Assessing the within-herd prevalence of Coxiella burnetii milk-shedder cows using a real-time PCR applied to bulk tank milk     

Guatteo R  Beaudeau F  Joly A  Seegers H 《Zoonoses and public health》2007,54(5):191-194

Thirty-seven bulk tank milk (BTM) and individual milk samples of all contributing cows were tested for Coxiella burnetii detection by a real-time PCR assay and used to assess the relationship between the BTM PCR-response and (i) the within-herd prevalence of milk-shedder cows and (ii) the proportion of heavy milk-shedder cows. The within-herd prevalence of milk-shedder cows (i) was found to be significantly higher in herds with a positive BTM and (ii) increased significantly with the estimated titre in Coxiella burnetii obtained in positive BTM. The proportion of heavy milk-shedder cows among the milk-shedder cows increased significantly with an increased estimated titre in Coxiella burnetii in positive BTM. Therefore, a real-time PCR assay applied to BTM samples collected repeatedly over time appears to be a valuable tool to assess on a larger scale the status of herds towards Coxiella shedding, and to evaluate the efficiency of control actions aimed at controlling and/or preventing Coxiella shedding in dairy herds.  相似文献   

甜瓜黄斑病毒SYBR Green Ⅰ实时荧光定量PCR方法     

孙晓辉  潘睿婧  刘永光  王丹  石朝鹏  乔宁  孙作文  竺晓平 《植物病理学报》2023,53(1):119-125

为建立检测甜瓜黄斑病毒(melon yellow spot virus, MYSV)的SYBR Green Ⅰ实时荧光定量PCR(qPCR)方法。基于MYSV核衣壳蛋白基因保守序列设计qPCR特异性引物对,针对引物退火温度、引物浓度、特异性和敏感性进行系列优化。结果显示,优化后的qPCR方法最适退火温度为61.3℃,最适引物浓度为0.65μmol·L^-1,特异性强,灵敏度高,比PCR高100倍。以携带目的基因片段的重组质粒为标准品,构建的qPCR标准曲线循环阈值与模板浓度呈良好的线性关系,相关系数为0.999 7。实验样品验证表明建立的qPCR方法可用于MYSV的定量检测。  相似文献   

薄壳山核桃疮痂病菌TaqMan荧光定量PCR检测方法的建立及应用     

赵玉强  卓可儿  郭云  朱灿灿  王行军  陆小美  田艳丽  胡白石 《植物保护》2023,49(3):193-198

疮痂病是薄壳山核桃上最具毁灭性的病害,带菌植物材料是传播疮痂病的重要来源。准确、灵敏、快速的检测方法可为该病害流行规律调查和防控提供有力的依据。本文通过比较薄壳山核桃疮痂病菌Venturia effusa及其近似种之间的ITS序列差异,设计了特异性引物和TaqMan探针,建立了薄壳山核桃疮痂病菌的荧光定量PCR检测方法。特异性检测结果表明,该方法可以检测不同地区的薄壳山核桃疮痂病菌菌株,而对其近似种以及薄壳山核桃上的其他真菌均没有信号。本研究建立的检测方法对薄壳山核桃疮痂病菌DNA的最低检测限可达0.5 pg/μL。该方法用于田间样品检测时,检测时间仅需1 h,远快于常规的分离培养法。本研究建立的基于TaqMan探针的荧光定量PCR检测方法为薄壳山核桃疮痂病菌的快速检测和监测提供了有力工具。  相似文献   

鼠源细胞TaqMan实时荧光定量PCR检测方法的建立     

苏佳  赵炜  秦义娴  邓永  高月异  白洪旭  薛青红  陈晓春  吴华伟 《中国兽药杂志》2023,57(3):10-16

为对鼠源细胞进行鉴别检测,以鼠线粒体16S rRNA基因序列为靶位点设计特异性引物及探针,建立实时荧光定量PCR检测方法,并评价该方法的特异性及敏感性。结果显示,所建立的检测方法特异性好,针对鼠源细胞基因组荧光定量PCR扩增曲线良好,其他物种来源细胞基因组及生物制品原辅材料未出现特异性扩增曲线;敏感性高,基因拷贝数检出限度为45.3拷贝。本试验建立的荧光定量PCR检测方法能够有效地对鼠源细胞进行快速检测,为细胞质量控制提供了有效方法。  相似文献   

植物基因启动子的克隆方法及其应用   总被引：1，自引：0，他引：1  

尹辉　李丹张毅  李秋莉 《分子植物育种》2006,4(Z1):85-91

植物基因的表达调控已成为分子生物学研究热点,启动子是基因表达调控的重要顺式元件,启动子的克隆对于研究基因表达调控、构建基因工程载体、表达目的蛋白有着重要的意义。启动子克隆的方法很多,从常用的启动子陷阱技术筛选启动子到PCR方法的应用,此后相继问世的一些基于PCR的克隆启动子技术,如载体锚定PCR、反向PCR、接头PCR、交错热不对称PCR等,为克隆启动子提供了更可靠,更合理的方法。本文着重介绍了几种植物基因启动子的克隆方法,分析了它们的优缺点,并展望了今后的研究前景。  相似文献   

Determination of vc and mating types of Cryphonectria parasitica isolates by multiplex PCR and their genetic diversity in 13 chestnut-growing provinces of Turkey     

Deniz Çakar  Göksel Özer  Seçil Akıllı Şimşek  Salih Maden 《Forest Pathology》2023,53(3):e12813

Vegetative compatibility (vc) and mating types and genetic diversity of Cryphonectria parasitica isolates were determined using 183 isolates obtained from 215 infected chestnut trees growing in 13 provinces of Turkey. Based on the cultural aspects, 143 of these isolates were evaluated as virulent whereas the remaining 40 isolates were hypovirulent. When vc types of 183 isolates were classically differentiated, 135 of them matched to EU-1 (82.3%), 29 of them to EU-12 (17.6%) vc type, whereas 19 of them did not match to the two. When molecular vic markers were used, all the isolates were assigned to two EU vc types; 149 to EU-1 (81.4%) and 34 (18.5%) to EU-12. Of the majority of the isolates, 134 (73.2%) had mating-type MAT-1, while 44 (24%) isolates had MAT-2 and 5 (2.8%) isolates had both mating types. The population analysis based on two DNA marker systems, Inter-Primer Binding Site and Start Codon Targeted Polymorphism, showed no intraspecific genetic variation among the C. parasitica isolates. The prevalence of two dominant vc types revealed by this study shows that biological control with hypovirulent EU-1 and EU-12 isolates will be significant for the country. The results might be helpful to chestnut breeders carrying out resistance breeding studies to manage this disease based on hypovirulence attributed to Cryphonectria hypovirus 1.  相似文献   

茶树咖啡因合成酶基因RNA干涉表达载体构建   总被引：11，自引：3，他引：8  

张广辉  梁月荣  陆建良  董俊杰 《茶叶科学》2006,26(4):243-248

咖啡因合成酶（TCS）是茶树咖啡因生物合成途径中的一个关键酶,催化7-甲基黄嘌呤转变为可可碱和可可碱转变为咖啡因。抑制TCS基因表达是培育低咖啡因茶树的最有效途径。用RT-PCR方法扩增茶树TCS基因的两个cDNA片段,并克隆入T-载体中。干涉载体和TCS基因片段的T克隆经限制性内切酶两次双酶切与连接,将两个TCS基因片段分别反向重复插入到干涉载体pFGC5941,构建了TCS基因的RNA干涉载体,分别命名为pFGC5941-TCS02和pFGC5941-TCS03。经PCR和DNA测序获得验证。TCS基因RNA干涉载体的构建为培育低咖啡因茶树奠定了基础。  相似文献   

A serological and molecular study on Francisella tularensis in rodents from Hamadan province,Western Iran     

《Comparative immunology, microbiology and infectious diseases》2020

Introduction and purposeTularemia is a zoonotic disease, the most important hosts of which are rodents. Endemic regions and reservoirs of F. tularensis are not well-researched areas in Iran. The present study aimed to study F. tularensis infection in the rodent populations of western Iran.Materials and methodsSamples were collected in different areas of Kabudar Ahang County in Hamadan province (west of Iran) from 2014 to 2017. Tularemia serological and molecular tests were conducted using the tube agglutination test and Real-time PCR method tracking the ISFtu2 gene. Positive serum samples were evaluated for cross-reactivity with brucellosis.ResultsA total of 433 rodents, collected from 33 localities, were included in the study. The most abundant species belonged to the Persian jird (Meriones persicus; 75.5%), and Libyan jird (Meriones libycus; 10.1%). Among the studied samples, three (0.74 %) were seropositive and five (1.15%) were PCR positive. Seropositive samples were two M. persicus and one M. libycus, and PCR positive rodents were four M. persicus and one M. vinogradovi. Tularemia seropositive samples showed no cross-reactivity with brucellosis.ConclusionGiven the presence of infection in rodents with tularemia agent in the studied area, it is crucial to elucidate the risks of rodent exposure to tularemia for physicians, health personnel and the general population.  相似文献   

柑橘黄龙病菌浓度对琼中绿橙果实品质的影响     

李丹阳  黄霖霄  杨毅 《中国果业信息》2021,50(2)

为了定量衡量黄龙病菌量对琼中绿橙果实品质的影响,本实验以采自海南琼中橘园中感染了柑橘黄龙病的琼中绿橙为实验材料,用实时荧光定量PCR相对定量的方法测定果实中柑橘黄龙病菌的浓度,同时对果实品质的8个外在指标（果实纵经、横径、果形指数、单果重、果皮率、残渣率、种子数量、种子重量）和5个内在指标（果汁率、可食率、可溶性固形物含量、可滴定酸含量、固酸比）进行测定,研究黄龙病菌浓度对琼中绿橙果实品质的影响。结果表明：随着琼中绿橙果实中柑橘黄龙病菌浓度的增大,果实外观品质变化明显的指标包括果实纵经、果形指数、单果重（p<0.05）,其中果实纵经呈现明显下降趋势,从64.97mm下降到57.44mm,相对较少11.59%;果形指数呈现明显上升趋势,从0.83上升到0.94,相对增多13.25%;单果重呈现下降趋势,从166.35g下降到109.66g,相对较少34.08%;果实横径、果皮率、残渣率、种子数量、种子重量等指标变化不明显。随着琼中绿橙果实中柑橘黄龙病菌浓度的增大,果实内在品质变化明显的指标为可溶性固形物含量,可溶性固形物含量呈现明显下降趋势（P<0.05）,从9.65%下降到8.19%,相对减少15.13%;果汁率、可食率、可滴定酸含量和TSS/TA没有明显变化。结论：在黄龙病菌浓度增加的情况下,琼中绿橙在果实外观、内在品质等多个指标呈现下降趋势。研究结果为定量评价黄龙病对柑橘果实的影响提供参考。  相似文献   

3种含氯消毒剂对柑橘黄龙病菌室内防治效果研究     

王贤达  胡菡青  林雄杰  范国成 《中国果业信息》2016,45(6)

柑橘黄龙病是柑橘生产上最具毁灭性的病害,目前没有防治特效药也没有较好的抗性品种。本文以含氯消毒剂作为柑橘黄龙病菌防治的候选药剂开展药剂筛选试验。将染病接穗分别浸泡3种含氯消毒剂再嫁接到健康枳壳砧木上,利用定量PCR技术定量分析药剂处理前后黄龙病菌含量的变化,建立柑橘黄龙病菌药剂防治效果评价体系。试验结果表明,漂白粉（CH）不适合利用嫁接技术进行候选药剂筛选;二氯异氰尿酸钠（NaDCC）和三氯泡腾消毒片（TCCA）的抑菌效果随处理浓度的降低而减弱,在有效氯含量为5000mg/L时,病菌减退率最高,分别为99.1%和99.5%,相对防效最佳,分别达到了97.9%和98.8%,与对照药剂盐酸四环素(TET)的抑制效果相当,说明NaDCC和TCCA可以作为柑橘黄龙病化学防治的候选药剂。  相似文献