单克隆抗体ELISA检测犊牛粪便中冠状病毒抗原

用两株单克隆抗体混合后包被ＥＬＬＩＳＡ微孔板，加牛冠状病毒抗原，加经白陶土处理过的兔抗牛冠状病毒免血清，再加上辣根氧化物酶标记的羊抗兔ＩｇＧ抗体，加底物质显色间接ＥＬＩＳＡ检测牛冠状病毒抗原获得成功。用此方法检测细胞培养的牛冠状病毒上清液，其敏感度高出血凝试验（ＨＡ）８倍，从武汉市附近３个奶牛场采集犊牛腹泻粪便１０５份，用单抗ＥＬＩＳＡ检测牛冠状病毒抗原，共检出阳性３６份，并与血凝及血凝抑制试验，     

压力实时监测法在管道泄漏检测中的应用

检测输油管道漏油的方法很多，介绍了通过压力实时监测确定输油管道漏油点方法，以及实际应用情况。实践证明，通过压力实时监视确定输油管道漏油点的方法在实际输油运行中是切实可行的。     

迪卡配套系猪RYR1的PCR分析及其利用的研究

试验从鸡东县永安猪场随机抽取迪卡配套系猪B系和E系42头，通过PCR扩增技术和酶切鉴定．采用克隆技术进行测序，来检测是否含有氟烷基因序列。经过以上技术检测这42头猪均没有氟烷基因。     

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     

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 .     

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.     

Seasonal distribution of phytoplasmas in Australian grapevines

The distribution and persistence of phytoplasmas were determined in Australian grapevines. Phytoplasmas could be detected using the polymerase chain reaction (PCR) from shoots, cordons, trunks and roots throughout the year, and phytoplasmas appear to persistently infect Australian grapevines from year to year. Phytoplasmas were not always detected in samples from the same sampling area from one sampling period to the next. Phytoplasma detection by PCR was improved by sampling from shoots, cordons and trunks, especially during October (early spring). The diseases expressed by the 20 grapevines used in the distribution and persistence studies were monitored. Australian grapevine yellows disease (AGY) was expressed by 17/20 grapevines at some time during the study, whilst only 4/20 and 15/20 grapevines expressed restricted growth disease (RG) and late season leaf curl disease (LSLC), respectively. All grapevines with RG and LSLC also had AGY. The three diseases were persistently expressed in some grapevines and remission of disease was observed in others. The results of PCR detection in the same grapevines indicated that phytoplasmas were more frequently detected in AGY-affected grapevines that also expressed RG and LSLC compared with grapevines expressing AGY alone. Phytoplasmas were detected in symptomless plant material but less frequently compared with AGY-affected material.     

刺梨及其近缘种PCR实验体系的建立与优化

以刺梨及其近缘种月季为试材,进行了RAPD-PCR实验参数的确立和优化试验。结果表明,刺梨及月季25μL反应体系的最优组成为2.5μL10×反应缓冲液,2mmol/LMg2+,0.2mmol/LdNTP,1.6mg/L模板DNA,0.4μmol/L随机引物和1.2UTaqDNA多聚酶。经PCR扩增验证,此反应体系亦适宜于刺梨的部分近缘种,可有效用于RAPD分析;通过将退火温度提高至50℃或采用“Touchdown”扩增程序,并在50μL反应体系中适当增加特异引物对浓度(1.0μmol/L),模板DNA(4.0mg/L)和TaqDNA多聚酶(3.0U/管)的使用量,建立起适合于刺梨特异DNA片段检测及回收的特异PCR扩增实验体系,为刺梨的分子克隆奠定了技术基础。