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。     

蔬菜连作障碍防治的研究动态

本文论述了引起蔬菜连作障碍的物理原因和生物原因,论述了包含培育抗性品种、作物嫁接、轮作倒茬、土壤消毒、土壤改良和施肥管理、化学防治、生物防治、植物检疫几种防治措施等方面的内容。本文还汇总了国内外近年来对蔬菜连作障碍的研究动态和进展,为进一步研究提供了大量的资料和参考文献。     

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.     

马铃薯块茎淀粉组分高效检测体系的建立及应用

马铃薯块茎淀粉组分(直链/支链淀粉比AM/AP)影响淀粉特性以及决定其在食品和其他工业上的应用。AM/AP的测定对其合理利用以及选育特异淀粉组分的专用品种等具有重要意义。采用96孔板双波长法对马铃薯不同AM/AP比率标准样品进行测试,发现当测试样品AM/AP比率超过制作标准曲线的AM/AP比率(33.3%)时,测试结果明显高估直链淀粉含量。基于96孔板的碘染分析,建立了马铃薯AM/AP比率和最大吸收峰下波长的标准曲线:y=601.88x^0.0215,R²=0.9999。该方法进一步提高了测试准确度和应用范围。利用该方法测试了198个马铃薯品种(系)以及高直链淀粉组分的转基因块茎,明确了收集的马铃薯品种(系)的直链淀粉组成范围为17.4%～33.3%。新建立的马铃薯淀粉组分测试方法为高效准确筛选遗传育种材料以及改良淀粉组分株系提供了方便。     

Taqman探针实时定量PCR检测猪伪狂犬病毒

选取猪伪狂犬病毒(PRV)基因组中gE基因,设计特异性引物和Taqman探针,利用实时定量PCR来定量检测PRV.利用PCR技术扩增猪伪狂犬病毒gE基因80 bp片段,并克隆到pMD18-T载体上,阳性重组质粒命名为pgE80.pgE80质粒进行10倍系列稀释,作为荧光PCR检测的标准模板,定量拷贝数,并绘制标准曲线.以提取的PRV、猪圆环病毒2型(PCV2)、猪细小病毒(PPV)、猪瘟病毒(CSFV)、猪繁殖与呼吸障碍综合症病毒(PRRSV)和传染性胃肠炎病毒(TGEV)的DNA作为模板,进行特异性检测.该实时定量PCR方法,标准曲线斜率为-0.37,R2=0.992,可以检测到20个拷贝数.PRV能被扩增出S型荧光曲线,而其它病毒均未能扩出.用PRV强毒肌肉注射仔猪,定量PCR比普通PCR更能灵敏而特异地检出呼吸道排毒和血液带毒.建立的实时定量PCR方法可用于临床样品快捷、准确、方便地检测.     

河南省烟草病毒的小RNA测序检测

To identify viruses in Henan tobacco-planting areas, from 2015 to 2017 and 2019, 288 symptomatic tobacco samples were collected and then subjected to small RNA sequencing. Results showed that at least 7 viruses were detected from these samples which including four previously reported viruses, cucumber mosaic virus (CMV), tobacco mosaic virus (TMV), potato virus Y (PVY) and tobacco vein banding mosaic virus (TVBMV). Other three viruses, wild tomato mosaic virus (WTMV), brassica yellows virus (BrYV), and cycas necrotic stunt virus (CNSV) were firstly detected in Henan province. However, tobacco etch virus (TEV) and tobacco ringspot virus (TRSV) were not detected from these samples. In addition, CMV, TMV, PVY, TVBMV, and BrYV were the dominant viruses infecting tobacco in Henan Province.     

玉米细菌性枯萎病菌的环介导恒温扩增(LAMP)检测方法

为了提高口岸和基层实验室检疫和监测玉米细菌性枯萎病菌的准确性和工作效率,利用环介导恒温扩增技术(LAMP),根据内切葡聚糖酶(EGase)基因前导序列,设计2个内引物和2个外引物,对玉米细菌性枯萎病菌进行快速检测。结果表明,使用玉米细菌性枯萎病菌的近缘种或引致相似症状的病原菌菊欧文氏菌玉米致病变种Erwinia chrysanthemi pv.zeae、玉米内州萎蔫病菌Clavibacter michiganensis subsp.nebraskensis、燕麦假单胞菌Pseudomonas avenae、杓兰欧文氏菌Erwinia cypripedii检测其特异性,仅玉米细菌性枯萎病菌有扩增。LAMP检测灵敏度达到2 pg DNA,为普通PCR的100倍;与其它检测方法相比,LAMP方法检测时间短,效率高,不仅降低了设备投入,易于操作,而且具有较高的灵敏度和特异性,适合玉米细菌性枯萎病菌的现场检疫和大规模检测。     

马铃薯Y病毒云南昭通烟草分离物的检测及年度间差异比较

利用DAS-ELISA检测试剂盒检测昭通烟草脉斑病样品,结果表明存在马铃薯Y病毒O株系和马铃薯Y病毒N株系两个不同株系病毒。利用Sprimer和M4引物扩增、克隆、测序,得到长度为1 771 bp目的片段,该片段包含病毒的外壳蛋白基因序列、3′ UTR序列以及部分Nib基因序列;序列分析表明与湖南HN 2分离物（GenBank No. GQ200836）和美国NE 11分离物（GenBank No. DQ157180）核苷酸序列相似性均为98%,系统进化分析表明其具有较近的亲缘关系。