Molecular investigation on the presence of canine parvovirus in Egypt

Canine parvovirus type 2 (CPV-2) causes a highly contagious gastroenteritis disease of dogs and wild canids. To investigate the CPV-2 prevalence in Dakahlia Governorate, Egypt, a total of 50 fecal swabs were collected from suspected diseased dogs during 2016–2017. Out of 50 collected samples, 35 samples (70 %) presented positive results for CPV-2 using immuno-chromatography (IC) as a rapid test. CPV-2DNA was detected in 42 samples (84 %) by using polymerase chain reaction (PCR). The frequencies of CPV-2 were significantly higher in German shepherd breed (46 %; 23/50) and in age groups less than 6 months (76%; 38/50). We evaluated the breed, age, sex, rapid test results and clinical signs as predictors for classification of animal status into infected and not infected. The best predictors for classification process were rapid test result and clinical signs. Both CPV-2b and CPV-2c subtypes were detected by CPV2-VP2 gene sequences analysis. Deduced amino acid sequences alignment showed substitutions at 3 sites (Arg453Pro, Ala574Glu and Gln457Leu). Further investigations are needed to reveal the genetic and antigenic relation between field and vaccinal strains of CPV-2 in Egypt.     

Serologic and molecular survey of horses to Coxiella burnetii in East of Iran a highly endemic area

There are few reports about Q fever in horse populations worldwide. This study aimed to detect the C. burnetii infection by serologic and molecular confirmation using commercial ELISA kit and real-time PCR in the East of Iran a region highly endemic. A total of 177 blood samples and 115 vaginal swabs were randomly collected from horses in East of Iran. The sera samples were analyzed for anti C.burnetii Ig G antibodies by a commercial ELISA kit and nucleic acid extraxted from vaginal samples were used to determine the C. burnetii DNA by real-time PCR assay. Antibodies were detected in 5.64 % (10/177) of sera samples and C. burnetii DNA was detected in 7.82 % (9/115) of horse vaginal samples. There was no significant difference in seroprevalence in different sex, age and breed groups. Our study showed that horses could be considered as a mild potential reservoir of C. burnetii which may be effective on horse health status. However, additional studies are needed to assess whether the horse could be considered as a relevant transmission risk indicator for Q fever.     

Real-time RT-qPCR assay to detect sequences in the Piscine orthoreovirus-1 genome segment S1 associated with heart and skeletal muscle inflammation in Atlantic salmon

During the last decade, Piscine orthoreovirus was identified as the main causative agent of heart and skeletal muscle inflammation (HSMI) in Atlantic Salmon, Norway. A recent study showed that PRV-1 sequences from salmonid collected in North Atlantic Pacific Coast (NAPC) grouped separately from the Norwegian sequences found in Atlantic Salmon diagnosed with HSMI. Currently, the routine assay used to screen for PRV-1 in NAPC water and worldwide cannot differentiate between the two groups of PRV-1. Therefore, this study aimed at developing a real-time polymerase chain reaction (RT-qPCR) assay to target the PRV-1 genome segments specific for variants associated with HSMI. The assay was optimized and tested against 71 tissue samples collected from different regions including Norway, Chile and both coast of Canada and different hosts farmed Atlantic Salmon, wild Coho Salmon and escaped Atlantic Salmon collected in British Columbia, West Coast of Canada. This assay has the potential to be used for screening salmonids and non-salmonids that may carry PRV-1 potentially causing HSMI.     

基于qPCR和LAMP技术的马铃薯晚疫病菌快速检测方法

马铃薯晚疫病菌（Phytophthora infestans）能侵染多种茄科植物,它引起的马铃薯晚疫病,是马铃薯生产中的第一大病害。为了开发能在田间快速检测马铃薯晚疫病病原的方法,利用P. infestans T30-4基因组测序数据的contig 1.18131,设计qPCR和LAMP引物,优化扩增条件后得到引物的特异性和灵敏度,最后通过检测田间收获薯块,比较形态学传统方法、qPCR及LAMP的差异。特异性检测结果发现,qPCR和LAMP仅在含有P. infestans DNA模板的体系有阳性扩增,在寄主和其他微生物DNA中均无扩增;在优化的条件下,qPCR和LAMP的检测下限可达1×10 ^-6ng/μL,在有寄主和其他微生物DNA存在的条件下,引物的灵敏度没有显著差异。利用两种快速方法对在大理、丽江及昆明3个地区田间收获薯块上检测发现,qPCR和LAMP方法得到的检出率差异极为不显著（P=0.420）,两种快速检测方法和形态学鉴定方法检出率差异极显著（P=0.009）。在大理、丽江及昆明3个地区的薯块中,两种分子检测方法检出率均比形态学方法高。其中,qPCR检测方法比形态学方法分别提高了12.00%、2.00%、8.70%;LAMP检测方法比形态学方法分别提高了11.30%、2.00%、8.70%。     

不同地区楼葱栽培种的性状与品质分析

为了研究不同地区楼葱的特性,试验对我国不同地区12个楼葱栽培种的数量性状和品质性状进行了测定分析。14个数量性状的相关分析结果显示,主成分分析数量指标后,单株质量、株高、假茎质量、花葶高、叶形指数和假茎指数影响分类;相关性分析说明主要数量指标间具有极显著相关性(P<0.01)。聚类分析后,遗传距离为3.1时可将楼葱种质根据所得主要数量性状分为4个类群。测定不同地区楼葱6个品质指标后,相关性分析结果表明,紧实度与蛋白质含量、干物质率、香辛油含量、总糖含量、游离氨基酸总量存在着极显著正相关或相关性,主成分分析表明主成分反映了89.99%的原有信息量,楼葱的干物质率与紧实度为主要成分;应用灰色关联度分析了品质与数量性状的关系,假茎直径、出叶孔间距、单株质量这3个数量性状对楼葱品质的影响居前3位。研究表明楼葱种质材料具有极其丰富的遗传多样性,宁夏同心2个地区黄水桥HSQ、石羊圈SYJ的楼葱种质距其他种质资源间亲缘关系最远且干物质率含量及紧实度最高。     

High vegetative compatibility diversity of Cryphonectria parasitica infecting sweet chestnut (Castanea sativa) in Britain indicates multiple pathogen introductions

Chestnut blight, caused by Cryphonectria parasitica, was identified in Devon, UK, in December 2016. Intensive surveys detected the disease at further sites in Devon (seven), Berkshire (one), Dorset (one), Derbyshire (four) and a cluster of eight sites in southeast London. Over 570 survey samples were tested, and 227 were positive for C. parasitica by isolation and real-time PCR. A total of 227 isolates were tested for mating type, and 197 screened for vegetative compatibility group (VCG) and compared with VCGs known from mainland Europe. The same isolates were also screened for the presence of Cryphonectria hypovirus 1 (CHV-1). Eleven VCGs were identified within the UK population. Five corresponded to already known European VCGs but six were unique. The European VCGs mainly came from the Devon, Dorset, Berkshire and Derbyshire disease outbreaks, whilst unique VCGs were almost exclusively from the southeast London cluster. Both mating types were detected, but only one mating type was present at each site, with the exception of a single Devon site. Perithecia of C. parasitica were never observed at any site. CHV-1 was found in seven isolates from three different locations and was always subtype-I, which has limited hypovirulence. Therefore, although CHV-1 is associated with C. parasitica at some outbreaks, it probably has limited impact on virulence. The diversity of VCGs and their distribution at outbreak sites, together with findings of CHV-1, suggests C. parasitica has been introduced to the UK multiple times over at least two decades through international plant trade.     

Detection of Leishmania infantum DNA by real-time PCR in saliva of dogs

This study developed a real-time quantitative PCR (qPCR) assay to detect L. infantum kinetoplast DNA (kDNA) in canine saliva. The qPCR showed an efficiency of 93.8%, a coefficient of correlation of 0.996 and a detection limit of 0.5 fg/reaction (0.005 parasites), although it detected until 0.25 fg/reaction (0.0025 parasites). When samples from 12 dogs experimentally infected with L. infantum were collected, L. infantum kDNA was detected at 16-weeks post-infection (wpi) in 41.7% and 91.7% of saliva and bone marrow samples, respectively, and at 47-wpi in 75% of both samples. L. infantum kDNA can be detected by qPCR in canine saliva, with lower sensitivity in the early stages of infection and a lower parasite load estimation compared to bone marrow. However, saliva had similar sensitivities to bone marrow in the later stages of the infection and could be used to detect L. infantum kDNA being aware of its limitations.     

2个抗虫棉的外源Bt基因分子鉴定及其染色体定位

以中美2个抗虫棉品种GK19与33B为试验材料,利用检测中美Bt基因的特异性引物,分别对抗虫棉亲本GK19和33B进行PCR扩增,并通过SSR分子标记技术对其Bt基因进行分子鉴定与染色体定位,旨在从外源基因转化事件的视角探究中美转基因抗虫棉差异的分子基础。结果表明, GK19为中国转Bt基因抗虫棉, 33B为美国转Bt基因抗虫棉; GK19的Bt基因被定位在棉花Chr.20上,共16对SSR多态性标记与其Bt基因连锁,两侧的分子标记为NAU3907和NAU2579,其遗传距离分别为2.4 cM和1.5 cM; 33B的Bt基因被定位在棉花Chr.26上,共20对SSR多态性标记与Bt基因连锁,目标Bt基因位于标记NAU460和dc40260之间,其遗传距离分别为3.6 cM和2.0 cM。以上结果表明GK19和33B属于不同的遗传转化事件。     

金花茶组叶片生化成分评价及其最佳采收期研究

本研究以26份来源于6种金花茶组植物、不同表型防城金花茶和不同月份采收的防城金花茶的成熟叶片为材料,建立同时测定芦丁、槲皮素、木犀草素、山奈酚4种黄酮成分的HPLC法,以及优化了测定茶多酚、总黄酮、总多糖和总皂苷成分含量的紫外分光光度法,基于以上8种成分含量,结合主成分综合评分和聚类分析的化学计量学方法,对不同来源金花茶叶片生化成分进行综合评价。结果表明：8种成分含量在不同来源金花茶叶片中存在差异,其中无名金花茶8种成分含量均较高,四季金花茶、防城金花茶次之;8年生1—12月份采收的防城金花茶叶片中,总皂苷、总黄酮和总多糖含量均表现为升-降-升-降的变化规律,并在10月含量达到最高;5年生不同种（S1~S14）主成分综合评分顺序为：无名金花茶>四季金花茶>防城金花茶（花蕾大、花多、不抗寒、花黄且大、尖果）>显脉金花茶>凹脉金花茶>小果金花茶;8年生不同月份（S15~S26）主成分综合评分顺序为：10月采>2月采>3月采>4月采>5月采>7月采>6月采>1月采>8月采>12月采>11月采>9月采,其中无名金花茶、四季金花茶及防城金花茶中花蕾大、花多、花黄且大等表型的金花茶叶片与2—4月份和10月份采集的8年生金花茶叶片的多指标综合评分排序位列前10;聚类分析结果将供试26份样品分为5类。综上所述,金花茶叶片的生化成分与金花茶植物种类和采收期有关,无名金花茶、四季金花茶和防城金花茶中花蕾大、花多、不抗寒、花黄且大的表现型的综合生化成分较高,初步确定防城金花茶叶片的适宜采收期为2—4月份和10月份,所建立的多指标定量结合化学计量学分析方法为金花茶组植物叶片的生化成分评价提供依据。     

牛星状病毒EvaGreen实时荧光定量PCR检测方法的建立及应用

牛星状病毒(BAstV)是我国新发的犊牛腹泻病原,本试验的目的是建立检测BAstV的Real-time PCR方法。根据BAstV流行株的ORF1a基因序列设计引物,通过优化反应条件和体系,成功建立基于EvaGreen检测BAstV的Real-time PCR方法。结果表明,该检测方法的Ct值与标准品模板在1.36×10¹~1.36×10⁸拷贝/μL线性关系良好,相关系数R²=0.999,扩增效率为93.79%;该方法可特异性检出BAstV,对犊牛腹泻其他相关病原呈阴性;最低检测下限为13.6拷贝/μL;批间和批内的变异系数均小于2%,重复性好。对2017年9月至2019年5月采自河南省的221份犊牛腹泻样本进行检测,BAstV的检出率为18.1%(40/221),采样场阳性率为100.0%(14/14)。本试验所建方法灵敏度高、特异性强、稳定性好,为BAstV的检测和流行病学调查提供了有力手段。