of APV infection in China were provided with technical supports by the established method. 全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202103019&v=2vpJqQNi66ENu4TMbnp5wITesOgSWVYX3GNv8UqvwAK%25mmd2FHFW9r%25mmd2BJ4RMfJbfgtfmNo
2021-03-16
七类消毒剂对非洲猪瘟病毒荧光定量PCR检测结果的影响
为评价戊二醛、酚、含碘类等常用消毒剂消毒后对非洲猪瘟病毒荧光定量PCR检测结果的影响,基于畜禽栏舍、运载工具、器具消毒及皮肤黏膜消毒目的,按消毒剂说明书推荐选择不同工作浓度,分别与不同滴度的非洲猪瘟病毒培养物于20 ℃条件下作用30 min后,采用荧光定量PCR方法检测作用后产物。结果显示,与对应的阳性对照组相比,含氯类(二氯异氰尿酸钠)、过硫酸氢钾类、二氧化氯类消毒剂,消毒后对荧光定量PCR检测结果影响最显著,检测Ct值显著上升或检测不到;戊二醛类、含碘类(主要成分聚维酮碘)消毒剂,核酸降解能力相对较弱,检测Ct值稍有上升;酚类、季铵盐类、含碘类(主要成分碘、磷酸、硫酸)类消毒剂,检测Ct值基本无变化。本研究评价了7类常用消毒剂消毒对非洲猪瘟病毒荧光定量PCR检测结果的影响,可为防控实践中科学、客观评价分析消毒效果提供技术参考。The Influence of Seven Categories of Disinfectants on the Test Results of ASFV by the Fluorescent Quantitative PCRIn order to evaluate the influence of common disinfectants including glutaraldehyde,phenol and iodine on the test results of African swine fever virus(ASFV),the disinfectants with different concentrations were used according to the corresponding instructions and based on the purpose of disinfecting pens,vehicles,instruments and skin mucous membrane to react respectively with the cultures of ASFV with different titers at 20 ℃ for 30 min,then the products were tested by fluorescent quantitative PCR. The results showed that,compared to the positive control group,test results of using chlorine(sodium dichloroisocyanurate),potassium bisulfate and chlorine dioxide compound disinfectants were significantly affected,the Ct value significantly increased or failed to be detected;for glutaraldehyde and iodine compound disinfectants(mainly contained povidone iodine),the capability of nucleic acid degradation was relatively lower,and the Ct value slightly increased;for phenol,quaternary ammonium and iodine compound disinfectants(mainly contained iodine,phosphoric acid and sulfuric acid),the Ct value generally remained the same. In conclusion,the influence of seven categories of disinfectants on the test results of ASFV by fluorescent quantitative PCR was evaluated,which could provide some technical references for scientific and objective evaluation and analysis of disinfection effect in practice.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202103018&v=2vpJqQNi66F8aSTvSBXTPV1Y3MmmScXJxElc9z63bfIKqOPCK4wm6NDvdOQjdWxT
2021-03-16
畜禽产品中四环素类药物残留色谱质谱检测技术及前处理方法研究进展
四环素类药物是一类广谱抗菌药物,在畜牧业中得到广泛应用。该药物的不合理使用甚至滥用,导致环境及动物源性食品中此类药物残留超标,从而威胁公共卫生安全。液相色谱法及液相色谱串联质谱法是检测畜禽产品中四环素类药物残留的主要方法。这类方法的难点及关键步骤是样品的前处理,也是目前研究的热点。本文就目前动物源性食品中四环素类药物残留色谱及质谱分析法,特别是样品前处理方法的研究进展进行综述。近年来新净化材料、新提取剂及新提取方法不断出现,提升了提取及净化效果,减少了试剂用量,提高了工作效率,并能够同时实现多类药物的提取和检测,有力保障了食品和环境安全。Research Progress on the Detection of Tetracycline Residues in Livestock and Poultry Products by LC-MS and Study on Pre-treatment MethodsTetracyclines,a class of broad-spectrum antibiotics,has been widely applied in animal husbandry. However,their residues in environment and animal-derived food would exceed the standard in the event of any unreasonable or abuse use,thus pose a threat to public health safety. Liquid chromatography(LC)and liquid chromatography-mass spectrometry(LC-MS)were the main methods to detect tetracycline residues in livestock and poultry products,and their difficulty and key step was pre-treatment of samples that was also a hotspot under study. In the paper,the LC and LC-MS for detection of tetracycline residues in animal-derived food,especially the research progress of sample pre-treatment methods,were summarized. With the emerging new purification materials,extraction agents and methods,the safety of food and environment was strongly safeguarded through improving the effectiveness of extraction and purification,reducing the dosage of reagents,increasing work efficiency as well as simultaneously extracting and detecting multiple drugs.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202103017&v=2vpJqQNi66HD98xaKRkUCe2%25mmd2FvY%25mmd2BdoLqNpkBm9OA0N3AHjQbEicL7krHGw89vj4Hl
2021-03-16
非洲猪瘟自然弱毒株演变进程
自2018年非洲猪瘟(African swine fever,ASF)传入中国以来疫情得到了有效控制。但2021年以后,我国的ASF流行情况出现了新的变化,临床中出现了“自然变异株”。这些毒株可能导致感染后临床症状不典型,容易与其他疫病混淆等问题。这与国外的流行演变规律一致,即当ASF在一个国家或地区流行较长时间后,其临床表现将由急性发病转变为缓慢发病或出现新的临床表现。经过多年的观察积累,国外已经对该病毒的流行规律特别是自然弱毒株的演变进程有了大量记载,但国内目前尚无这方面的描述。为此,就ASF自然弱毒株的演变进程进行综述,从而提出加强血清学诊断产品研发、加快产业化报批进程、适时开展血清学监测追溯等建议,以期为我国ASF科学防控提供参考。An Overview of Evolution Concerning Natural Attenuated Strains of ASFVThe outbreak of African swine fever(ASF)has been effectively controlled since African swine fever virus(ASFV)was introduced into China in 2018. But its prevalence has changed since 2021,that is,“natural variants”are clinically emerging. The strains may cause atypical clinical symptoms,and could be confused with other animal diseases. All of which is consistent with the evolution rule in other countries,that is,when ASF is prevalent in a country or region for a long time,its clinical symptoms will transfer from acute morbidity to slow occurrence or new symptoms are emerging. The prevalence rule of the virus,especially the natural attenuated strains,has been largely documented in other countries,except China. Therefore,the evolution process of the natural attenuated strains was summarized in the paper,and some suggestions were hereby put forward,such as further developing serological diagnosis products,speeding up the approval process for industrialization as well as carrying out serological surveillance and tracing as appropriate,with a view to providing some references for scientific prevention and control of ASF in China.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202103015&v=2vpJqQNi66GkJtuoa4GJnJM%25mmd2BMvT6D%25mmd2Fr5%25mmd2FMlDSyjfqrg20KKTyGmToW8Ifn3RGbxw
2021-03-16
动物新型冠状病毒流行现状
新型冠状病毒(SARS-CoV-2)感染导致的新冠肺炎(COVID-19)疫情自2019年12月以来,已在全球221个国家和地区传播,导致1亿多人感染,230多万人死亡。SARS-CoV-2除了对公共卫生产生极大影响之外,全球已有24个国家和地区报告470余起动物感染疫情,涉及犬、猫、水貂、狮子、老虎等多种动物。因此,在同一健康框架下,应高度关注动物感染SARS-CoV-2的早期预警。本文描述了动物感染SARS-CoV-2的国际流行特点,对其流行现状和感染动物种类进行了综合分析,评估了当前国内防控动物SARS-CoV-2感染面临的形势,并提出了针对性的防控建议,为国内COVID-19联防联控和养殖业健康安全提供了借鉴。The Prevalence Status of SARS-CoV-2 in AnimalsCorona virus disease 2019(COVID-19),caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),has been spreading in 221 countries and regions since September 2019,leading to more than 100 million persons infected and 2.3 million died. In addition to its considerable effect on public health,more than 470 outbreaks in animals were also reported in 24 countries and regions,involving canines,cats,minks,lions,tigers and other animals. Therefore,early warning of animal infection with SARS-CoV-2 should be highly concerned under the“one-health”framework. In the paper,the international prevalence characteristics of SARS-CoV-2 in animals were described,the prevalence status and species of infected animals were comprehensively analyzed,then the conditions for prevention and control of SARS-CoV-2 in animals in China was evaluated,and specific suggestions for prevention and control of SARS-CoV-2 were put forward,hoping to provide some references for joint prevention and control of the virus as well as the health and safety of livestock industry.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202103014&v=2vpJqQNi66HWgQ%25mmd2Bz2bqFmH%25mmd2BkG3SOxiQ%25mmd2B0l5c2HLERzRt0YyzkiRpu7LgOLgcxdxs
2021-03-16
RNA病毒重组及发生我国部分地区禽戊型肝炎病毒分子流行病学分析
为了解我国鸡群中禽戊型肝炎病毒(avain hepatitis E virus,aHEV)的分子变异情况,对2018年从山东、河南、河北、辽宁、吉林、黑龙江、陕西、山西、江苏等地疑似aHEV感染鸡群中采集的679份肝脏、脾脏病料样品进行RT-PCR检测,从中选取PCR阳性产物,对其ORF1基因进行序列测定与遗传进化分析。结果显示:从病料中检出阳性样品37份,阳性检出率为5.45%;选取的15个试验毒株之间的ORF1基因同源性为90.1%~100%,与目前报道的4种基因型同源性均小于90%,其中与基因 1 型的澳大利亚株和韩国株同源性为85.5%~89.0%,与基因2型的美国原型株和美国无毒株同源性为85.5%~88.2%,与基因3型的欧洲株和中国株同源性为86.3%~89.7%,与基因 4 型的匈牙利株和中国台湾株同源性为86.4%~88.5%。ORF1基因进化树分析发现,所分离的aHEV均属于戊型肝炎B种,不在目前所划分的基因型分支上,形成独立的基因分支,不属于已报道的4个基因型。结果表明,目前在我国流行的aHEV是一种新基因型病毒,这为我国aHEV分子流行病学分析提供了补充和依据。Molecular Epidemiological Analysis on Avian Hepatitis E Virusin Some Regions of ChinaIn order to investigate the variation status of avian hepatitis E virus(aHEV)in chicken in China,679 lesion samples of livers and spleens collected from the chicken suspected of being infected with aHEV in Shandong,Henan,Hebei,Liaoning,Jilin,Heilongjiang,Shaanxi,Shanxi,and Jiangsu provinces in 2018 were tested by RT-PCR,from which,positive samples were selected,and their ORF1 genes were sequenced and analyzed for genetic evolution. The results showed that 37 positive samples were detected out with the positive rate of 5.45%;the homology of ORF1 genes among 15 selected strains ranged from 90.1% to 100%,and the homology with the four genotypes previously reported was lower than 90%,specifically,that with genotype 1(Australian and the Korean strains)was from 85.5% to 89.0%,that with the genotype 2(American prototype and avirulent strains)was from 85.5% to 88.2%;that with the genotype 3(European and Chinese strains)was from 86.3% to 89.7%;and that with the genotype 4(Hungarian and China Taiwan strains)was from 86.4% to 88.5%. It was found that,by phylogenetic tree analysis of ORF1 genes,all the isolated aHEV strains belonged to genotype B of hepatitis E,which was not located in current genotype branch,but a new independent branch outside of the four reported genotypes. In conclusion,the aHEV strain prevalent in China was a new genotype virus,which provided a supplement and basis for molecular epidemiological analysis on aHEV.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202103001&v=2vpJqQNi66EYoeXG8NysKNTLG%25mmd2BSLPAV%25mmd2F9kJo6Jm0oxCBbOGcNz8zXyVXnrg3VoTk
2021-03-16
规模猪场密闭连廊及相关设施的设计和应用
自2018年非洲猪瘟传入我国以来,生猪养殖业遭受了沉重打击。建设规模猪场密闭连廊,可有效切断非洲猪瘟等动物疫病的传播途径。广西横县通过设计和应用密闭连廊连接生活区、猪舍、物资熏蒸房、出(进)猪台及死猪出口等端口,使猪舍防疫关口前移,增加了防疫纵深,实现了多层阻断抵御病原传播。本文介绍了密闭连廊及相关设施的设计要点和使用流程,以期为规模猪场落实各项生物安全措施,防控非洲猪瘟等动物疫病提供参考。Design and Application of Enclosed Corridors and Related Facilities in Scale Swine FarmsPig production industry has been seriously damaged by African swine fever(ASF)in China since 2018 when it was introduced into China. The spreading routes of animal diseases including ASF could be effectively blocked through the construction of enclosed corridors in scale swine farms. In Heng County of Guangxi,the living area,pens,material fumigation room,loading/unloading platform,discharge outlet for dead pigs and other ports were connected by the enclosed corridors,by which the gateway to pens for disease prevention and control was moved forward to go deep into the prevention and control measures,so as to block the spread of pathogens through such multi-layer blocking. In the paper,the design considerations and application process of enclosed corridors and related facilities were introduced for the purpose of providing some references for implementation of various bio-security measures,prevention and control of ASF and other animal diseases in scale farms. 全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202102014&v=2vpJqQNi66EkqWZmAe7NlkUZ08tJo2iW7N0Pl6GHCHEh%25mmd2FGT1fyLtm0bdV5pRcOW%25mmd2F
2021-02-26
超高效液相色谱-质谱法测定猪尿中15种β-受体阻断剂残留
为建立一种快速测定猪尿中15种β-受体阻断剂残留的超高效液相色谱-质谱(UPLC-MS/MS)确证方法,本试验优化了分子印迹固相萃取(MISPE)净化条件及色谱、质谱条件。猪尿样品经离心后加入MISPE柱上样,依次经水、乙腈淋洗,10%乙酸甲醇洗脱,N2吹干,0.1%甲酸水-乙腈(v:v=9:1)复溶;采用ESI正离子模式在多反应监测模式下进行离子扫描,外标法定量。结果显示,猪尿中15种药物在0.1~10 μg/L范围内具有良好的线性关系(R2≥0.996),检出限和定量限分别为0.03和0.10 μg/L;各药物回收率在76.7%~109.2%之间,批内、批间变异系数分别在2.2%~18.7%、2.1%~19.7%之间。结果表明,本方法操作简单快速、灵敏度高、特异性好。Determination of 15 β-blocker Residues in Pig Urine Using Ultra-high Performance Liquid Chromatography-Mass SpectrometryIn order to establish an ultra-high performance liquid chromatography-mass spectrometry(UPLC-MS/MS)to rapidly determine 15 kinds of β-blocker residues in pig urine,the conditions for purification of molecular imprinted solid phase extraction(MISPE)as well as chromatographic and mass spectrometry were optimized in the study. Pig urine samples were centrifuged and added in the MISPE column,then rinsed with water and acetonitrile successively,eluted with 10% acetic acid methanol,dried with N2,and redissolved with 0.1% formic acid water acetonitrile(V:V=9:1);ion scanning was carried out by ESI positive ion mode under multi-reaction monitoring mode(MRM),which was quantified by external standard method. The results showed that the 15 drugs were with a good linear relationship within 0.1~10 μg/L(R2≥0.996),and the limits of detection(LOD)and quantification(LOQ)were 0.03 and 0.10 μg/L,respectively;the recovery rate of all the drugs ranged from 76.7% to 109.2%,and the coefficients of variation ranged 2.2%~18.7% and 2.1%~19.7%,respectively. It was concluded that the method was simple,rapid,sensitive and specific.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202102025&v=2vpJqQNi66GojmWBcc%25mmd2B6miRpG%25mmd2Bo8GsLrhOfHgYqNYW69EExMBPYDl2RICFk9Kmq4
2021-02-26
三株牛支原体的致病性比较
为检测从内蒙古发病牛场分离到的3株牛支原体(HS2019、HSZ2019、HSS2019)的致病性,对其进行本动物回归试验,通过观察攻毒后的临床症状、病理变化,以及应用实时荧光定量PCR确定组织器官中支原体载量,分析3株支原体的毒力。结果显示,3株牛支原体回归牛体后均使试验牛出现体温升高、咳嗽、呼吸困难等临床症状,解剖可观察到试验牛肺部损伤以及肺脏与胸腔粘连的病理变化,其中HS2019株较其他两株引起的症状与病变更为明显,组织脏器中的载菌量最高。结果表明,这3株支原体均有致病性,其中HS2019株致病性最强,可作为今后疫苗研制的预备菌株。本研究既为国内疫苗的研制提供了菌株资源,也为今后牛支原体的免疫攻毒试验提供了评价标准。Comparison of the Virulence of Three Strains of Mycoplasma bovis In order to test the virulence of three strains of Mycoplasma bovis(HS2019,HSZ2019 and HSS2019)isolated from an infected farm in Inner Mongolia,an animal regression test was carried out,the number of Mycoplasma in tissue and organs was determined by observing clinical symptoms and pathological changes after virus challenge as well as by real-time fluorescent quantitative PCR,then the virulence of the three strains was analyzed. The results showed that such clinical symptoms as increasing body temperature,cough and dyspnea appeared in experimental cattle after the regression test of the three strains. The lung lesions as well as adhesion between lung and thoracic cavity and other pathological changes could be observed after anatomy,especially HS2019 strain,by which more obvious symptoms and lesions were caused,and the volume of Mycoplasma in tissues was maximum. In conclusion,the three strains of Mycoplasma were all pathogenic,especially HS2019 strain whose virulence was the strongest,which could be used as the preparation strain for vaccine development in the future. By the study,strain resources were provided for the development of vaccines,and evaluation criterion for immune challenge test of Mycoplasma bovis in the future was also provided.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202102023&v=2vpJqQNi66FfOvHvmWPuBUnH9uxLqtYVTp8wOiuUbXQ9dmslaZtj8%25mmd2BtumdMbS7yB
2021-02-26
裂谷热病毒Gn蛋白主要抗原区的串联表达和多克隆抗体制备
裂谷热(Rift Valley fever,RVF)是由裂谷热病毒(Rift Valley fever virus,RVFV)引起的一种烈性人兽共患传染病。RVFV囊膜蛋白Gn可诱导产生中和抗体,是RVFV检测方法和疫苗研究的重要抗原靶标。本研究通过分析蛋白抗原位点信息,构建包含Gn蛋白两个主要抗原区域的重组表达载体,随后将质粒转化至BL21感受态细胞,以IPTG诱导重组蛋白表达并优化蛋白表达条件,通过Western Blot鉴定重组蛋白;将重组蛋白免疫BALB/c小鼠,制备多克隆抗体,并以ELISA、Western Blot、IFA检测多克隆抗体的反应性。结果显示:诱导表达的Gn重组蛋白分子质量约为45 kDa;蛋白表达条件优化为IPTG终浓度0.25 mmol/L,诱导时间5 h;Western Blot鉴定发现蛋白成功表达。通过ELISA测定小鼠三免后血清抗体,结果发现抗体效价大于1:51 200;Western Blot检测显示,制备的多抗血清能与重组蛋白发生反应;进一步的IFA检测结果表明,制备的多克隆抗体可与真核质粒转染细胞中表达的Gn蛋白反应。本研究获得的Gn重组蛋白及制备的多克隆抗体为后续RVFV检测方法的建立奠定了基础。Tandem Expression of the Major Antigenic Areas of RVFV Gn Protein and the Preparation of Polyclonal Antibodies Rift Valley fever(RVF)is a virulent zoonotic disease caused by Rift Valley fever virus(RVFV). The envelope protein Gn of RVFV is an important antigen target for the detection method development and vaccine research,as it could induce the production of neutralizing antibodies. In the study,the recombinant expression vector containing two major antigen areas of Gn protein was constructed through analyzing the information of protein antigen sites,then the plasmid was transformed into BL21 cells,the recombinant protein expression was induced by IPTG,followed by optimization of relevant conditions,the recombinant protein was identified by Western Blot and then vaccinated into BALB/c mice to prepare the polyclonal antibodies that were tested by ELISA,Western Blot and IFA. The results showed that the molecular weight of the induced Gn recombinant protein was about 45 kDa;the expression conditions were optimized as follows:the final concentration of IPTG was 0.25 mmol/L with the induction time of 5 h;and the protein was successfully expressed as identified by Western Blot. The antibody titer in vaccinated mice was higher than 1:51 200 as tested by ELISA;it was shown by Western Blot that,the prepared polyclonal antiserum could react with the recombinant protein;and the polyclonal antibodies could react with the Gn protein expressed in eukaryotic transfection cells as detected by IFA. In conclusion,future development of RVF detection methods was provided with a basis by the expression of Gn protein and preparation of polyclonal antibodies. 全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202102022&v=2vpJqQNi66GGZ3UELIBPZGPh607vmumyimkuiumm5ssFBue57bzwY4ZSGwS2FGnC
2021-02-26
1型、2型牛病毒性腹泻病毒通用RT-PCR检测方法的建立与应用
为建立一种快速检测1型、2型牛病毒性腹泻病毒(BVDV)的通用RT-PCR方法,根据GenBank上收录的64株1型、2型BVDV以及1株猪瘟病毒(CSFV)的全基因组序列,应用Primer 6.0软件设计针对5'-UTR区域的1型、2型BVDV特异性通用引物对,扩增目的片段,并对该方法进行特异性、敏感性、重复性试验及利用该方法开展临床样品检测。结果显示:扩增的目的片段长度约为302 bp;该方法的灵敏度为2.09×102 copies/μL,无非特异性扩增,且重复性良好。对采自山东省发病牛场的13份鼻腔棉拭子和9份牛血清临床样品进行检测,发现有8份鼻腔棉拭子和8份血清为BVDV阳性,随机抽取4份阳性样品送测序,发现3份样品毒株为1型BVDV,1份样品还需进一步鉴定。本研究建立的RT-PCR方法可实现对1型、2型BVDV核酸的特异性检测,也可用于该病的流行病学调查研究。Establishment and Application of a General RT-PCR Assay for Detection of BVDV Type 1 and 2In order to establish a general RT-PCR assay to rapidly detect bovine viral virus(BVDV)type 1 and 2,the general primer was designed based on 5'UTR sequences of BVDV type 1 and 2 by Primer 6.0 software according to the analysis of complete gene sequence of 64 strains of BVDV type 1and 2 and one strain of classical swine fever virus(CSFV)registered in GenBank,and the target fragment was amplified,then the specificity,sensitivity and reproducibility of the RT-PCR method were evaluated,and the clinical samples were tested. The results showed that the amplified target fragment was about 302 bp;the detection limt of the method was 2.09×102 copies/μL,with no nonspecific amplification but good reproducibility. 13 nasal swabs and 9 bovine serum samples collected from an infected farm in Shandong Province were detected by the established method,it was found that 8 nasal swabs and 8 sera were positive against BVDV,then 4 of the positive samples were randomly selected to carry out virus gene sequencing and comparison,it was found that virus of 3 samples could be identified as BVDV type 1,and the other 1 sample still needed further analysis. In conclusion,specific detection of nucleic acids of BVDV 1 and 2 could be realized by the RT-PCR assay established in the study,which could also be used in epidemiological investigation of BVDV.全文下载链接:https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202102020&v=2vpJqQNi66ECPxin0tV%25mmd2BwXuEDdY2ncWQTI3QnKz94n6p6pDeO9uI1raFF86me8bE
2021-02-26