VSV的RT-PCR快速检测方法的建立

选取水泡性口炎病毒 N基因序列 ,设计 1对引物 ,建立检测水泡性口炎病毒的 RT- PCR方法。对VSV各毒株进行检测 ,结果均为阳性 ,而对反刍动物病毒性疾病相关病毒进行检测 ,结果均为阴性。结果表明所建立的 RT- PCR技术可用于水泡性口炎的诊断和流行病学调查     

单抗酶联试剂盒与PCR方法快速检测沙门氏菌的比较

本文对单抗酶联试剂盒与PCR方法快速检测沙门氏菌进行了比较。将沙门氏菌与大肠杆菌以不同比例混合后 ,分别用ELISA和PCR法检测 ,在 1∶1 0 0的比例时 ,用这 2种方法均能检测出阳性样品 ;在 1∶1 0 0 0的比例时 ,用ELISA法检测为阴性 ,而用PCR法则能检测出。检测鲜牛奶、龙虾、虾仁、熟食制品等样品 ,2种方法的符合率达 97 6 %。对冻虾仁、人粪便样品的前增菌、选择性增菌、后增菌过程进行同步检测 ,在样品的前增菌液 ,直接ELISA法检测的OD值小于 0 5 ,而PCR法扩增能出现特异条带 ,经国标方法验证为阳性 ,证实PCR法的敏感性优于直接ELISA法 ,而在选择性增菌液和M 肉汤后增菌液中 ,2种方法均能检出阳性样品。     

JEV、PPV、PRRSV、PRV多联PCR的应用研究

用 JEV、PRRSV二联 PCR,PPV、PRV二联 PCR以及这 4种病毒 4联 PCR对来自内蒙、广州、广西、天津、北京、吉林病料进行检测 ,共检了 1 4 6份病料。其中 PRRSV阳性 7份 ,PPV阳性 1 2份 ,PRV阳性 2 1份 ,PRV和 PPV混合感染 4份。并对 5份人工接种 JEV小鼠病料检测 ,其中 4份为阳性。随后对部分阳性 PCR扩增产物进行点杂交和核苷酸测序鉴定 ,证实了 PCR扩增准确性。对内蒙 PRRSV阳性扩增带测序结果显示 ,我国流行 PRRSV为美洲型 ,在扩增片段的核苷酸序列上有 3个碱基差异     

鸡新城疫病毒B_(95)株的RT-PCR检测方法研究

本试验设计了一对B95 株的特异引物NDV -P1/P2,探讨了对其进行RT-PCR检测的可行性。结果证明 ,用该引物可以扩增出一条310bp的特异核酸带 ,敏感性为0.0043ng/ul。     

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.     

应用PCR技术检测空肠弯曲菌及结肠弯曲菌的研究

本研究根据空肠弯曲菌及结肠弯曲菌的16s rRNA基因相同保守序列来设计引物,建立了一种PCR检测方法,该方法具有较强的特异性及灵敏性,其对空肠弯曲菌及结肠弯曲菌的最低检测限度为5 CFU/mL,它无需增菌培养过程可以直接对样品进行检验,在对鸡肉样品空肠弯曲菌及结肠弯曲菌的检测中取得了较好的效果.     

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 .