中华蜜蜂囊状幼虫病毒检测方法研究进展

中蜂囊状幼虫病是一种由中蜂囊状幼虫病毒引起的严重威胁中蜂健康的传染性疾病。目前由于该病尚无有效的药物治疗方式,因此,预防与及时检测对于遏制该病传播显得尤为重要。本文主要从临床诊断、分子生物学和免疫学方面总结中蜂囊状幼虫病检测与鉴定的方法,包括形态诊断、RT-PCR技术、实时荧光定量PCR、巢式PCR、胶体金免疫快速诊断技术、ELISA、血清学技术和基因芯片等,为中蜂囊状幼虫病毒检测方法的改进和优化提供参考。     

绵羊BMP15基因B2突变可视化检测技术的建立

骨形态发生蛋白15（bone morphogenetic protein 15,BMP15）基因突变对绵羊排卵率、产仔数以及繁殖力有很大影响,本研究旨在建立快速可视化检测BMP15基因B2突变的技术。将改良的扩增阻滞突变系统（amplification refractory mutation system,ARMS）与核酸染料SYBR Green Ⅰ结合,针对BMP15基因B2突变设计ARMS特异性引物,使引物下游3'端的最后一个碱基为A,并在下游引物3'端的第3位碱基处设计额外的错配,以提高引物的特异性;经ARMS PCR扩增后,向产物中加入SYBR Green Ⅰ,通过肉眼观察PCR管内的颜色变化来检测BMP15基因的B2突变。经测序发现所采集的样本均为野生型,因此,利用重叠延伸PCR构建BMP15基因B2突变型模板,测序结果显示BMP15基因B2突变型模板构建成功。ARMS PCR结果显示,ARMS特异性引物能够只扩增突变型模板,而不会扩增野生型模板,且扩增效果良好。ARMS PCR扩增后加入SYBR Green Ⅰ发现已知野生型模板与阴性对照一致,呈SYBR Green Ⅰ原始颜色橙黄色,而已知突变型模板呈亮绿色,且色差明显,肉眼可辨。用建立的可视化检测BMP15基因B2突变的技术检测50个小尾寒羊基因组DNA （随机向其中20个样本中加入构建的BMP15基因B2突变型模板）,将可视化检测的突变型样本编号与混合样本时记录的编号对比,结果表明该技术能够准确检测绵羊BMP15基因的B2突变,且准确率高达100%。将构建的BMP15基因B2突变型的模板进行梯度稀释,对本试验可视化检测体系的灵敏度进行检测,发现ARMS可视化检测技术的灵敏度达到0.006 ng/μL。因此,本研究建立的技术能够可视化地检测绵羊BMP15基因B2突变,且准确率高,有望为高繁殖力绵羊的选育提供技术支持。     

气肿疽梭菌CctA基因的原核表达及其间接ELISA检测方法的建立

研究旨在原核表达气肿疽梭菌细胞毒素A （CctA）基因,用纯化的重组蛋白建立其间接ELISA检测方法,a。利用大肠杆菌密码子的偏爱性优化CctA基因序列,克隆至原核表达载体pET-28a （+）,双酶切鉴定原核表达质粒pET28a-CctA并测序。将重组质粒转入大肠杆菌,经IPTG诱导得到高表达的重组CctA包涵体蛋白。包涵体蛋白变性后经镍（Ni）柱纯化,SDS-PAGE检测其纯化效果。以豚鼠抗气肿疽梭菌抗血清为一抗,用Western blotting方法检测重组CctA蛋白的反应原性。用棋盘滴定法建立间接ELISA检测方法,以复性后的重组CctA蛋白作为检测抗原,摸索抗原包被浓度、封闭液的种类及浓度、抗体的最适稀释度和反应条件等。选用3批气肿疽灭活疫苗免疫豚鼠后采集血清,同时用攻毒和间接ELISA两种方法验证疫苗的免疫效力。质粒双酶切结果显示,得到大小约853 bp的条带,与预期相符,且测序结果正确。SDS-PAGE结果表明,成功表达并纯化大小为35 ku的重组CctA蛋白。Western blotting结果显示,豚鼠抗气肿疽梭菌抗血清与重组CctA蛋白具有良好的反应原性。建立的间接ELISA法的最适条件为：抗原的包被浓度为0.5 μg/mL,于4 ℃包被过夜;封闭液选择10%胎牛血清,37 ℃孵育2 h;二抗的稀释度为1：8 000;室温避光显色10 min后终止反应,测定D_{450 nm}值。当P/N>4.6时,间接ELISA法检测结果与豚鼠攻毒试验结果拟合度较好。本研究成功表达CctA基因并纯化了重组CctA蛋白,建立的以重组CctA蛋白为检测抗原的间接ELISA检测方法,有望成为气肿疽灭活疫苗免疫效果验证的替代方法。     

基于标记的极半径极值红枣形状识别方法

形状是分级的最重要参数之一,本文采用标记法对红枣形状进行了识别。通过图像预处理获取红枣二值图像,通过边界追踪获取目标边界笛卡尔坐标,并将其转化为极坐标,对目标图像进行缩放旋转使均值圆成为基线,切割的4部分边界曲线能完整表达。对边界曲线进行多项式拟合,获取极值点坐标,将其映射回被拟合曲线上,获取对应极值点坐标。若两极小极半径差值大于阈值,则红枣畸形;若两极大极半径附近区域极半径过渡平缓,判红枣为规整,否则为较规整。取53粒红枣进行检测,其中16粒畸形,17粒较规整,20粒规整。检测结果表明:畸形枣识别准确率达100%,较规整枣的识别准确率94%,规整枣识别准确率95%,可基本满足红枣分级系统精度的要求。     

Establishment and Application of a Multiplex RT-PCR Assay for Detection of PEDV,TGEV and PRoV

In this study,a multiplex RT-PCR assay was established to differentially detect porcine epidemic diarrhea virus (PEDV),porcine transmissible gastroenteritis virus (TGEV) and porcine rotavirus (PRoV) after optimization of the reaction conditions.Three pairs of primers PEDV-N,TGEV-M and PRoV-VP6 were designed for specifically amplifying PEDV N gene,TGEV M gene and PRoV VP6 gene,respectively.The assay could specifically amplify PEDV,TGEV and PRoV,but not classical swine fever virus (CSFV),porcine foot and mouth disease virus (FMDV),pseudorabies virus (PRV),porcine parvovirus (PPV) and porcine circovirus type 2 (PCV2).The detection limits of PEDV,TGEV and PRoV standard recombinant plasmids were 1.41×10³,1.41×10² and 1.41×10³ copies/μL,respectively.The repeated reaction under the same conditions obtained uniform results.The assay was used to detect a total number of 190 clinical samples,of which 42 (22.11%) samples were positive for PEDV,58 (30.53%) samples for TGEV and 34 (17.89%) samples for PRoV,and there were mixed infection among these viruses.The results indicated that this multiplex RT-PCR assay had the advantages of sensitivity,specificity and repeatability and provided a useful tool for differential detection and epidemiological investigation of PEDV,TGEV and PRoV.     

鸡毒支原体研究概况

鸡毒支原体(MG)是对养禽业危害很大的支原体,主要导致禽类慢性呼吸道疾病(CRD),以禽的结膜炎、产蛋率及饲料转换率下降、屠宰率下降等为主要特征。MG可通过垂直和水平传播方式在鸡群中传播,每年给全球家禽产业带来巨大经济损失。随着对MG细胞表面抗原黏附素蛋白(pMGA)和PvpA、GapA的结构与功能研究的深入,K株、TG5株等MG疫苗研究也取得较大进展。由于抗生素的滥用,MG基因中也发生耐药突变,产生了QRDRs等抗药结构,导致MG在耐药性上也出现新的特点。论文主要对国内外MG的疫苗开发、耐药情况和检测技术等进行综述,旨在对家禽MG的综合防控提供借鉴。     

Habitat use and home range of black-backed jackals (Canis mesomelas) on farmlands in the Midlands of KwaZulu-Natal,South Africa

Black-backed jackals (Canis mesomelas) are an abundant mesopredator on farmlands (croplands and rangelands) across South Africa. Given their adaptability in a changing anthropogenic landscape, knowledge of spatial movements can provide important ecological information on the species. We captured, collared and monitored five black-backed jackals in the Midlands of KwaZulu-Natal to determine their home range and habitat use in farmlands. The 95% fixed kernel (95% FK) home range sizes were large, showing a marked seasonal variation in movement. One adult male dispersed 150?km over two seasons (winter and spring) and thereafter settled into a home range that it maintained. Transient home ranges of males and juveniles were larger than resident home ranges. The mean resident home ranges (95% FK) across seasons for adult males, adult female and juvenile males were 11.4 ± 4.3 km², 5.6 ± 0.36?km² and 2.15 ± 0.45?km², respectively. The mean transient home ranges (95% FK) of all the seasons for adult and juvenile males were 1 181.93 ± 575.81 km² and 104.27 ± 35.64?km², respectively. Adult jackal preferred croplands in spring, summer and autumn but avoided croplands in winter. Variable habitat use and large home ranges in our study confirmed the species’ ability to adapt to agricultural areas.