Development of Xanthomonas fragariae populations and disease progression in strawberry plants after spray‐inoculation of leaves

Xanthomonas fragariae is the causative agent of angular leaf spot disease of strawberry. Greenhouse experiments were conducted using a X. fragariae isolate tagged with a green fluorescent protein (GFP) for detailed population dynamic studies in and on leaves after spray‐inoculation. The GFP‐tagged bacteria were monitored with dilution plating of leaf washings and leaf extracts, and analysis of intact leaves using a non‐invasive monitoring system called PathoScreen, based on laser radiation of fluorescent cells in plant tissues and signal recording with a sensitive camera. PathoScreen was also used to monitor bacteria grown on an agar medium after leaf printing. During the first 3 days after inoculation, bacterial populations washed off leaves rapidly decreased by at least a factor of 1000, after which populations remained stable until 14 days post‐inoculation (dpi), when symptoms first started to appear. Thereafter, populations increased to a level of 10¹² colony‐forming units (CFU) g^?1 of leaf material or higher. Similarly, densities in leaf extracts were low during the first 3 days after inoculation, at a level of 100–1000 CFU g^?1 of leaf tissue. Gradually populations increased to a level of 10⁹–10¹² CFU g^?1 at 28 dpi. Higher densities of epiphytic populations were found on the abaxial side than on the adaxial leaf side during the first 2 weeks after inoculation. After spray‐inoculation of leaves, bacterial populations released from infected plants remained low until symptoms appeared, after which plants became highly infectious, in particular under high humidity.     

CSFV及美洲型、欧洲型PRRSV多重qRT-PCR鉴别检测方法的建立及应用

根据猪瘟病毒(CSFV)以及猪繁殖与呼吸综合征病毒(PRRSV)基因组序列特点,设计3对特异性引物和3条TaqMan探针,经过反应条件的优化,建立了能同时检测并区分CSFV以及美洲型、欧洲型PRRSV的多重TaqMan荧光定量RT-PCR(qRT-PCR)方法。该方法具有特异性强、敏感性高、重复性好的特点,可以特异扩增CSFV和PRRSV,而与猪的其它常见病毒无交叉反应;对CSFV及美洲型、欧洲型PRRSV3种重组质粒标准品的检出下限均为2.68拷贝/μL;组内及组间重复试验的变异系数均小于1.5%。应用该方法对253份临床疑似样品进行检测,结果病原阳性106份,其中20份为CSFV阳性,86份为美洲型PRRSV阳性,11份为CSFV和美洲型PRRSV混合感染,未检测到欧洲型PRRSV。试验表明,研究建立的多重qRT-PCR方法可用于CSFV和PRRSV的快速鉴别检测及流行病学调查。     

TaqMan荧光定量RT-PCR检测猪脂蛋白酯酶mRNA方法的建立

【目的】克隆猪cDNA,作为猪LPL mRNA定量检测的标准品,建立检测方法。【方法】用RT-PCR,从猪背最长肌的总RNA中逆转录扩增LPL的cDNA,将纯化的LPL cDNA与pGM-T载体进行连接,转化宿主菌TOP10,提取重组质粒DNA,PCR鉴定并测序分析,对质粒标准进行实时荧光定量PCR 检测。纯化质粒并检测260 nm吸光度,确定原液的重组质粒拷贝浓度并以此制备荧光定量PCR梯度浓度标准品。【结果】建立了猪LPL mRNA实时定量PCR检测方法,特异性好,检测灵敏度达103拷贝,线性范围为1×103 ～1×1010拷贝,阈值与PCR体系中起始模板量的对数值之间有着良好的线性关系（R2＝0.9871）。【结论】成功克隆了LPL实时荧光PCR定量标准,且TaqMan荧光定量RT-PCR的方法可对猪背最长肌LPL mRNA的表达进行准确定量。     

EHP、SHIV双重TaqMan实时荧光定量PCR检测方法的构建及应用

本研究根据GenBank中已有的虾肝肠胞虫(EHP)和虾血细胞虹彩病毒(SHIV)基因的保守序列,设计特异的引物和探针.建立了快速诊断EHP和SHIV的双重TaqMan实时荧光定量PCR检测方法,并对其特异性、敏感性和稳定性进行检测.结果表明:该方法检测限可达10 copies/μL,其敏感性是SYBR Green r...     

Molecular diagnosis and direct quantification of cereal cyst nematode (Heterodera filipjevi) from field soil using TaqMan real-time PCR

Heterodera filipjevi continues to be a major threat to wheat production worldwide. Rapid detection and quantification of cyst nematodes are essential for more effective control against this nematode disease. In the present study, a TaqMan-minor groove binder (TaqMan-MGB) probe-based fluorescence quantitative real-time PCR (qPCR) was successfully developed and used for quantifying H. filipjevi from DNA extracts of soil. The primers and probe designed from the obtained RAPD-SCAR marker fragments of H. filipjevi showed high specificity to H. filipjevi using DNA from isolates-confirmed species of 23 Heterodera spp., 1 Globodera spp. and 3 Pratylenchus spp. The qPCR assay is highly sensitive and provides improved H. filipjevi detection sensitivity of as low as 4^–3 single second-stage juvenile (J2) DNAs, 10^–3 female DNAs, and 0.01 μg μL^–1 genomic DNAs. A standard curve relating to the threshold cycle and log values of nematode numbers was generated and validated from artificially infested soils and was used to quantify H. filipjevi in naturally infested field soils. There was a high correlation between the H. filipjevi numbers estimated from 32 naturally infested field soils by both conventional methods and the numbers quantified using the qPCR assay. qPCR potentially provides a useful platform for the efficient detection and quantification of H. filipjevi directly from field soils and to quantify this species directly from DNA extracts of field soils.     

猪腹泻病毒一步法多重TaqMan荧光定量RT-PCR检测法的建立及应用

【目的】建立一种可同时检测猪流行性腹泻病毒（PEDV）野毒株、猪A群轮状病毒（GARV）、猪德尔塔冠状病毒（PDCoV）、猪阿尔法冠状病毒（SADS-CoV）及猪捷申病毒（PTV）5种猪腹泻病毒的一步法多重TaqMan荧光定量RT-PCR检测法。为猪腹泻病的快速诊断和流行病学调查提供高效灵敏的工具。【方法】对 PEDV 多个基因型毒株ORF3基因比对分析,以PEDV野毒株为模板,对疫苗株ORF3基因稳定缺失区域设计特异性探针,并在两端保守区域设计上下游引物;在靠近GARV G3、G4、G5和G9型 NSP5基因5′端保守碱基区域设计引物及探针,并加入简并碱基。同时,分别选择PDCoV M基因、PTV 5′UTR序列、SADS-CoV N基因等保守基因设计特异性引物及探针,用于多重荧光定量PCR方法的建立。对引物、探针浓度和退火温度进行优化;用RStudio参照代码绘制ROC曲线,确定检测方法的敏感度值、特异度值及曲线下面积AUC,并计算Youden指数,最终确定检测临界值;从阳性核酸中扩增靶基因,并克隆至pEASY-T1载体。通过体外转录,获得5种标准品分别命名为：cRNA-PEDV、cRNA-GARV、cRNA-PDCoV、cRNA-PTV和cRNA-SADS-CoV。对检测方法的敏感性、特异性和重复性等进行评估;并与同类方法对临床样本的检测符合率进行比较。【结果】得到了5种病原检测的最佳引物、探针浓度和最佳退火温度。根据ROC曲线确定PEDV、GARV、PDCoV、PTV和SADS-CoV临界CT值分别为：35.78、34.25、34.98、34.60和35.70;5种病原的检测下限均可达到1×10² copies/μL,标准曲线线性关系良好,扩增效率在96.3%—104%之间;该方法对PEDV CV777疫苗株、PEDV AJ1102疫苗株、猪传染性胃肠炎病毒（TGEV）、猪瘟病毒（CSFV）、猪伪狂犬病毒（PRV）、猪繁殖与呼吸障碍综合征病毒（PRRSV）、猪霍乱沙门氏菌（S.choleraesuis）、多杀性巴氏杆菌（P.multocida）、大肠杆菌（E.coli）、猪链球菌（S.suis）和葡萄球菌（S.aureus）等多种菌毒株均不检出,具有良好的特异性;经重复性检验,组内变异系数在0.22%—3.08%之间,组间变异系数在0.89%—4.0%之间;对242份临床样本检测并与同类方法的检测结果进行比较,符合率分别为：PEDV 97.9%、GARV 98.8%、PDCoV 100%、PTV98.3%和SADS-CoV100%,Kappa值均大于0.9。对PEDV野毒株的检测准确性高于同类方法。此外,对临床样本检测结果显示,当前四川省腹泻猪群中尚无SADS-COV检出;但PEDV、GARV、PDCoV和PTV仍持续流行,其总体阳性率分别达到：10.7%（26/242）、13.6%（33/242）、18.2%（44/242）和14.5%（35/242）,且各腹泻病原之间存在不同形式和程度的混合感染,使感染猪腹泻病情加剧。故需进一步加强几种猪腹泻病毒在本地区猪群中流行情况调查和遗传变异规律研究,为制定更具针对性的防控措施提供依据。【结论】本研究成功建立了一种同时检测PEDV野毒、PDCoV、SADS-CoV和 GARV、PTV多基因型的一步法多重TaqMan荧光定量RT-PCR,为猪腹泻病的快速鉴别诊断和流行病学调查提供了一种高效灵敏的工具。     

扁桃拟茎点霉TaqMan荧光定量PCR检测方法的建立与应用

扁桃拟茎点霉(Phomopsis amygdali)引起的桃缢缩溃疡病是桃生产上重要的病害之一。带菌苗木及接穗是病害向无病区传播的主要途径。严格的检疫措施是保证无病区健康生产的关键。准确、灵敏、快速的检测方法是严格执行检疫措施及研究病害发生规律的有力工具。将扁桃拟茎点霉组蛋白H3(histone H3)基因与其近缘种比对发现,该基因特异性强,适合作为分子检测的靶标。针对histone H3基因为靶标,设计了特异性引物和Taq Man探针,建立了扁桃拟茎点霉的荧光定量PCR检测方法。结果发现,该方法只能特异识别扁桃拟茎点霉。检测灵敏度可达4×10~1copies·uL~(-1),比常规PCR检测方法高1 000倍;孢子检测最低限达2个孢子,比常规PCR检测方法高10倍,且仅需1 h即可完成检测。该方法用于田间样品检测,扁桃拟茎点霉的阳性检出率达86%,高于分离培养法和常规PCR检出结果。本研究建立的基于Taq Man探针的荧光定量PCR检测体系可用于田间对扁桃拟茎点霉的快速检测。     

3种根萤叶甲实时荧光PCR检测技术研究

根萤叶甲属(Diabrotica)被我国列为进境植物检疫性有害生物,该属中的玉米根萤叶甲(D.virgifera virgifera)、十一星根萤叶甲(D.undecimpunctata)、巴氏根萤叶甲(D.barberi)是北美的主要农业害虫。为了建立一种快速的分子鉴定方法以鉴定这3种根萤叶甲,本研究从田间采集的成虫样品中获得其部分mtDNA COI序列,与Gen Bank中的相关序列进行比对,设计筛选出3种根萤叶甲的特异性引物和TaqMan探针,并进行实时荧光PCR特异性和灵敏度检测。特异性检测结果表明,用目标种根萤叶甲DNA和其特异性探针和引物进行实时荧光PCR反应时,在30个循环反应内(Ct值30)有近S型扩增曲线出现,同时其他种均无荧光信号增长。此外,灵敏度结果显示,玉米根萤叶甲和巴氏根萤叶甲可检测的最小DNA模板浓度,即实时荧光PCR反应的灵敏度为0.1 ng/μL,十一星根萤叶甲为0.01 ng/μL。     

Development and Application of Duplex Real-time RT-PCR Assay for Detection of H1 and H3 Subtype Swine Influenza Viruses

To establish a rapid, accurate method to diagnose and detect H1 and H3 subtype swine influenza viruses (SIV) at the same time, the specific primers and TaqMan probes were designed according to the conserved region of the HA gene of H1 and H3 subtype SIV. A duplex Real-time RT-PCR assay was developed for detection of H1 and H3 subtype SIV. The results showed that the Real-time RT-PCR could detect 10² copies/μL of H1 and H3 subtype SIV, the sensibility was well. Coefficient of variation of Ct value between repeating groups were all below 5%, the repeatability was favorable. The results were negative for the detection of H4, H5, H7, H9 subtypes SIV, classical swine fever virus, porcine reproductive and respiratory syndrome virus, foot and disease virus and pseudorabies virus, the specificity was fine. One sample was H1 subtype SIV, and one sample was H3 subtype SIV, by the established assay, the positive rate was 1.16%. The method was highly accurate, rapid, sensitive and specific, and could provide a method for rapid detection and epidemiological investigation of H1 and H3 subtype SIV.     

Stand-scale transpiration estimates in a Moso bamboo forest: (I) Applicability of sap flux measurements

The applicability of sap flux (F_d) measurements to bamboo forests has not been studied. This study was undertaken to establish an optimal and effective design for stand-scale transpiration (E) estimates in a Moso bamboo forest. To this aim, we validated F_d measurements in Moso bamboos in a cut bamboo experiment. In addition, we analyzed how sample sizes affect the reliability of E estimates calculated from F_d and conducting culm area (A_{S_b}). In the cut bamboo experiments, we found that F_d measurement using a 10 mm probe was a valid means of determining the water-use behavior of a Moso bamboo, although a specific correction was needed. Furthermore, we calculated E from stand A_{S_b} (A_{S_stand}) and mean stand F_d (J_S). Employing Monte Carlo analysis, we examined potential errors associated with sample size in E, A_{S_stand}, and J_S using an original dataset with A_{S_b} and F_d measured for 40 and 16 individuals, respectively. Consequently, we determined the optimal sample size for both A_{S_stand} and J_S estimates as 11. The optimal sample sizes for J_S were almost the same under different vapor pressure deficit and soil moisture conditions. The optimal sample size for J_S at the study site was less than that of a coniferous plantation in the same region probably owing to small individual-to-individual variations in sap flux in the Moso bamboo forest. Our study concludes that sap flux measurements are an applicable technique for assessing water use in Moso bamboo forests.