Rapidly quantitative detection of Nosema ceranae in honeybees using ultra-rapid real-time quantitative PCR

BackgroundThe microsporidian parasite Nosema ceranae is a global problem in honeybee populations and is known to cause winter mortality. A sensitive and rapid tool for stable quantitative detection is necessary to establish further research related to the diagnosis, prevention, and treatment of this pathogen.ObjectivesThe present study aimed to develop a quantitative method that incorporates ultra-rapid real-time quantitative polymerase chain reaction (UR-qPCR) for the rapid enumeration of N. ceranae in infected bees.MethodsA procedure for UR-qPCR detection of N. ceranae was developed, and the advantages of molecular detection were evaluated in comparison with microscopic enumeration.ResultsUR-qPCR was more sensitive than microscopic enumeration for detecting two copies of N. ceranae DNA and 24 spores per bee. Meanwhile, the limit of detection by microscopy was 2.40 × 10⁴ spores/bee, and the stable detection level was ≥ 2.40 × 10⁵ spores/bee. The results of N. ceranae calculations from the infected honeybees and purified spores by UR-qPCR showed that the DNA copy number was approximately 8-fold higher than the spore count. Additionally, honeybees infected with N. ceranae with 2.74 × 10⁴ copies of N. ceranae DNA were incapable of detection by microscopy. The results of quantitative analysis using UR-qPCR were accomplished within 20 min.ConclusionsUR-qPCR is expected to be the most rapid molecular method for Nosema detection and has been developed for diagnosing nosemosis at low levels of infection.     

猪圆环病毒2型灭活疫苗中病毒抗原含量实时荧光定量PCR检测方法的建立

依据GenBank公布的猪圆环病毒2型Cap基因序列,在保守区域设计特异性引物和TaqMan探针,优化反应体系,建立评价猪圆环病毒2型灭活疫苗中病毒含量的实时荧光定量PCR检测方法,对方法的特异性、敏感性和重复性进行试验,并验证灭活剂用量和灭活时间对检测结果的影响。结果显示：该方法只对猪圆环病毒2型基因有特异性扩增,其他3种对照病毒基因的扩增结果均为阴性;检测灵敏度达到10^2.0 TCID₅₀/mL,比普通PCR方法高100倍;方法的重复性好,对同一样品进行10次检测,变异系数为2.28%;不同灭活剂用量和灭活时间对结果的影响较小,不会因各厂家使用的灭活剂用量和灭活时间不同,影响疫苗对比实验的公平性。该研究成功建立了一种评价猪圆环病毒2型灭活疫苗中病毒含量的实时荧光定量PCR检测方法,用于猪圆环病毒2型灭活疫苗样品中病毒抗原含量的定量,其结果可以反映不同猪圆环病毒2型灭活疫苗样品中抗原含量差异,为研究猪圆环病毒2型灭活疫苗病毒抗原含量评估方法提供了新的思路。     

Object‐Based Mapping of Karst Rocky Desertification using a Support Vector Machine

Accurate and cost‐effective mapping of karst rocky desertification (KRD) is still a challenge at the regional and national scale. Visual interpretation has been utilised in the majority of studies, while an automated method based on pixel data has been investigated repeatedly. An object‐based method coupling with support vector machine (SVM) was developed and tested using Enhanced Thematic Mapper Plus (ETM+) images from three selected counties (Liujiang, Changshun and Zhenyuan) with different karst landscapes in SW China. The method supports a strategy of defining a mapping unit. It combined ETM+ images and ancillary data including elevation, slope and Normalized Difference Vegetation Index images. A sequence of scale parameters estimation, image segmentation, training data sampling, SVM parameters tuning and object classification was performed to achieve the mapping. A quantitative and semi‐automated approach was used to estimate scale parameters for segmenting an object at an optimal scale. We calculated the sum of area‐weighted standard deviation (WS), rate of change for WS, local variance (LV) and rate of change for LV at each scale level, and the threshold of the aforementioned index that indicated the optimal segment level and merge level. The KRD classification results had overall accuracies of 85·50, 84·00 and 84·86 per cent for Liujiang, Changshun and Zhenyuan, respectively, and kappa coefficients are up to 0·8062, 0·7917 and 0·8083, respectively. This approach mapped six classes of KRD and offered a visually appealing presentation. Moreover, it proposed a conceptual and size‐variable object from the classification standard of KRD. The results demonstrate that the application of our method provides an efficient approach for the mapping of KRD. Copyright © 2012 John Wiley & Sons, Ltd.     

基于高斯自适应拟合的苹果目标分割方法研究

苹果目标的准确识别是苹果机械化采摘需要解决的关键问题之一。为此,基于YUV颜色空间模型,提出了一种结合色差分量与高斯自适应拟合算法的苹果目标分割方法。该方法采用首先将苹果目标由RGB颜色空间转换至YUV颜色空间,并利用色差分量V建立果实与背景分割的高斯分布拟合模型,根据拟合结果自动获取分割阈值,以实现自然场景下苹果目标的准确分割。为了验证文中算法的有效性,利用多幅图像进行了试验并与Otsu自适应阈值分割算法进行了比较。试验结果表明,采用文中算法得到的苹果果实的平均检出率达87.08%,识别率领先Otsu算法9.91%。因此,对于着色度较为均匀的苹果目标,采用高斯自适应拟合方法可以有效提高其识别率。     

基于MatLab和DSP的哈密瓜纹理分割与分类

以新疆特色水果哈密瓜为研究对象,提出一种利用Matlab辅助DSP提取哈密瓜纹理的方法。该方法首先利用Matlab把待处理的图像转换为数据文件(dat文件),然后利用DSP强大的运算能力分割哈密瓜表面纹理特征,并将分割结果同样以dat文件传递给Mat Lab,最后利用Mat Lab强大的数据分析与图像显示能力建立哈密瓜分类模型。实验结果表明,该方法能够有效地对哈密瓜表面纹理进行分割与分类,分类准确率为88.10%。该方法不但能够缩短DSP系统的开发周期,且能够为今后开发基于DSP的哈密瓜品质实时检测系统奠定基础。     

金针菇异戊烯基焦磷酸异构酶基因的克隆及表达分析

PCR扩增了金针菇(Flammulina velutipes)异戊烯基焦磷酸异构酶基因(FvIDI)的DNA全长和开放阅读框序列.该基因的ORF全长753 bp,DNA全长923 bp,含有3个内含子和4个外显子,编码250个氨基酸,金针菇异戊烯基焦磷酸异构酶相对分子质量为28450,等电点为5.05.采用实时荧光定量PCR技术研究FvIDI基因在不同发育时期和不同温度下的表达量,结果显示,FvIDI基因在采收期菌盖中表达量最高,4℃诱导处理2～4 h和37℃诱导处理2～6 h的表达量明显高于常规温度21℃处理.     

Effects of Alternative Molting Programs and Population on Layer Performance: Results of the Thirty-Fifth North Carolina Layer Performance and Management Test

A study involving 7 white-egg and 3 brown-egg commercial layer strains was conducted at the North Carolina Piedmont Research Station to assess the effects cage population 3-bird vs. 4-bird cages and alternative molt programs on performance through 113 wk of age (791 d). The flock was divided into 3 groups; not molted (NM), maintained in continuous production; molted using feed restriction (FR), an industry-type 14-d feed withdrawal molting program initiated at 463 d of age; and molted using a nonfasted/anorexic program (NF), in which an ad libitum supply of a low protein, low energy molt feed was provided. Productivity, egg size, and egg quality were monitored at 28-d periods throughout the test providing the performance data for the white-egg and brown-egg strains with regard to these management factors. Detailed results by strain are available on the following Web site: http://www.ces.ncsu.edu/depts/poulsci/tech_info.html#layer. The performance for the 3-bird or 4-bird cage groups for either type of layer was similar, except that birds in 4-bird cages had higher feed consumption in the first production phase than did the birds in 3-bird cages. In the same time period the white-egg birds in the 3-bird cages had a greater percentage of cracked eggs and a smaller percentage of Grade A large eggs than those in 4-bird cages. No differences were observed in egg income for the 2 population sizes for either type of layer. However, due to their higher feed consumption, the white egg birds in the 4-bird cages had higher feed cost per hen for the first cycle, which was offset by the egg income component due to their increased production rate. The molted layers outperformed the nonmolted layers for both types of layers in terms of overall income over feed costs. The FR molted hens, under a program that has traditionally been used by commercial producers, outperformed NF-molted hens, indicating that further refinements are needed to make the NF program economically competitive with the FR molting program.     

甜瓜黄斑病毒SYBR Green Ⅰ实时荧光定量PCR方法

为建立检测甜瓜黄斑病毒(melon yellow spot virus, MYSV)的SYBR Green Ⅰ实时荧光定量PCR(qPCR)方法。基于MYSV核衣壳蛋白基因保守序列设计qPCR特异性引物对,针对引物退火温度、引物浓度、特异性和敏感性进行系列优化。结果显示,优化后的qPCR方法最适退火温度为61.3℃,最适引物浓度为0.65μmol·L^-1,特异性强,灵敏度高,比PCR高100倍。以携带目的基因片段的重组质粒为标准品,构建的qPCR标准曲线循环阈值与模板浓度呈良好的线性关系,相关系数为0.999 7。实验样品验证表明建立的qPCR方法可用于MYSV的定量检测。     

一种农业电网远程监测系统的设计

基于当前嵌入式的流行，设计了一套嵌入式网络服务器，对其设计方案及主要芯片端口和功能进行介绍，将其应用到农业电网远程监测系统中的网络通信，给出了系统的功能框图，所涉及到的技术有数据采集技术、实时网络通讯技术等等。     

The Shortcoming and Improvement of the Project Break-Even Analysis Method

This paper points out the shortcoming of the existing project break-even analysis method. A new improved method-the dynamic project break-even analysis method has been put forward.