RT-PCR技术检测猪瘟病毒的应用研究

本试验应用反转录-聚合酶链式反应(RT-PCR)对猪瘟进行诊断应用研究.应用RT-PCR对来自广西不同地区的135份疑似猪瘟病料进行检测,84份诊断为阳性,阳性率62.2%.从百色、柳州地区等采集的健康猪扁桃体和淋巴结共276份,经RT-PCR检测,37份为阳性,阳性率为13.4%.其中健康猪扁桃体带毒较高,246份扁桃体中有35份阳性,占14.2%.采自柳州健康猪的26份淋巴结材料全为阴性,只有邕宁县的1份健猪淋巴结阳性.结果表明,RT-PCR技术可应用于猪瘟的临床诊断.     

Diagnosis of Congenital Cardiac Right-to-left Shunts with 99mTc-macroaggregated Albumin

Diagnosing right-to-left congenital cardiac shunts can be difficult. Cardiac catheterization and angiocardiography represent the traditional gold standard for diagnosis, but they are invasive. Nuclear scintigraphy using 99mTc-macroaggregated albumin (MAA) has been employed in humans as an alternate method of diagnosis. This study reviews eight dogs presented for evaluation of a suspect right-to-left cardiac shunt that were examined using 99mTc-MAA. In all, 2-4 mCi (74-148 MBq) of reduced particle 99mTc-MAA were injected IV in a cephalic vein and static images of the whole body, including right and left lateral, dorsal, and ventral views, were acquired for 60 s and stored into a 256 x 256 x 16 matrix. Shunt fractions were calculated. One dog with radiopharmaceutical distribution limited to the lungs did not have a shunt. Seven dogs had distribution of the radiopharmaceutical outside the pulmonary capillary bed, indicating bypassing of the pulmonary capillary circulation due to a right-to-left shunt. Four dogs had 99mTc-MAA within the brain. Three dogs that did not have brain uptake, but instead had a sharp cutoff of radioactivity at the level of the front limbs and neck, were diagnosed with reverse patent ductus arteriosus (PDA). The asymmetric distribution of the radiopharmaceutical is due to the location of the shunt, distal to the brachiocephalic trunk and left subclavian artery. Shunt fractions of dogs with extrapulmonary radioactivity ranged from 40% to 59%. Nuclear scintigraphy with 99mTc-MAA is a quick alternative method of diagnosing right-to-left cardiac shunts that permits quantification of shunt fraction. Distinguishing between reverse PDA and other right-to-left shunts may be possible based on the radiopharmaceutical distribution.     

应用聚合酶链式反应(PCR)技术检测野猪的氟烷基因

猪应激综合征(Procine Stress Syndrome,PSS)是指猪在应激因子的作用下发生恶性高热综合症(Malignant Hyperthermia Syndrome，MHS)。试验随机选取18头纯种野猪提取基因组DNA进行聚合酶链式反应，扩增得到RYR。基因特异片段，经过HhaⅠ酶切阳性鉴定可断定这18头猪的RYR1基因都没有发生突变，因此不存在猪应激综合征问题。     

西尼罗热病毒RT-PCR检测方法的建立

参考Genebank发表的西尼罗热病毒(West Nile virus，WNV)E糖蛋白基因序列，自行设计合成一对引物，对WNV进行RT—PCR扩增，产物经琼脂糖电泳分析，呈现一条约400bp的条带，将其克隆入pMD18-T—Vector载体中，并进行序列测定，与已发表的WNV基因比较发现，核苷酸的同源性为99．7％，证实为WNV的E基因，通过对样品多次检测，都能扩增出一条约400bp的条带，表明该方法比较稳定。     

禽呼肠病毒感染鸡胚成纤维细胞后Toll样受体mRNA转录水平的动态变化

为分析禽呼肠病毒(ARV)标准毒株S1133株感染鸡胚成纤维细胞(CEF)后对相关鸡Toll样受体(ChTLRs)mRNA转录水平的影响作用,利用实时荧光定量PCR,测定和分析ARV-S1133感染CEF后ARV结构蛋白σC和ChTLRs的mRNA转录水平变化情况。结果表明,ARV-S1133感染CEF 10h后,ARVσC蛋白的mRNA相对表达量开始迅速上升,在48h达到峰值;同时,感染CEF中的ChTLR3、ChTLR5、ChTLR7、ChTLR15和ChTLR21mRNA表达量发生显著变化,在感染72h内各个受体的mRNA表达量呈波浪式变化。5个不同滴度的ARV感染CEF 24 h后,ChTLR3、ChTLR5、ChTLR7、ChTLR15、ChTLR21mRNA转录水平与病毒滴度均呈正线性相关。上述结果表明,ARV感染后可诱导CEF ChTLR3、ChTLR5、ChTLR7、ChTLR15、ChTLR21的mRNA转录水平发生变化,可能与禽呼肠病毒的复制和致病机制相关。     

Rapid detection of contagious ecthyma by loop-mediated isothermal amplification and epidemiology in Jilin Province China

The aim of this experiment was to develop a loop-mediated isothermal amplification (LAMP) assay and to research the recent epidemiology of contagious ecthyma in Jilin Province, China, using the assay. A LAMP assay targeting a highly conserved region of the F1L gene was developed to detect contagious ecthyma virus (CEV). Three hundred and sixty-five cases from 64 flocks in 9 different areas of Jilin Province, China, from 2011 to 2014 were tested using the LAMP assay. The results showed that the sensitivity of the LAMP assay was 100 copies of the standard plasmid, which is 100-fold higher than the sensitivity of PCR. No cross-reactivity was observed with capripoxvirus, fowlpox virus, foot-and-mouth disease virus serotype O, foot-and-mouth disease virus serotype Asia I and bluetongue virus. The average positive rate was 19.73% (72/365), and the positive rate was highest in lambs aged 1–6 months. Our results demonstrated that CEV infection was very widespread in the flocks of Jilin Province and that the LAMP assay allows for easy, rapid, accurate and sensitive detection of CEV infection.     

行星轮系水稻钵苗移栽机构正反求设计方法研究

为了解决现有行星轮系水稻钵苗移栽机构较难同时具备理想的移栽轨迹和姿态的问题,提出了正向设计与局部轨迹微调的反求设计相结合的设计方法。以非圆齿轮行星轮系水稻钵苗移栽机构的设计为例,阐述该方法的实现过程。建立行星轮系水稻钵苗移栽机构设计要求,并分析其工作原理,在前期正向设计的基础上,局部调整其移栽静轨迹,通过反求设计进一步优化设计移栽机构。进行局部反求设计的运动学分析和运动学建模,基于Matlab平台开发计算机辅助设计软件,通过人机交互的方式得到移栽机构的机构参数,使其不仅满足移栽臂移栽的姿态要求,同时获得更理想的移栽工作轨迹。根据最终得到的机构参数设计移栽机构结构,完成三维建模、虚拟仿真试验,加工、装配移栽机构物理样机,完成了高速摄像运动学试验。将移栽机构理论计算、虚拟仿真、样机试验得到的移栽静轨迹比较,同时逐一将轨迹相关参数、取秧角、推秧角、角度差等设计目标参数与相应数值化目标进行对比,结果显示,均满足要求,验证了设计方法的正确性。与以往单一的正向设计、反求设计方法相比,该方法设计的移栽机构不仅具有更优的移栽工作轨迹,也可满足较好的移栽姿态要求。     

甜菜DAMD-PCR体系的建立及优化

为了建立甜菜DAMD扩增体系,以期利用DAMD引物应用于甜菜品种指纹图谱的构建及分子标记辅助育种。本实验利用单因素变量的方法对甜菜DAMD体系进行优化。同时选用12个甜菜品种,利用优化的体系对25条DAMD引物进行扩增。获得甜菜的最适DAMD体系:总体积为20μL,包含模板DNA 10~80 ng、0.75 U的DNA聚合酶、0.2μL的d NTPs(2.5 mmol/L each)以及2.0μL的引物(10μmol/L)。同时25条引物均扩增出了清晰条带,除了个别引物多态性较差外,其余引物多态性都非常的丰富,其中引物62H(-)就可以把实验中用到的12个甜菜品种全部区分开。由此可见,DAMD引物的扩增效率很高,并且扩增结果稳定,条带清晰,非常适合甜菜品种指纹图谱的构建及遗传多样性分析。     

苹果Hsf家族成员的序列特征、表达与进化分析

为全面了解苹果基因组中热激转录因子(Hsf)的序列特征及进化,采用生物信息学手段,在苹果全基因组水平上鉴定出50个MdHsf基因,并对其系统发育关系、序列特征、表达情况以及选择压力进行详细分析。系统发育与序列分析显示:与拟南芥和水稻相似,50个MdHsf基因可分为A、B、C 3个亚族;2个或多个MdHsf基因位于同一个末端进化支,说明该基因家族在苹果中发生了物种特异性扩增;尽管MdHsf基因的内含子数目和长度变异较大,但其蛋白的保守基序和功能结构域具有较高的保守性,这可能与功能约束有关。基于EST数目,可推知:除了MdHsfA2a和MdHsfA3a/b/c等14个基因没有相应的EST外,其余72%的基因都有转录活性。选择压力检测和结构建模分析显示:在36个MdHsf蛋白的选择压力检测中,位点模型未鉴定到正选择位点的存在;而在显著水平下(P0.05),分支-位点模型在d和e进化分支上,共检测到5个正选择位点,它们是28R、30L、35D、51M、67V,其中28R和30L位于Hsf结构域中,35D、51M和67V位于Hsf结构域之外,这说明除了MdHsfA4d/e和MdHsf C1a/b发生快速进化外,其他成员受控于纯净选择,具有高度保守性。综合以上研究结果,苹果基因组中存在多种热激转录因子,其蛋白的保守基序和功能结构域具有较高保守性,大多具有转录活性,在进化上该家族受纯净选择主导。