新疆库尔勒地区羊源产志贺毒素大肠杆菌及其耐药性分析

为了解新疆库尔勒地区羊源产志贺毒素大肠杆菌（Shiga toxin-producing Escherichia coli,STEC）毒力基因的分布情况及其耐药性,对新疆库尔勒地区羊肉生产环节（养殖场、定点屠宰场和市场）的282份样品进行STEC的分离鉴定,并检测毒力基因的编码情况,进一步检测了17种常用抗生素的耐药情况。结果表明：羊肉生产环节样品中检出72株STEC,检出率为25.5%。72株STEC中编码毒力基因stx1+stx2、stx1、stx2、rfbE、eae、hly的数量分别为22、45、2、0、4、51个。其中,羊养殖场中存在较多的非O157 STEC,检出率为37.3%;stx以stx1为主,检出率达到了652%。72株STEC中以β-内酰胺类、氨基糖苷类、大环内酯类抗生素耐药为主,STEC菌株的多重耐药比较严重,最多可耐9种抗生素,多重耐药情况主要是耐1种和耐2种抗生素。研究表明,羊肉生产环节中羊养殖场存在大量的非O157 STEC菌株,且耐药情况比较严重,务必预防源头污染。     

海南辣椒、樱桃番茄病毒病调查及病原dot-ELISA鉴定

[目的]调查和鉴定海南省辣椒和樱桃番茄病毒病发生情况和病毒种类,明确病毒病发生为害程度及毒源种类.[方法]2018—2019年,通过5点取样法对海南省10个市县的辣椒和樱桃番茄进行病毒症状类型、发病率和病情指数统计.采集辣椒和樱桃番茄主产区病毒病样本593份,通过斑点酶联免疫吸附法(dot-ELISA)对黄瓜花叶病毒(...     

建兰花叶病毒广东分离物的全基因组序列分析

【目的】建兰花叶病毒(Cymbidium mosaic virus,CymMV)是最主要的兰花病毒病原之一,其广泛传播对兰花产业发展造成严重威胁。探究CymMV遗传信息和进化,为广东省兰花病毒病的监测预警和兰花抗病毒病基因工程提供重要科学依据。【方法】对采自广州地区的墨兰疑似病毒病叶片进行CymMV的RT-PCR及DA-SELISA检测鉴定,利用相关分子生物学软件对CymMV分离物进行基因组序列组装、注释、系统进化及选择压力分析。【结果】首次在广东省发现2个CymMV分离物GZV013和ZC-29,基因组全长均为6227 nt,编码5个功能蛋白;GZV013与台湾分离物M2的核苷酸序列一致性为97.03%;ZC29与南京分离物NJ-1的核苷酸序列一致性为97.11%;CymMV各分离物的全基因组序列一致性为86.85%～98.31%,且多样性种群的分化与寄主种类和地理隔离关系密切;CymMV基因组每个区域均受到负选择的影响,并符合中性进化模型;编码RdRp、TGB1和TGB2的基因在基因组中变异率最高。【结论】GZV013与台湾分离物M2的亲缘关系最近,ZC29与南京分离物NJ-1的亲...     

Fluorescence localization of epidermal cathepsins L and B in the Japanese eel

Histochemical localization of proteolytic activities in the dorsal epidermis of Japanese eel was demonstrated by fluorescent microscopy utilizing 4-methoxy-2-naphthylamide (4M$\beta$NA) derivatives as substrates and 5-nitrosalicylaldehyde as a trapping agent. Carbobenzoxy-L-phenylalanyl-L-arginyl-4M$\beta$NA (Cbz-Phe-Arg-4M$\beta$NA) and Cbz-Arg-Arg-4M$\beta$NA were used for direct detection of cathepsins L and B activities, respectively, in fresh frozen sections and unfixed cells of the eel epidermis. The fluorescing areas, where Cbz-Phe-Arg-4M$\beta$NA was hydrolyzed by cathepsin L, were shown in mucus secretory cells and club cells and broadly around skin surface. The fluorescing areas due to Cbz-Arg-Arg-4M$\beta$NA hydrolysis by cathepsin B were localized similarly in these tissues. The fluorescing intensity for both catheptic activities in mucus secretory cells was higher than that in club cells, where small fluorescing granules were distributed. These results indicate that eel cathepsins L and B are stored in epidermal secretory cells at different levels and probably serve as defense factors before or after secretion by these cells. Abbreviations: Cbz – carbobenzoxy; 4M$\beta$NA – 4-methoxy-2-naphthylamide; NSA – 5-nitrosalicylaldehyde.     

Generation and characterization of aptamers against grass carp reovirus infection for the development of rapid detection assay

Grass carp reovirus (GCRV) causes devastating viral haemorrhagic disease in farmed grass carp (Ctenopharyngon idellus). As novel molecular probes, aptamers have been widely applied in rapid diagnosis and efficient therapies against virus or diseases. In this study, three single‐stranded DNA (ssDNA) aptamers were selected against GCRV‐infected CIK cells via SELEX (systematic evolution of ligands by exponential enrichment technology). Secondary structures predicted by MFOLD indicated that aptamers formed stem‐loop structures, and GVI‐11 had the lowest ΔG value of ?30.84 KJ/mol. Three aptamers could specifically recognize GCRV‐infected CIK cells, with calculated dissociation constants (Kd) of 220.86, 176.63 and 278.66 nM for aptamers GVI‐1, GVI‐7 and GVI‐11, respectively, which indicated that they could serve as specific delivery system for antiviral therapies. The targets of aptamers GVI‐1, GVI‐7 and GVI‐11 on the surface of GCRV‐infected cells could be membrane proteins, which were trypsin‐sensitive. Furthermore, FAM‐labelled aptamer GVI‐7 could be applied to detect GCRV infection in vivo. It is the first time to generate and characterize aptamers against GCRV‐infected cells. These aptamers have great potentials in development of rapid diagnosis technology and antiviral agents against GCRV infection in aquaculture.     

Production of polyclonal antiserum against recombinant VP28 protein and its application for the detection of white spot syndrome virus in crustaceans

The VP28 gene of white spot syndrome virus (WSSV) was cloned into pRSET B expression vector. The VP28 protein was expressed as a protein with a 6-histidine taq in Escherichia coli GJ1158 with NaCl induction. Antiserum was raised against this recombinant-VP28 protein in rabbits and it recognized VP28 protein in naturally and experimentally WSSV-infected shrimp, marine crabs, freshwater prawns and freshwater crabs. The antiserum did not recognize any of the other known WSSV structural proteins. Various organs such as eyestalks, head muscle, gill tissue, heart tissue, haemolymph, tail tissue and appendages were found to be good materials for detection of WSSV using the antiserum and detection of WSSV was successful in experimentally infected Penaeus monodon and P. indicus at 12 and 24 h post-infection (p.i.), respectively. The antiserum was capable of detecting WSSV in 5 ng of total haemolymph protein from WSSV-infected shrimp.     

白斑综合征病毒环介导等温扩增快速检测方法的建立

根据对虾白斑综合征病毒(WSSV)囊膜蛋白VP28基因保守序列,利用Primer Explorerv 4.0软件设计了4条引物,建立了白斑综合征病毒环介导等温扩增快速检测方法,对反应温度和反应时间等参数进行了优化,同时将建立的LAMP检测方法与巢式PCR进行了比较分析。结果表明,LAMP最适反应在64℃恒温条件60min内完成,凝胶电泳呈现梯型条带;反应体系中添加SYBR Green I荧光染料后,绿色的阳性结果明显区别于橙色阴性结果。LAMP方法的最低检出限为100拷贝/μL,灵敏度较巢式PCR高100倍,而且LAMP方法在1h内即可完成检测,操作简单,无需复杂仪器,肉眼可直接观察检测结果。用建立的LAMP方法对临床发病南美白对虾样品进行了检测,结果表明,LAMP方法适合对虾白斑综合征病毒的现场快速检测。     

弧菌属PCR检测方法的建立与应用

根据弧菌属细菌(Vibrio spp.)共有的rpo A基因序列,比对和设计了一对PCR引物,建立了能够快速而准确地检测弧菌属细菌的通用PCR检测方法,该方法对靶标DNA的检测灵敏度为58 fg/μL,对菌液的检测灵敏度为2.7×102cfu/m L,能够区分该属细菌与其它属的细菌,具有极高的特异性。使用建立的方法对分离自南美白对虾的病原菌进行了检验,并结合16S r DNA序列分析,结果显示待检测病原均为弧菌属的细菌,与测序结果一致,说明该检测方法可用于弧菌属细菌的检测和诊断。     

梨叶片中苹果褪绿叶斑病毒的引物原位标记检测

 选用带苹果褪绿叶斑病毒的香梨叶片,制备成适合进行原位RT-PCR的石蜡切片。设计特异引物,利用引物原位标记技术对切片组织中的病毒进行了检测研究,结果表明,PRINS-FITC染色法和PRINS-Rhodamine染色法均能获得良好的检测效果,有病毒部位分别显示绿色荧光和红色荧光,而且荧光出现的位置与原位RT-PCR检测的结果一致。由此证明引物原位标记技术可用于果树病毒的原位检测。     

我国食品中农药残留研究进展

随着人们安全健康意识的提高,食品中农药残留问题更加受到重视。为此,对样品预处理研究进展及农药残留快速检测技术研究进展进行综述,并简单介绍各种方法的优缺点。