Comparison of white spot syndrome virus quantification of fleshy shrimp Fenneropenaeus chinensis in outdoor ponds between different growing seasons by TaqMan real‐time polymerase chain reaction

In the present study, we used TaqMan real‐time polymerase chain reaction to quantify and compare infection of white spot syndrome virus (WSSV) with shrimp production of Fenneropenaeus chinensis cultured in outdoor ponds along the west coast of the South Korea. In 2007, a total of 60 specimens in summer and 116 specimens in autumn were collected from 12 growing‐out ponds and 12 harvest ponds respectively. Pond harvest data were obtained from farmers. Of the summer samples, all specimens were WSSV positive, with a wide range of 12.4–7.0 × 10⁷ (mean 7.5 × 10⁶) copies ng^?1 DNA; shrimp production was 1.7 metric tonnes per hectare (mt ha^?1). Of the 116 autumn‐sample specimens, 81 (69.8%) were WSSV positive; WSSV infection had been decreased dramatically, to 0–7.2 (mean 3.5) copies ng^?1 DNA. Shrimp production of autumn ponds was 2.1 mt ha^?1. Statistical analysis indicated that the difference in WSSV infections detected in summer and autumn was highly significant (P<0.01). In summer, seven ponds (58.3%) with low‐WSSV infection loads (0–1000 WSSV copies ng^?1 DNA) had shrimp production of 2.7 mt ha^?1; the others had shrimp production of only 0.2 mt ha^?1. The mean shrimp production between the two infection levels showed a highly statistically significant difference (P<0.01).     

Biological control of potato soft rot caused by Dickeya solani and the survival of bacterial antagonists under cold storage conditions

Dickeya and Pectobacterium are responsible for causing blackleg of plants and soft rot of tubers in storage and in the field, giving rise to losses in seed potato production. In an attempt to improve potato health, biocontrol activity of known and putative antagonists was screened using in vitro and in planta assays, followed by analysis of their persistence at various storage temperatures. Most antagonists had low survival on potato tuber surfaces at 4 °C. The population dynamics of the best low-temperature tolerant strain and also the most efficient antagonist, Serratia plymuthica A30, along with Dickeya solani as target pathogen, was studied with TaqMan real-time PCR throughout the storage period. Tubers of three potato cultivars were treated in the autumn with the antagonist and then inoculated with D. solani. Although the cell densities of both strains decreased during the storage period in inoculated tubers, the pathogen population was always lower in the presence of the antagonist. The treated tubers were planted in the field the following growing season to evaluate the efficiency of the bacterial antagonist for controlling disease incidence. The potato endophyte S. plymuthica A30 protected potato plants by reducing blackleg development on average by 58.5% and transmission to tuber progeny as latent infection by 47–75%. These results suggest that treatment of potato tubers with biocontrol agents after harvest can reduce the severity of soft rot disease during storage and affect the transmission of soft rot bacteria from mother tubers to progeny tubers during field cultivation.     

校正温度和盐度对单电容传感器的影响

Several newly developed capacitance sensors have simplified real-time determination of soil water content.Previous work has shown that salinity and temperature can affect these sensors,but relatively little has been done to correct these effects.The objectives of this study were to evaluate the effect of media temperature and salinity on the apparent water content measured with a single capacitance sensor (SCS),and to mitigate this effect using a temperature dependent scaled voltage technique under laboratory conditions.A column study was conducted containing two media:pure deionized water and quartz sand under varying water contents (0.05 to 0.30 cm³ cm^-3) and salinity (0 to 80 mmol L^-1).Media temperature was varied between 5 and 45℃ using an incubator.The SCS probes and thermocouples were placed in the middle of the columns and were logged at an interval of 1 minute.There was strong negative correlation between sensor reading and temperature of deionized water with a rate of -0.779 mV ℃^-1.Rates of SCS apparent output were 0.454 and 0.535 mV ℃^-1 for air in heating and cooling cycles,respectively.A similar positive correlation with temperature was observed in sand at different water contents.The SCS probe was less sensitive to temperature as salinity and water content increased.Using a temperature-corrected voltage calibration model,the effect of temperature was reduced by 98%.An analytical model for salinity correction was able to minimize the error as low as ± 2% over the salinity level tested.     

Seasonal dynamics and virus translocation of Grapevine leafroll‐associated virus 3 in grapevine cultivars

Grapevine leafroll‐associated virus 3 (GLRaV‐3) is associated with grapevine leafroll disease, one of the most economically important viral diseases of grapevines. This disease impacts on both vine health and grape quality; reduction in yield, brix and wine colour are among its detrimental effects. Many methods, including serological and molecular procedures, have been developed for the detection of GLRaV‐3; however, there is no PCR‐based assay available to quantify virus populations within plant tissues. A real‐time RT‐PCR assay with TaqMan probe was developed for specific and reliable quantitative detection of GLRaV‐3 in infected tissues. The designed primers and probes target the conserved sequence in the RNA‐dependent RNA polymerase (RdRp) domain of the viral genome to prevent amplification of most subgenomic and defective RNAs. This protocol was used to examine the seasonal dynamics and translocation of GLRaV‐3 in field‐grown grapevines. The results showed that the virus spread quickly from trunks to new growing shoots and leaves early in the growing season, and most samples still harboured detectable virus during late summer and autumn. The seasonal progress of one GLRaV‐3 isolate was compared in four grapevine cultivars (Chardonnay, Cabernet Sauvignon, Italia and Thompson Seedless). Within cultivars there was little variability in the distribution and translocation of GLRaV‐3, except for in Thompson Seedless. This quantitative detection assay will be a valuable tool for GLRaV‐3 diagnosis, disease monitoring and population ecology studies.     

猪瘟病毒TaqMan实时荧光定量PCR的建立与应用

为了建立一种既能检测野毒,又能检测疫苗毒的猪瘟病毒TaqMan实时荧光定量PCR方法,经过对GenBank中所有瘟病毒属成员高度保守的5’端非翻译区序列比对分析后,设计出1对TaqMan Real-time RT-PCR引物、1条TaqMan探针和3条RNA标准品制备引物。猪瘟病毒石门毒株经RNA标准品制备引物RT-PCR扩增后,再经T7 RNA聚合酶体外转录制备包含检测目的片断序列的猪瘟病毒RNA标准品。通过最佳引物、探针浓度的筛选及反应条件的优化,建立了猪瘟病毒TaqMan实时荧光定量PCR检测方法。该方法批内及批间重复试验变异系数均低于2%,特异性试验仅能检测出猪瘟强毒及疫苗毒株,最低浓度检测极限为1 X 102 copies/μL,上机检测时间少于60 min,建立的标准曲线斜率(Slope)为:-3.97,截距(Intercept)为:47.41,相关系数(R2)为:0.999779。运用该方法对3份猪瘟临床组织样品及6家企业生产的5种细胞苗及2种脾淋苗进行定量检测,结果提示:临床病料含有的病毒拷贝数差异不大,而疫苗产品每头份含有的病毒拷贝数差异较大。所建立的方法具有特异、快速、灵敏、可重复性和线性关系好的特点,不仅适合于猪瘟临床样品的早期检测,也适用于疫苗生产过程中的质控及疫苗制品的效价评估。     

Prevalence and genotyping of bovine Cryptosporidium species in the Mediterranean and Central Anatolia Region of Turkey

The prevalence of Cryptosporidium species in calves and heifers with relation to diarrhea from several herds was investigated in this study. Fecal samples were collected from 135 and 120 pre-weaned calves and 79 and 130 heifers raised in the Central Anatolia (CAR) and Mediterranean Regions (MR) of Turkey, respectively. A total of 86 post-weaned calves in CAR were also included in the study. For diagnostic comparison, all samples were examined by microscopic examination, SSU rRNA nested PCR and TaqMan real-time PCR for the presence of oocyst and Cryptosporidium DNA. In total, 102 (34.0 %) and 93 (37.2 %) of the examined samples from CAR and MR were found positive for Cryptosporidium DNA with both nested PCR and real-time PCR analyses, respectively with an overall prevalence of 35.5 %. The diagnostic sensitivity and specificity of microscopic examination were determined as 68.7 % and 100.0 % compared to molecular tools, respectively. RFLP and sequence analyses of the SSU rRNA from the PCR products revealed that 138 (70.8 %) out of 195 positive isolates were C. parvum further confirming the species-specific real-time PCR results. Among the remaining 57 (29.2 %) positive isolates, 30 (15.4 %) and 27 (13.8 %) were characterized as C. ryanae and C. bovis, respectively. C. parvum was the dominant species in pre-weaned calves especially with diarrhea while C. bovis and C. ryanae were mostly found in post-weaned calves and heifers. The sequence analyses of the gp60 gene of C. parvum isolates revealed two subtypes (IIaA13G2R1, IIaA14G1R1) belonging to zoonotic family IIa, with IIaA13G2R1 being the most common in diarrheic calves.     

绵羊主效基因FecB的检测与肉用多羔核心群的建立

基于前期本实验室建立的绵羊多羔性状主效基因FecB高通量检测技术(TaqMan探针法),对10个绵羊群体的16 460只绵羊进行了多羔性状主效基因FecB的检测,结果发现小尾寒羊、湖羊以及滩羊的B等位基因频率与前人实验结果一致,有些群体经过长期选择已处于Hardy-Weinberg不平衡状态(P<0.05);本课题组与多个企业经过长期努力已建立了多个肉用多羔绵羊核心群,通过比较发现BB型绵羊产羔数极显著高于B+型,B+型极显著高于++型,且高繁殖力核心群3种FecB基因型小尾寒羊产羔数高于普通小尾寒羊群体。绵羊多羔性状主效基因FecB的检测和肉用多羔绵羊核心群的建立为绵羊群体改良提供了支撑。     

检测猪圆环病毒2型TaqMan荧光定量PCR方法的建立

针对猪圆环病毒2型(PCV2)ORF2的序列设计两对特异性引物及TaqMan探针,以感PCV2的PK-15细胞培养上清的DNA提取物为模板,分别扩增499 bp和131 bp的片段,建立了检测PCV2的TaqMan荧光PCR方法。结果表明:TaqMan荧光PCR检测PCV2的最佳探针浓度为0.5 pmol·μL-1;TaqMan荧光PCR两步法操作快捷,扩增131 bp的引物PCF1101/PCR1232检测敏感性高(3.17×102拷贝·μL-1),重复性好。在诸多检测样品中,除检测到PCV2检测指标出现阳性外,猪呼吸与繁殖障碍综合征病毒(PRRSV),日本乙型脑炎病毒(JEV),猪伪狂犬病毒(PRV),猪瘟病毒(CSFV)及PK-15细胞等检测指标均为阴性,表明特异性强;与普通PCR的检测结果(2/10)比较,本法的敏感性和对临床样品的阳性检出率(3/10)更高。上述研究表明,本方法快速、敏感、特异,具有很高的重复性,且可实时监测,能有效检测PCV2。     

PDCoV与TGEV双重实时荧光定量PCR检测方法的建立及初步应用

试验旨在建立快捷、高效而准确的鉴别诊断猪德尔塔冠状病毒（Porcine deltacoronavirus,PDCoV）与传染性胃肠炎病毒（Transmissible gastroenteritis virus,TGEV）的双重实时荧光定量PCR方法。通过绘制双重实时荧光定量PCR的标准曲线,检验该方法的特异性、敏感性和重复性,并对临床样品进行检测。结果显示,双重实时荧光定量PCR方法的循环阈值与PDCoV和TGEV质粒拷贝数的对数之间存在良好的线性关系,且对应的相关系数分别为R_（P）²=0.9994和R_（T）²=0.996;能特异性地检测PDCoV和TGEV,而与PEDV、PRV、PRRSV、CSFV和RV无交叉反应,具有较强的特异性;检验PDCoV与TGEV质粒标准品的最低检测限度分别达到2和20拷贝/μL,且分别比常规RT-PCR高1 000和100倍,具有较高的敏感度;PDCoV与TGEV的批内和批间重复性检测的Ct均值基本相同,且变异系数（CV）均<2%,具有较好的重复性。用该方法对114份仔猪腹泻样品检测结果显示,PDCoV和TGEV的阳性率分别为5.6%（6/114）和8.8%（10/114）,混合感染检出率为4.6%（5/114）,比常规RT-PCR具有更高的检出率和敏感性。结果表明,本试验建立的双重实时荧光定量PCR方法具有特异性强、灵敏度高、重复性和稳定性好等优点,适用于病毒早期诊断和批量临床样品检测,为疾病防控、流行病学调查及相关性研究提供了技术支持及数据参考。     

小麦－簇毛麦杂种染色体的DNA原位杂交研究

以生物素标记的簇毛麦基因组ＤＮＡ作探针与小麦－簇毛麦杂种双二倍体及异附加系染色体进行原位杂交，旨在探索一种鉴定小麦中簇毛麦染色质的分子细胞遗传学方法。首先试验了标记的簇毛麦ＤＮＡ与小麦ＤＮＡ之含量比对原位杂交效果的影响，发现探针混合液中不加未标记的小麦ＤＮＡ时，获得的原位杂交结果较理想。在八倍体小麦－簇毛麦双二倍体（２ｎ＝５６）中，１４对簇毛麦染色体出现明显的棕褐色原位杂交信号，而小麦染色体不显标记，使这２个物种的染色体很易相互区分开来。进一步用生物素标记的簇毛麦ＤＮＡ与小麦－簇毛麦６Ｖ染色体附加系体细胞杂交，也可检测出其中１对附加的６Ｖ染色体。由于该方法标记的簇毛麦ＤＮＡ覆盖各簇毛麦染色体的整个染色体臂，由此，用其鉴定小片段小麦－簇毛麦染色体易位是有效的。