高位池养殖过程凡纳滨对虾携带WSSV情况的动态变化

为了更好地预防对虾白斑综合征(WSS)的暴发,探讨该病毒病的流行规律,笔者针对养殖过程中对虾的携带WSSV情况展开调查。调查于2010年7月-2010年11月广东省汕尾市红海湾养殖场进行,从10口凡纳滨对虾高位养殖池中随机抽取6口进行跟踪采样。收集指标包括对虾生长状况、基本环境指标、浮游微藻种群结构和对虾病毒携带量等。本文重点报道利用实时定量PCR-TaqMan探针法检测6口精养池塘对虾体内WSSV的携带量变化情况,检测结果显示:①1-3号虾池苗种携带WSSV,其波动范围在1.3×103~1.7×104copy/g之间;②对虾在养殖过程中均带毒,鳃组织中的平均病毒携带量(2.3×109copy/g)多于肌肉组织中的平均病毒携带量(3.2×108copy/g),且变化趋势一致,但没有显著性差异(P>0.05);③在整个养殖过程中对虾WSSV携带量总体呈现波动上升的趋势,期间各池出现过数次高值。前期WSSV拷贝数的波动范围在1.3×103~3.0×107copy/g之间,后期上升到1.5×106~1.2×1011copy/g,使得某些池塘养殖对虾WSS暴发。调查结果说明:1)对虾携带WSSV可以进行养殖生长;2)WSSV在对虾体内的含量是变化的,且其变化存在着一定的规律性;3)这种变化规律主要体现在带毒量随着养殖时间的进行及外界水环境中某些主要因子的变化而变化,如:养殖时间越长,带毒量越高;养殖环境中某些关键环境因子的改变,如:温差较大,不良藻相转换,天气骤变等均可引起对虾体内病毒含量较大的波动。鉴此,作者提出,构建并维持良好的浮游微藻的群落结构,注意有害藻相改变时保持养殖水体环境稳定,对环境突变前后都做好应对对虾应激的措施等可以极大程度地减少WSS暴发的可能。本研究通过对WSSV的密切跟踪,旨在更好的反映其在养殖环境下的动态变化规律,以及受各种环境因子影响的情况,从而为预防对虾WSS提供依据和参考。     

TaqMan探针荧光RT-PCR检测狂犬病病毒

根据GenBank已公布的狂犬病病毒(rabies virus,RV)核蛋白(N)基因序列设计并合成一对特异性的引物和探针,建立基于TaqMan探针的荧光RT-PCR检测狂犬病病毒方法。对狂犬病疫苗提取核酸后进行RT-PCR扩增,将目的条带切胶回收,克隆测序,重组质粒作为标准阳性对照。对建立的TaqMan探针荧光RT-PCR方法做灵敏度、特异性、重复性及稳定性试验。结果显示,该方法可以达到10拷贝/μL的灵敏度,可以将狂犬病病毒与犬瘟热病毒、犬细小病毒、犬腺病毒、犬冠状病毒和犬副流感病毒分开,方法重复性好,稳定可靠。     

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.     

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.     

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.     

Biochemical, molecular, and cytogenetic technologies for characterizing 1RS in wheat: A review

Chromosome arm 1RS of rye ( Secale cereale L.), when transferred to wheat ( Triticum sp.), significantly influences variety performance, because it carries genes for resistance to disease and insect pathogens. Inserted into wheat, 1RS also promotes haploid production, affects end-product quality, and sometimes affects yield. Therefore, its detection by breeders and geneticists is important. The entire 1RS arm is present in chromosome substitutions and in Robertsonian translocations involving chromosomes 1A, 1B, or 1D of wheat. In recombinant lines, a segment of 1RS has been exchanged with a segment of a group-1 wheat chromosome. Determining the wheat chromosome arm involved in a translocation, the source of rye chromatin, and the amount of 1RS chromatin introduced is necessary for a complete characterization of the introgressed segment. Biochemical, molecular, and cytogenetic technologies are described which enable such a characterization of 1RS in wheat. Examples of using gel electrophoresis, high-performance liquid chromatography, monoclonal antibodies, rye-specific molecular probes, RFLP and PCR assays, chromosome banding, in situ hybridization, and flow cytometry are provided. A comparison of these technologies is made and the advantages and disadvantages of each technology are discussed relative to modern wheat breeding efforts. This revised version was published online in July 2006 with corrections to the Cover Date.     

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。     

Improved detection and identification of the sudden oak death pathogen Phytophthora ramorum and the Port Orford cedar root pathogen Phytophthora lateralis

Early detection provides the best way to prevent introduction and establishment of alien plant pathogens. Amplification of DNA by PCR has revolutionized the detection and monitoring of plant pathogens. Most of those assays rely on the amplification of a fraction of the genome of the targeted species. With the availability of whole genomes for a growing number of fungi and oomycetes it is becoming possible to compare genomes and discover regions that are unique to a target organism. This study has applied this pipeline to develop a set of hierarchical TaqMan real-time PCR detection assays targeting DNA of all four Phytophthora ramorum lineages, and a closely related species, P. lateralis. Nine assays were generated: three targeting DNA of all P. ramorum lineages, one for each lineage of P. ramorum, one for P. lateralis and one targeting DNA of P. ramorum and P. lateralis. These assays were very accurate and sensitive, ranging from 98.7% to 100% detection accuracy of 2–10 gene copies of the targeted taxa from pure cultures or inoculated tissues. This level of sensitivity is within the lowest theoretical limit of detection of DNA. It is expected that these assays will be useful because of their high level of specificity and the ease with which they can be multiplexed because of the inherent flexibility in primer and probe design afforded by their lack of conservation in non-target species.     

Detection and quantification of Aeromonas schubertii in Channa maculata by TaqMan MGB probe fluorescence real‐time quantitative PCR

Aeromonas schubertii is a major epidemiological agent that threatens cultured snakeheads (Channidae) and has caused great economic losses in fish‐farming industries in China in recent years. In present study, a specific TaqMan minor groove binder (MGB) probe fluorescence real‐time quantitative PCR (qPCR) assay was developed to rapidly detect and quantify A. schubertii. A pair of qPCR primers and a TaqMan MGB probe were selected from the rpoD gene, which were shown to be specific for A. schubertii. A high correlation coefficient (R² = 0.9998) in a standard curve with a 103% efficiency was obtained. Moreover, the qPCR method's detection limit was as low as 18 copies/μl, which was 100 times more sensitive than that of conventional PCR. The detection results for the A. schubertii in pond water and fish tissue were consistent with those of the viable counts. Bacterial load changes detected by qPCR in different tissues of snakeheads infected with A. schubertii showed that the gills and intestines may be the entry for A. schubertii, and the spleen and kidney are major sites for A. schubertii replication. The established method in present study should be a useful tool for the early surveillance and quantitation of A. schubertii.