猪细小病毒检测技术研究进展

近年来,随着猪细小病毒分子生物学的研究进一步深入,各种新型疫苗的成功研制,为猪细小病毒病的防制带来希望。但目前为止猪细小病毒的检测技术以传统方法为主,大量研究发现猪细小病毒常与其他病原微生物混合感染,为了根除本病,必须提高诊断和防疫水平。文章就该病诊断方法的研究进展做一综述。     

桃品种间质地、水分及细胞壁组分的比较

为了探索桃果实的贮藏品质,利用生物力学、核磁共振影像、理化检测等分析手段,以北方晚熟桃品种为试材,研究桃品种间和贮藏期间质地、水分和细胞壁组分之间的差异及相互关系。结果表明:硬度从大到小依次是‘蟠桃’‘青州蜜桃’‘中华寿桃’和‘肥城桃’,‘蟠桃’硬度大,T_(23)弛豫时间长,水溶性果胶质量分数高,但纤维素质量分数低,而‘肥城桃’硬度最小,果胶、半纤维素和纤维素质量分数低,T_(23)弛豫时间短,自由水较多。相关性分析显示,桃果肉硬度与T_(23)、可溶性果胶、共价结合型果胶和半纤维素呈显著正相关,而粘附性与离子结合型果胶、半纤维素和纤维素呈显著负相关。T_(22)与T_(20)、T_(21)和T_(23)均呈显著正相关,T_(22)与可溶性果胶和半纤维素显著正相关,T_(23)与可溶性果胶、共价结合型果胶和半纤维素均呈显著正相关,桃品种间硬度的差异是细胞壁组分和水分共同起作用的结果。并且,通过贮藏试验进一步表明,‘肥城桃’果实硬度下降伴随着纤维素和半纤维素的降解,水溶性果胶显著增加,果实内紧密结合水(T_(20))逐渐消失,而自由水(T_(23)和M_(23))显著增加。     

AcMNPV chiA基因表达产物对棉铃虫围食膜的破坏及对Bt和NPV的增效作用

采用PCR方法扩增出苜蓿银纹夜蛾(Autographa californica)核型多角体病毒(AcMNPV)几丁质酶基因(chiA)编码区1.6kb全长片段,并将该片段分别克隆至原核表达载体pET30a和杆状病毒BactoBac表达系统转移载体pFastBac中,分别在大肠杆菌(Escherichia coli)BL21(DE3)和草地贪夜蛾(Spodoptera frugiperda)细胞系Sf-9中进行了表达。SDS-PAGE分析表明,在大肠杆菌和昆虫细胞中均有效表达了60kD的蛋白。将表达产物饲喂5龄棉铃虫(Helicoverpa armigera)幼虫后取其围食膜,扫描电镜结果显示,围食膜结构遭到破坏形成大量孔洞。生物测定结果表明,以上两种表达产物对苏云金芽孢杆菌(Bt)和核型多角体病毒(NPV)均具有增效作用。以AcMNPVChiA在大肠杆菌和细胞系Sf-9中的表达产物分别与BtCry2Ac蛋白混合饲喂棉铃虫初孵幼虫,增效率分别为33.4%和54.5%,其LT50较对照处理分别缩短了17.8和20.6h;当AcMNPVChiA在大肠杆菌和细胞系Sf-9中的表达产物分别与甘蓝夜蛾(Mamestra brassica)核型多角体病毒(MbNPV)混合处理棉铃虫初孵幼虫时,其LT50与对照比较分别缩短了16.6和22.4h。     

Rapid detection of potato late blight using a loop-mediated isothermal amplification assay

Potato late blight caused by Phytophthora infestans is one of the most destructive plant diseases that threaten global food security. Early and effective diagnosis of P. infestans is required before disease management decisions are made. Here, we developed a quick protocol to detect P. infestans based on a loop-mediated isothermal amplification(LAMP) assay. The P. infestans specific multiple copy DNA sequences(PiSMC), a transposon-like element, provides an ideal target for molecular detection of this pathogen. We designed highly specific and sensitive primers allowing effective LAMP detection of the pathogen at 64℃ in 70 min. In the validation assay, all 15 P. infestans isolates collected from China, Europe and South America could be positively detected, but results of the other 9 Phytophthora species infecting different plants, fungal and bacterial plant pathogens tested were negative. The detection limit of this assay is 1 pg P. infestans DNA. Moreover, the LAMP-PiSMC assay is able to detect P. infestans from infected leaves, tubers and soil. Taken together, this study reports the development of a specific and sensitive LAMP-PiSMC assay for early diagnosis of potato late blight.     

Development of a reverse-transcription loop-mediated isothermal amplification assay to detect avian influenza viruses in clinical specimens

In recent years, the avian influenza has brought not only serious economic loss to the poultry industry in China but also a serious threat to human health because of the avian influenza virus(AIV) gene recombination and reassortment. Until now, traditional RT-PCR, fluorescence RT-PCR and virus isolation identification have been developed and utilized to detect AIV, but these methods require high-level instruments and experimental conditions, not suitable for the rapid detection in field and farms. In order to develop a rapid, sensitive and practical method to detect and identify AIV subtypes, 4 specific primers to the conserved region of AIV M gene were designed and a loop-mediated isothermal amplification(RT-LAMP) method was established. Using this method, the M gene of H1–H16 subtypes of AIV were amplified in 30 min with a water bath and all 16 H subtypes of AIV were able to be visually identified in presence of fluorescein, without cross reaction with other susceptible avian viruses. In addition, the detection limit of the common H1, H5, H7, and H9 AIV subtypes with the RT-LAMP method was 0.1 PFU(plaque-forming unit), which was 10 times more sensitive than that using the routine RT-PCR. Further comparative tests found that the positivity rate of RT-LAMP on detecting clinical samples was 4.18%(14/335) comparing with 3.58%(12/335) from real-time RT-PCR. All these results suggested that the RT-LAMP method can specifically detect and identify AIV with high sensitivity and can be considered as a fast, convenient and practical method for the clinic test and epidemiological investigation of AIV.     

牧草病害诊断系统的知识表示——《中国草业开发与生态建设》专家系统病害诊断

针对牧草病害诊断的特点,结合框架知识表示的分层特点和规则表示法的过程性和不确定性,提出二者相结合的表示方法.该知识表示包括基础库和动态库,前者用来存放已经确定的数据,后者用来存放诊断的中间结果.基础库中又包括基础数据库、基础知识库和媒体库,数据库是一个关系数据库,根据牧草病害诊断的特点,采用分层建库的方式.知识库采用规则方式,按照病害名称建立.媒体库主要是辅助诊断而使用的图片、视频数据.动态库包括动态数据库和动态知识库,动态库的结构与基础库中的基础数据库、基础知识库一样.     

Diagnostic accuracy of digital photography and image analysis for the measurement of foot conformation in the horse

Reasons for performing study: Rigorous evaluation of practicable methods for the objective assessment of foot conformation has not been performed. Objectives: To assess the practicability, precision and accuracy of the process of obtaining measurements of horses' feet using photography and image processing software. Methods: Precision study: Lateral photographs of horses' feet were obtained twice by 2 veterinary surgeons (image acquisition ‐ IAc). Photographs were analysed by 2 masked veterinary surgeons on 2 occasions (image analysis ‐ IAn). Measurements were compared within and between operators for self and non‐self acquired photographs. Agreement indices (AIs) and 95% limits of agreement (LOA) were calculated for the IAn process alone and for the combined IAc + IAn processes. Accuracy study: Measurements obtained from lateral photographs were compared with those obtained from lateromedial radiographs. AIs and 95% LOA were calculated for each measurement. Results: Precision study: Mean intra‐ and interoperator AIs for the IAn process alone were ≥0.90 while those for the combined IAc + IAn processes were ≥0.89 for all measurements. Similar mean AIs and 95% LOA were calculated regardless of image origin. The 95% LOA for hoof angle, heel height/toe height% and coronary band angle for all comparisons were within target values. Accuracy study: Mean AIs were ≥0.89 for all measurements. The 95% LOA for heel height/toe height% and coronary band angle were within target values. Conclusions: Excellent precision was identified within and between operators regardless of image origin. High levels of accuracy were also identified, especially for heel height/toe height% and coronary band angle, indicating that photography and radiography may be used interchangeably. Potential relevance: Acquisition and analysis of photographic images is an appropriate method for the objective measurement of foot conformation, both in clinical and research settings.