Indexing of Citrus Viroids by Imprint Hybridisation

A method based on the hybridisation of tissue imprints was developed for routine indexing of citrus viroids. For maximum sensitivity and reliability, the inoculation of Citrus medica (Etrog citron) as a viroid amplification host is required. Hybridisation against Digoxigenin-labelled RNA- or DNA-probes followed by detection of viroid-probe hybrids using anti-DIG-alkaline phosphate conjugate and the chemiluminescence substrate CSPD was suitable for the detection of all citrus viroids with the same sensitivity as other available methods. The overall process is extremely simple and allows quick analysis of large numbers of samples by easily trained personnel and minimum equipment.     

Characterization of a pothos (Scindapsus aureus) virus with unusual properties

A virus for which the name of pothos latent virus (PoLV) is proposed, was isolated by inoculation of sap from symptomless plants ofScindapsus aureus. PoLV had isometric particlesc. 30 nm in diameter, a monopartite genome consisting of a non polyadenylated, single-stranded RNA moleculec. 4,300 nucleotides in length, constitutingc. 17% of the particle weight, and a single type of coat protein subunit with aM _r ofc. 40,000 Daltons. The biological properties (host range reactions) of PoLV resembled those ofTombusviridae for it infected most of the artificial hosts locally, inducing symptoms recalling those elicited by several species of the above family. Like tombus- and carmoviruses, PoLV had two subgenomic RNAs which, however, differed in size from those of both genera. The dsRNA pattern was also distinctly different. Cytopathological features recalled those of tombusviruses except for the lack of multivesicular inclusion bodies. PoLV was serologically related to, but distinct from twoCarmovirus (i.e., galinsoga mosaic and Ahlum waterborne viruses) and threeTombusvirus species (i.e. eggplant mottled crinkle, Sikte waterborne and Lato river viruses). Thus, PoLV had properties somewhat intermediate between those ofTombusvirus andCarmovirus genera but bridged the two taxa through the serological relationship with some of their species. The taxonomic position of PoLV is still undetermined. It must await the results of molecular investigations now underway.     

Detection of Colletotrichum coccodes from soil and potato tubers by conventional and quantitative real-time PCR

Colletotrichum coccodes is the causal agent of the potato blemish disease black dot. Two PCR primer sets were designed to sequences of the ribosomal internal transcribed spacer (ITS1 and ITS2) regions for use in a nested PCR. The genus-specific outer primers (Cc1F1/Cc2R1) were designed to regions common to Colletotrichum spp., and the species-specific nested primers (Cc1NF1/Cc2NR1) were designed to sequences unique to C . coccodes . The primer sets amplified single products of 447 bp (Cc1F1/Cc2R1) and 349 bp (Cc1NF1/Cc2NR1) with DNA extracted from 33 European and North American isolates of C. coccodes. The specificity of primers Cc1NF1/Cc2NR1 was confirmed by the absence of amplified product with DNA of other species representing the six phylogenetic groups of the genus Colletotrichum and 46 other eukaryotic and prokaryotic plant pathogenic species. A rapid procedure for the direct extraction of DNA from soil and potato tubers was used to verify the PCR assay for detecting C. coccodes in environmental samples. The limit of sensitivity of PCR for the specific detection of C. coccodes when inoculum was added to soils was 3·0 spores per g, or the equivalent of 0·06 microsclerotia per g soil, the lowest level of inoculum tested. Colletotrichum coccodes was also detected by PCR in naturally infested soil and from both potato peel and peel extract from infected and apparently healthy tubers. Specific primers and a TaqMan fluorogenic probe were designed to perform quantitative real-time (TaqMan) PCR to obtain the same levels of sensitivity for detection of C. coccodes in soil and tubers during a first-round PCR as with conventional nested PCR and gel electrophoresis. This rapid and quantitative PCR diagnostic assay allows an accurate estimation of tuber and soil contamination by C. coccodes .     

新疆棉田根际土壤真菌荧光PCR技术定量及其时空动态分析

【目的】研究新疆棉花黄萎病病株根际土壤真菌数量的时空动态变化,及棉花根际土壤真菌与黄萎病病原菌数量的相关性。【方法】用荧光定量PCR方法,测定新疆不同植棉区及其不同生育时期棉田根际土壤真菌总量和棉花黄萎病病原菌数量进行测定,分析棉花黄萎病病株根际土壤真菌数量在不同植棉区和不同生育时期的变化趋势,及棉花根际土壤真菌与棉花黄萎病病原菌数量的相关性。【结果】新疆不同植棉区的棉花在不同生育期其病株根际土壤真菌数量表现出不同变化趋势。哈密棉花病株吐絮期根际土壤真菌最大值达 6.16×10⁴ copies/g FRW;库尔勒棉花病株根际土壤真菌在苗期达到最大为4.23×10⁴ copies/g FRW;阿拉尔棉花病株根际土壤真菌在苗期至蕾期逐渐增加,随后逐渐减小,在吐絮期达到最小值为1.41×10^-4copies/g FRW。北疆精河和东疆哈密棉花根际土壤真菌数量与黄萎病病原菌数量的PCC分别高达0.989和0.993,呈显著正相关。南疆图木舒克棉花根际土壤真菌数量与黄萎病病原菌数量的PCC为0.880,呈正相关。【结论】新疆棉花黄萎病病株根际土壤真菌含量较高,在不同采样区和不同生育期,其根际土壤真菌总量均呈波动性变化。从棉花的四个生育期(苗期、蕾期、花铃期、吐絮期)来看,棉花病株根际土壤真菌数量最大值出现在吐絮期。新疆棉花病株根际土壤真菌数量的空间变化是东疆大于南疆大于北疆。在不同生态区,棉花根际土壤真菌和棉花黄萎病病原菌之间表现为正相关,而在不同发育期则表现为负相关。     

脊尾白虾不同发育期mtDNA拷贝数变化特征分析

为了解脊尾白虾(Exopalaemon carinicauda)在不同发育期线粒体基因组(mtDNA)拷贝数的变化特征,本研究共采集其8个时期(受精卵期、溞状幼体Ⅰ期、溞状幼体Ⅱ期、溞状幼体Ⅲ期、溞状幼体Ⅳ期、溞状幼体Ⅴ期、仔虾期和成虾期)的DNA样品,利用TaqMan实时荧光定量PCR技术,对上述各发育期单个细胞中的mtDNA拷贝数进行了测定。检测结果显示,脊尾白虾受精卵期、溞状幼体Ⅰ期、溞状幼体Ⅱ期、溞状幼体Ⅲ期、溞状幼体Ⅳ期、溞状幼体Ⅴ期、仔虾期和成虾期的mtDNA拷贝数的均值分别为2366、2648、2644、2873、3559、9948、6452和8872。进一步统计分析结果显示,mtDNA拷贝数与发育时间存在正相关性,相关系数为0.83。研究表明,随着脊尾白虾不断生长,其单个细胞中mtDNA拷贝数总体呈上升趋势。     

First Report on the Frequency and Molecular Detection of Neuropathogenic EHV-1 in Turkey

There has been an increase in outbreaks of neuropathogenic equine herpesvirus-1 (EHV-1) in the United States and Europe. However, the presence and frequency of neuropathogenic EHV-1 in Turkish horses are not known at present. This study aimed to investigate the frequency of EHV-1 and neuropathogenic strains of EHV-1 in the Marmara Region of Turkey. Samples were analyzed for the presence of EHV-1 and neuropathogenic EHV-1 by real-time PCR TaqMan probe assays. Overall detection rate of EHV-1 was 45.5% (51 of 112). The detection rates were 70.5% (24 of 34) in aborted fetuses, 53.3% (8 of 15) in neonatal deads, 66.6% (4 of 6) in foals, 40% (2 of 5) in dead mares, and 25% (13 of 52) in living mares. Overall detection rate of neuropathogenic EHV-1 was 7.8% (4 of 51), and the real-time PCR results were confirmed by sequencing. Neuropathogenic strains of EHV-1 were detected in the brain and lung of two mares with neurological disease but without a history of abortion, in the brain of a foal that died of respiratory disorder, and in the nasal swab from a mare with a history of abortion. On histopathology, nonpurulent meningoencephalitis, hemorrhages, and vasculitis were seen in the brain. In conclusion, results of this study indicated, for the first time, that the neuropathogenic EHV-1 is circulating in the Marmara Region of Turkey. The results of this study also show that the current risk for non-neuropathogenic strains is high, whereas risk for the neuropathogenic EHV-1-G₂₂₅₄ strain seems to be low. As outbreaks of EHV-1 continue in the Marmara region of Turkey, surveillance for neuropathogenic EHV-1 genotype should be maintained.     

H1、H3亚型猪流感病毒双重实时荧光定量RT-PCR检测方法的建立及应用

为建立一种快速、准确地检测H1和H3亚型猪流感病毒（swine influence virus,SIV）的方法,根据H1和H3亚型SIV血凝素（hemagglutinin,HA）基因保守序列,分别设计2对特异性引物和2条TaqMan探针,建立双重实时荧光定量RT-PCR方法。结果显示,该方法敏感性高,可检测到最低拷贝数为10²拷贝/μL;重复性良好,重复孔Ct值的变异系数均在5%以下;特异性好,除H1和H3亚型SIV外,H4、H5、H7、H9亚型SIV、猪瘟病毒、猪繁殖与呼吸综合征病毒、口蹄疫病毒、伪狂犬病病毒检测均为阴性;在田间样品中成功检出1株H1和1株H3亚型SIV,检出率为1.16%。该方法准确、快速、灵敏、特异,可为H1、H3亚型SIV的快速鉴别检测及流行病学调查提供有效的技术手段。     

应用TaqMan real-time PCR研究BSV在带毒香蕉组培苗中传递

由香蕉线条病毒(Banana streak virus,BSV)引起的香蕉线条病,严重影响了香蕉产量及种质资源交流。本研究根据BSV ORF Ⅲ基因保守序列设计引物和探针,建立了BSV的实时荧光定量PCR检测方法。该法检测BSV时,与黄瓜花叶病毒(Cucumber mosaic virus,CMV)和香蕉束顶病毒(Banana bunchy top virus,BBTV)无交叉反应;不论是检测质粒DNA还是香蕉病株总DNA,其灵敏度都比普通PCR高,检测质粒DNA的灵敏度比普通PCR高100倍,检测香蕉病株总DNA的灵敏度比普通PCR高10倍,且具有良好的重复性。利用建立的实时荧光定量PCR方法检测我国主栽的不同香蕉品种‘大蕉’、‘粉蕉’和‘粤优抗一号’香蕉组培苗中BSV的浓度,发现不同品种BSV传递规律不同。‘大蕉’第3代、第9代组培分化芽中BSV含量较原代显著减少;‘粉蕉’中BSV含量随继代数增加表现为先增加后减少;‘粤优抗一号’中BSV含量随继代数增加呈增加趋势。BSV在‘粉蕉’、‘大蕉’和‘粤优抗一号’第10代到第12代组培苗中以顶部第1片或(及)第2片叶中含量最高。直接结合PCR分析初步表明不同品种在原代和组培至第12代,其植株中的BSV均为游离状态。本文通过对带毒香蕉组培苗中BSV的含量监测,明确了BSV在不同品种带毒香蕉组培苗中的传递和分布规律,为研究BSV与寄主互作及BSV检测提供了理论依据。     

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。     

猪博卡病毒实时荧光定量PCR检测方法的建立

根据GenBank公布的猪博卡病毒(PBoV)序列,通过VP1/2基因设计引物和Taq Man探针建立实时荧光定量PCR检测方法。建立的方法与PPV、PRRSV及PCV均无交叉反应,具有较高特异性,在107 copies/mL～101 copies/mL模板范围内具有良好的线性关系,所制作的标准曲线相关系数为0.997,最低可检测到101copies/mL的阳性质粒。说明所建立的PBoV实时荧光定量PCR检测方法具有灵敏度高、特异性好和精确性高等优点。