应用生物素标记的NDV－cDNA探针检测感染尿囊液中NDV－RNA的初步研究

本文应用聚合酶链反应（ＰＣＲ）技术从构建的新城疫病毒（ＮＤＶ）ｃＤＮＡ文库中扩增含编码Ｆ糖蛋白前体──Ｆｏ酶切位点序列的３５９ｂｐ的Ｆ蛋白基因ｃＤＮＡ片段。将此３５９ｂｐｃＤＮＡ片段经光敏生物素标记后,即成ＮＤＶ－ｃＤＮＡ探针。该探针能特异性地从感染的尿囊液中检测出ＮＤＶ强毒株和疫苗毒株的基因组ＲＮＡ,而不与ＩＢＤｖ－ｄｓＲＮＡ、ＡＩＢｖ－ｓｓＲＮＡ、ＥＤＳ７６－ｄｓＤＮＡ、ＭＤＶ－ｄｓＤＮＡ,ＦＰＶ－ｄｓＲＮＡ及ＡＩＬＶ－ｄｓＤＮＡ发生交叉杂交反应。试验结果表明：尽管该探钎含有编码Ｆｏ蛋白酶切位点序列的碱基顺序,但它还是不能把ＮＤＶ的强、弱毒株区分开。这说明ＮＤＶ强、弱毒株比区域内的碱基存在着相当大的同源性。不过,此探针对ＮＤＶ来说具有特异性,这就为ＮＤＶ的诊断技术开创了基因水平检测的新途径。     

Genetic mapping and utilization of quantitative trichome-mediated insect resistance in potato

Introgression of trichome-mediated insect resistance from the wild speciesSolanum berthaultii has become a major focus of the potato improvement program at Cornell University during the past twelve years. Several quantitative characters are involved in this resistance which is effective against a wide range of pest types. Correlative biochemical assays have been developed to assay specific components of the resistance, and the effects of the resistance on the target pests have been studied. Quantitative laboratory assays and specific measurements of insect behavior and biology have increased the precision of selection and enable the investigation of the genetic control of the resistance.We are currently using restriction fragment length polymorphisms (RFLPs) for genetic mapping of factors controlling the trichome traits fromS. berthaultii. Backcrosses to both the wild and the cultivated species parents have been evaluated for phenotypes contributing to the resistance mechanism, including trichome density, sucrose ester and polyphenol oxidase production by the trichomes, and the enzymatic browning reaction responsible for insect entrapment. Genetic maps are being developed for these progenies, using RFLP markers previously mapped in potato. Field and greenhouse trials under insect infestations are also being conducted with the mapping progeny. Our goal is to locate genes responsible for quantitative insect resistance by correlating RFLP variation at mapped loci with the trichome phenotypes and insect resistance. Genetic markers for these traits will be useful in transfer of the effective wild chromosomal segments into and among tetraploid potatoes, and for a better understanding of the resistance mechanism.     

安徽省鸡免疫抑制性疾病的流行病学调查

采集了安徽省6个主要养鸡地区65群鸡的185只病鸡共986份组织样品，对可引起免疫抑制性疾病的5种最常见病毒进行了PCR检测。结果，传染性腔上囊病病毒（IBDV）、鸡传染性贫血病毒（CIAV）、马立克氏病病毒（MDV）、禽白血病病毒（ALV）和网状内皮增生症病毒（REV）的群阳性率和个体阳性率分别为73．08％和40．91％、46．15％和25．95％、41．54％和17．84％、18．46％和7．57％、13．85％和4．86％，其中被检鸡之二重或多重混合感染的总阳性率为24．33％。调查结果证实，免疫抑制性疾病在安徽省的商业鸡群中普遍存在，并与鸡群疾病多而复杂、损失大相关。     

高效液相色谱法定量分析甲氨基阿维菌素苯甲酸盐

研究用HPLC定量分析甲维盐的方法。色谱条件如下:150mm×4.6mm不锈钢柱,用Hypersil,C185μm粒径,孔径300A;流动相:甲醇+水=80+20;流速1.0mL/min;紫外检测器波长254nm。本方法在1～8μg进样范围内和峰高呈线性。相关系数值(r)为0.9997。甲维盐分析方法的回收率为99.42%。     

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 .     

Detection of airborne inoculum of Leptosphaeria maculans and Pyrenopeziza brassicae in oilseed rape crops by polymerase chain reaction (PCR) assays

The potential use of DNA-based methods for detecting airborne inoculum of Leptosphaeria maculans and Pyrenopeziza brassicae , both damaging pathogens of oilseed rape, was investigated. A method for purifying DNA from spores collected using Hirst-type spore samplers and detecting it using polymerase chain reaction (PCR) assays is described. For both pathogens, the sensitivities of the DNA assays were similar for spore-trap samples and pure spore suspensions. As few as 10 spores of L. maculans or P. brassicae could be detected by PCR and spores of both species could be detected against a background of spores of six other species. The method successfully detected spores of P. brassicae collected using spore traps in oilseed rape crops that were infected with P. brassicae. Leptosphaeria maculans spores were detected using spore traps on open ground close to L. maculans -infected oilseed rape stems. The potential use of PCR detection of airborne inoculum in forecasting the diseases caused by these pathogens is discussed.     

陕甘宁老区脆弱生态环境定量评价——以榆林、延安两市为例

位于黄土高原中部的陕甘宁老区生态环境极为脆弱 ,近年来由于气候、人类开发资源等自然和人为原因 ,使生态环境的脆弱程度升高。选定年降水量、年均温、蒸发量等 8个指标 ,定量评价各县 1 970 - 2 0 0 0年的脆弱度状况 ,结果表明榆林、延安两市生态环境整体脆弱 ,脆弱度存在空间差异但差异不明显 ,时间段上的波动幅度不大。陕甘宁老区脆弱生态环境具有不稳定性 ,对外界干扰较敏感。     

应用Nested PCR技术检测柑桔木虱及其寄主九里香的柑桔黄龙病带菌率

应用PCR及Nested PCR技术检测柑桔木虱及其寄主九里香的结果表明:PCR只可检测最低2头带菌木虱,Nested PCR可检测到单个带菌木虱。100头带菌木虱中,单虫检出率为96％。检测田间重、中等、轻病的柑桔园内的木虱,其带菌率依次为87％、53％和21％。在病芦柑上饲菌不同天数的木虱均能检测出带菌,其饲菌时间最短为1d。城市九里香叶片及在其叶上取食的木虱单虫,均能用Nested PCR检测出病原。饲菌木虱接种九里香及芦柑健苗,在植株尚未表现症状时,常规PCR难检测出病原,但用Nested PCR则能检测到病原,说明九里香不仅是木虱的寄主,而且是黄龙病病原的隐症寄主。     

番木瓜环斑病毒株系的分子生物学方法鉴定

 以PRSV株系特异性引物对PRSV的PRSV126(PRSV日本分离物)、Ys、Vb和Sm等株系进行RT-PCR方法鉴定,引物PR21/PR22能把Ys从Vb和Sm中鉴定出来,PR300/PR301则能把Vb从Ys、Sm和PRSV126中鉴定出来;用限制性内切酶Hae Ⅱ、Sau3A I和Hinf I对PRSV的PRSV126、Ys、Vb和Sm等株系进行单酶切RT-PCR-RFLP分析,Hinf I能把PRSV126与Ys、Vb和Sm鉴别开来,Sau3A I能把Ys与Vb和Sm鉴别开来,Hae Ⅱ则能把Ys与PRSV126、Vb和Sm鉴别开来;以P1/P2为引物,对Vb、Ys和Sm株系进行RT-PCR-RFLP-SSCP分析,结果能一次把三者较好地区别开来。     

Identification of ALS inhibitor‐resistant Amaranthus biotypes using polymerase chain reaction amplification of specific alleles

Summary Polymerase chain reaction (PCR) amplification of specific alleles (PASA) was adapted as a molecular marker‐based method for the rapid detection of point mutations in Amaranthus retroflexus and Amaranthus rudis leading to ALS inhibitor resistance. Two pairs of primers were designed for the specific amplification of alleles of the ALS gene of susceptible and resistant biotypes. The allele‐specific primer matched the desired allele, but mismatched the different allele at its 3′ end. Differentiation was carried out by comparison of the amplified DNA fragments in gel electrophoresis after PASA‐PCR. In A. rudis, differentiation was possible with one PCR and genomic DNA as probe. A ‘nested’ PCR was necessary for the differentiation of sensitive and resistant A. retroflexus. PASA is useful for the identification of resistant weed biotypes and also as a monitoring tool to map resistance occurrence and distribution. Advantages include the fast and clear separation of those plants with and without mutations at an early stage of development, its easy and consistent performance and quick results compared with existing resistance detection tests. These advantages, when combined with management strategies, enable further activities to reduce herbicide resistance.