喀斯特炭疽菌TaqMan探针实时荧光PCR检测方法的建立

为寻求喀斯特炭疽菌的快速检测方法,利用TaqMan探针实时荧光定量PCR技术,以喀斯特炭疽菌基因组中的ACT基因为靶序列设计并筛选特异性引物探针,通过特异性、灵敏度、重复性试验建立喀斯特炭疽菌TaqMan探针实时荧光PCR检测方法。结果表明:建立方法的特异性较强、灵敏度较高、重复性稳定性较好,最低检测限为0.2pg DNA/反应,标准曲线的线性关系良好,相关系数为0.998。该方法操作简便,可用于进出境口岸喀斯特炭疽菌的检疫。     

Epidemiology of dark leaf spot caused by Alternaria brassicicola and A. brassicae in organic seed production of cauliflower

In organic seed production of Brassica vegetables, infections by Alternaria brassicicola and A. brassicae can cause severe losses of yield and seed quality. Four field experiments with or without artificial inoculation with A. brassicicola were conducted in organically managed seed‐production crops of cauliflower cv. Opaal RZ in 2005 and 2006 in the Netherlands. The development of A. brassicicola and A. brassicae on pod tissues and developing seeds was followed and seed quality was assessed. Alternaria brassicicola was externally present on 1·2% of the seeds 14 days after flowering and observed internally within 4 weeks after flowering. In both seasons, seed colonization by the pathogen increased slowly until maturation but sharply increased during maturation. A similar pattern was found for the colonization of pod tissues by A. brassicicola as quantified by TaqMan‐PCR. The incidence of A. brassicicola on mature seeds reached 70–90%. Internal colonization was found for 62–80% of the seeds. External and internal seed colonization by A. brassicae was much lower, with incidences below 3%. The quality of harvested seeds was generally low, with less than 80% of seeds able to germinate. Seed quality was not affected by warm water treatments. It was concluded that A. brassicicola and A. brassicae have the potential to infect pods and seeds soon after flowering. For the production of high quality seeds, producers must prevent such early infections. Therefore, new control measures are needed for use in organic cropping systems.     

大洋臀纹粉蚧和南洋臀纹粉蚧TaqMan实时荧光PCR检测方法

臀纹粉蚧属有多个种类是重要的农业害虫,大洋臀纹粉蚧(Planococcus minor(Maskell))和南洋臀纹粉蚧(Planococcus lilacius Cockerell)是我国有重要检疫意义的有害生物.这两种臀纹粉蚧经常从进口泰国和东南亚水果口岸检疫中截获,但形态学方法很难进行准确鉴定.本研究首次利用mtDNA COI基因设计了两条特异性探针,应用TaqMan实时荧光PCR方法对大洋臀纹粉蚧和南洋臀纹粉蚧进行了快速准确鉴定.     

Early Supplies of Available Nitrogen to Seed‐Row of a Canola Crop as Affected by Fertilizer Placement

Abstract

A field experiment was conducted at Star City (legal location SW6‐45‐16‐W2); Saskatchewan, Canada from May 2000 to June 2000, to measure nitrogen (N) and phosphorus (P) supply rates from fertilizer bands to the seed‐row of canola crop. Ion exchange resin membrane probes (PRS^TM) were used to measure N and P supply rates in four treatments [80 kg N ha^?1 of urea as side‐row band, 80 kg N ha^?1 of urea as mid‐row band, check/no N (side‐row)/P side‐row, check/no N (mid‐row)/seed placed P]. The treatments were arranged in a randomized complete block design with four replications. Two anion and cation exchange resin probes (PRS^TM) were placed in each plot in the seed‐row immediately after seeding and fertilizing. The probes were allowed to remain in the field for 2 days and replaced with another set of probes every 4 days for a total of 14 days until canola emerged. Ammonium‐N, nitrate‐N and P supply rates were calculated based on the ion accumulated on the probes. Urea side‐row band treatments (fertilizer N 2.5 cm to side of every seed‐row) had significantly higher cumulative available N supply rates than mid‐row band placement in which fertilizer N was placed 10 cm from the seed‐row in between every second seed‐row. No significant differences were observed in P supply rates. The higher N rates (120 kg N ha^?1) resulted in lower grain yield in side‐row banding than mid‐row banding possibly due to seedling damage. However, the earlier fluxes of N into the seed‐row observed with side‐row banding may be an advantage at lower N rates in N deficient soils.     

基于TaqMan MGB探针的可可花瘿病菌快速检测方法

可可花瘿病菌是一种我国进境植物检疫性真菌?本文根据可可花瘿病菌EF1α基因的保守序列, 设计并合成1对特异性的实时荧光PCR引物和1条TaqMan MGB探针, 建立了可可花瘿病菌的实时荧光PCR检测方法?特异性试验结果表明, 该检测方法能够特异性检出可可花瘿病菌; 实时荧光PCR优化反应条件为引物终浓度0.2 μmol/L, 探针终浓度0.6 μmol/L; 灵敏度试验结果表明, 20 μL反应体系中可可花瘿病菌DNA含量最低检测限为10 pg; 重复性试验结果表明, 该检测方法的重复性和稳定性良好; 接种试验样品检测结果表明, 该方法可用于疑似携带可可花瘿病菌样品的检测与初筛?本文建立的方法具有良好的灵敏性?特异性和应用性, 为可可花瘿病菌早期快速检测提供了一种有效手段?     

Implementation of an artificial reaction control in a TaqMan method for PCR detection of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> race 3 biovar 2

A previously published TaqMan PCR test for R. solanacearum race 3 biovar 2 was modified to enable both the validation of negative results and the confirmation of positive results in a closed-tube system. Negative results were validated through the use of a reaction control plasmid, designated pRB2C2, which was designed to generate a 94bp product using the same amplimers targeting the primary diagnostic 68bp sequence in R. solanacearum race 3 biovar 2 DNA. SYBR Green was included in the reaction mix to facilitate the identification of post-reaction products using melt peak analysis. The 94bp reaction control had a melt peak temperature of about 90°C, while the diagnostic target amplicon had a melt peak temperature of about 83°C; thus positive results could be easily confirmed and distinguished from the reaction control product. Addition of pRB2C2 at 100 copies per reaction had no effect on the sensitivity of the TaqMan assay for R. solanacearum race 3 biovar 2, and the modified assay successfully detected R. solanacearum race 3 biovar 2 in infected, asymptomatic tomato stems and leaves as well as in potato tubers and stems.     

Improved fireblight diagnostics using quantitative real-time PCR detection of Erwinia amylovora chromosomal DNA

Specific and sensitive TaqMan real-time PCR assays were developed targeting chromosomal DNA of Erwinia amylovora ( ams C gene and ITS region). These assays increased the reliability of detection of E. amylovora strains, regardless of their plasmid profile, and have the ability to differentiate between Erwinia spp. strains from Hokkaido, Erwinia pyrifoliae and Erwinia spp. isolated from necrotic pear blossoms in Spain. The assays were used for testing the efficiency of three different extraction methods to remove plant-based PCR inhibitors. Combined with an automated DNA-extraction method based on magnetic beads (QuickPick™), the real-time PCR assays reliably detected at least 10³ cells mL^−l ( c. four cells per reaction) of the pathogen from blighted woody plant material. In testing of symptomless samples, absolute quantification of E. amylovora before and after enrichment in liquid media provided proof of E. amylovora viability and its ability to multiply, including in cases when subsequent isolation in pure culture was unsuccessful.     

槟榔隐症病毒1型TaqMan实时荧光定量PCR检测方法的建立

为了建立一种快速、精准且能定量分析槟榔隐症病毒1型（Areca palm velarivirus 1, APV1）的检测方法。本研究参照GenBank中已登录的APV1全基因组序列,在p21蛋白基因保守区域和p60蛋白基因保守区域之间设计1对特异性RT-PCR引物,并构建阳性重组质粒;在p60蛋白基因保守区域部分设计荧光定量PCR特异性引物和TaqMan-MGB探针,利用阳性重组质粒,构建标准曲线,并对该检测方法的敏感性、特异性、稳定性及其应用效果进行验证。结果显示：该方法对阳性质粒标准品检测的敏感性达3.0×10¹ copies/μL级别,是常规RT-PCR敏感性的100倍;标准曲线显示,C_t值与拷贝数的对数呈线性关系,其标准曲线方程为y=-3.2748x+42.957,扩增效率为102%,相关系数R²为0.9992;该方法对APV1的检测具有较好的特异性,其他槟榔病原物及内生菌不会对本方法产生非特异性干扰;批内与批间重复性试验结果显示,该方法对APV1的检测具有较好的重复性和稳定性;利用该方法对海南万宁、琼海、定安、文昌等4个市（县）采集的181份疑似样品进行检测,阳性率分别为91.95%、86.95%、89.65%、73.68%,其阳性的病毒拷贝数最高达1.59×10⁷copies/μL,表明海南部分地区APV1病毒载量较高、流行情况比较严重。     

猪δ冠状病毒TaqMan荧光定量RT-PCR检测方法的建立与初步应用

本研究旨在建立一种灵敏、快速检测猪δ冠状病毒(porcine deltacoronavirus,PDCoV)的TaqMan荧光定量RT-PCR方法。参照GenBank中PDCoV有关基因序列,设计一对特异性引物用于扩增PDCoV M 基因。将测序正确的基因片段克隆入pMD18-T载体,构建重组质粒作为建立标准曲线的病毒模板。设计合成一对特异性引物与TaqMan探针,进行反应条件和反应体系的优化,建立快速检测PDCoV的TaqMan实时荧光定量RT-PCR方法,并进行该方法的灵敏性、特异性与重复性验证。结果表明,该方法能有效扩增1.0×10^1 ~1.0×10^9 拷贝·μL^-1 的PDCoV标准质粒,建立的标准曲线呈现良好的线性关系。该方法的检测灵敏度为1.0×10^1 拷贝·μL^-1;对猪流行性腹泻病毒、猪传染性胃肠炎病毒、猪伪狂犬病病毒等病原不发生交叉反应,具有很好的特异性;重复性试验结果显示变异系数(CV)小于1%,重复性良好。对2017—2018年期间收集的河南省不同猪场的100份腹泻病料进行检测,PDCoV的阳性检出率为23%(23/100),与PDCoV SYBR Green Ⅰ荧光定量RT-PCR检测方法的符合率为100%。人工感染PDCoV的仔猪,取攻毒后不同时间的粪便样品,应用本研究建立方法与常规RT-PCR方法对样品进行检测,TaqMan荧光定量RT-PCR检测方法的灵敏度远远优于常规RT-PCR。上述结果表明,本研究所建立的TaqMan荧光定量RT-PCR检测方法能够灵敏、特异地检测PDCoV,可用于临床PDCoV的检测。