基于qPCR和LAMP技术的马铃薯晚疫病菌快速检测方法     

祝菊澧  梁静思  张佩  王伟伟  林桐司骐  谢欣娱  苏瑞  唐唯 《作物杂志》2019,35(6):168-3311

马铃薯晚疫病菌（Phytophthora infestans）能侵染多种茄科植物,它引起的马铃薯晚疫病,是马铃薯生产中的第一大病害。为了开发能在田间快速检测马铃薯晚疫病病原的方法,利用P. infestans T30-4基因组测序数据的contig 1.18131,设计qPCR和LAMP引物,优化扩增条件后得到引物的特异性和灵敏度,最后通过检测田间收获薯块,比较形态学传统方法、qPCR及LAMP的差异。特异性检测结果发现,qPCR和LAMP仅在含有P. infestans DNA模板的体系有阳性扩增,在寄主和其他微生物DNA中均无扩增;在优化的条件下,qPCR和LAMP的检测下限可达1×10 ^-6ng/μL,在有寄主和其他微生物DNA存在的条件下,引物的灵敏度没有显著差异。利用两种快速方法对在大理、丽江及昆明3个地区田间收获薯块上检测发现,qPCR和LAMP方法得到的检出率差异极为不显著（P=0.420）,两种快速检测方法和形态学鉴定方法检出率差异极显著（P=0.009）。在大理、丽江及昆明3个地区的薯块中,两种分子检测方法检出率均比形态学方法高。其中,qPCR检测方法比形态学方法分别提高了12.00%、2.00%、8.70%;LAMP检测方法比形态学方法分别提高了11.30%、2.00%、8.70%。  相似文献   

Rapid detection of contagious ecthyma by loop-mediated isothermal amplification and epidemiology in Jilin Province China     

Kai WANG  Hongze SHAO  Zhihua PEI  Guixue HU 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2016,78(1):125-128

The aim of this experiment was to develop a loop-mediated isothermal amplification (LAMP) assay and to research the recent epidemiology of contagious ecthyma in Jilin Province, China, using the assay. A LAMP assay targeting a highly conserved region of the F1L gene was developed to detect contagious ecthyma virus (CEV). Three hundred and sixty-five cases from 64 flocks in 9 different areas of Jilin Province, China, from 2011 to 2014 were tested using the LAMP assay. The results showed that the sensitivity of the LAMP assay was 100 copies of the standard plasmid, which is 100-fold higher than the sensitivity of PCR. No cross-reactivity was observed with capripoxvirus, fowlpox virus, foot-and-mouth disease virus serotype O, foot-and-mouth disease virus serotype Asia I and bluetongue virus. The average positive rate was 19.73% (72/365), and the positive rate was highest in lambs aged 1–6 months. Our results demonstrated that CEV infection was very widespread in the flocks of Jilin Province and that the LAMP assay allows for easy, rapid, accurate and sensitive detection of CEV infection.  相似文献   

柑橘溃疡病菌LAMP实时浊度检测方法的建立及应用     

封立平  孔德英  孙涛  孙冬  纪瑛  王简 《热带作物学报》2015,36(6):1153-1160

以柑橘溃疡病菌特异性高的假定蛋白基因（登录号：AE008923.1）为靶标，设计并筛选4条引物来特异性识别靶标序列中的6个独立区域，建立柑橘溃疡病菌LAMP实时浊度检测方法。该方法利用实时浊度仪检测反应过程中产生的焦磷酸镁白色沉淀，实现对LAMP整个反应过程的实时监控。从镁离子浓度、甜菜碱浓度、dNTPs浓度、引物浓度和反应温度5个变量参数对 LAMP检测体系进行优化，并对方法的特异性、灵敏度、稳定性及实际样品检测效果进行评价。结果显示，该体系经优化后稳定性良好，可在66 ℃ 恒温条件下，60 min内完成检测；DNA检测下限可达0.02 ng/μL，灵敏度与荧光定量PCR方法相同，比常规PCR方法高1个数量级；能快速、准确地对田间疑似样品进行检测，与荧光定量PCR方法的检出情况一致，没有漏检。结果说明柑橘溃疡病菌实时浊度LAMP检测方法操作简便、所需时间短，特异性与灵敏度高，为柑橘溃疡病菌的快速筛查提供较好的途径。  相似文献   

饲料中动物源性成分微流控检测芯片的研究     

陈宇飞  杨柳  王磊 《饲料研究》2021,(4):92-95

试验以"LAMP+微流控"技术为基础,开发饲料动物源性成分检测芯片。针对牛、羊、猪、鸡的16srRNA靶序列,根据LAMP引物设计软件进行4种肉类LAMP引物设计。在LAMP优化试验、芯片优化测试、特异性验证试验和芯片应用测试中,进行最佳引物筛选测试和特异性测试。结果显示,筛选出的4种肉类最佳引物特异性强,最佳反应温度为65℃。芯片特异性验证试验的归一化荧光曲线显示,试验结果准确、特异性稳定。对饲料市场购买的16份样本中检测出的动物源性成分,采用中华人民共和国农业行业标准验证,符合率100%。微流控检测芯片的开发与应用为饲料中动物源性成分检测提供新方法,为饲料产品质量安全提供保障。  相似文献   

Development of a rapid assay for the diagnosis of Myxobolus cerebralis in fish and oligochaetes using loop-mediated isothermal amplification   总被引：1，自引：0，他引：1  

El-Matbouli M  Soliman H 《Journal of fish diseases》2005,28(9):549-557

A loop-mediated isothermal amplification assay was developed for the rapid detection of Myxobolus cerebralis in both fish and oligochaete hosts. The assay was optimized to amplify parasitic DNA by incubation with Bst DNA polymerase and a set of six specially constructed primers at 65 degrees C for 60 min. The amplification products were detected visually using SYBR Green I dye which gave identical results to gel electrophoresis analysis. Parasite DNA was detected from infected oligochaetes, and from the anal fin, caudal fin, dorsal fin and operculum of clinically infected fish. This 'Myxo-LAMP' assay has a detection limit similar to that of a polymerase chain reaction assay (10(-6)), but is more rapid and only requires a water bath for amplification and is therefore practical for simple and rapid diagnosis of infected tissue.  相似文献   

Development of a multiplex loop-mediated isothermal amplification assay to detect shiga toxin-producing Escherichia coli in cattle     

Hee-Jin Dong  Ae-Ri Cho  Tae-Wook Hahn  Seongbeom Cho 《Journal of veterinary science (Suw?n-si, Korea)》2014,15(2):317-325

A multiplex loop-mediated isothermal amplification (mLAMP) assay was developed for simultaneous detection of the stx1 and stx2 genes and applied for detection of shiga toxin-producing Escherichia coli (STEC) in cattle farm samples. Two target genes were distinguished based on T_m values of 85.03 ± 0.54℃ for stx1 and 87.47 ± 0.35℃ for stx2. The mLAMP assay was specific (100% inclusivity and exclusivity), sensitive (with a detection limit as low as 10 fg/µL), and quantifiable (R² = 0.9313). The efficacy and sensitivity were measured to evaluate applicability of the mLAMP assay to cattle farm samples. A total of 12 (12/253; 4.7%) and 17 (17/253; 6.7%) STEC O157, and 11 (11/236; 4.7%) non-O157 STEC strains were isolated from cattle farm samples by conventional selective culture, immunomagnetic separation, and PCR-based culture methods, respectively. The coinciding multiplex PCR and mLAMP results for the types of shiga toxin revealed the value of the mLAMP assay in terms of accuracy and rapidity for characterizing shiga toxin genes. Furthermore, the high detection rate of specific genes from enrichment broth samples indicates the potential utility of this assay as a screening method for detecting STEC in cattle farm samples.  相似文献   

基于LAMP的MVC模式电子商务网站的构建     

岳峻  邢如义  邱丽娟  梅树立 《中国农业大学学报》2003,8(6):66-68

提出了一种构建中小型连锁企业电子商务平台的软件设计方案。该方案采用应用服务器与数据库服务器相分离的B／W／D三层结构模型，用户通过中间层访问数据，从而具有更好的安全性和灵活性；使用自由软件组合Linux Apache MySQL PHP，使得商务网站的软件环境搭建成本大大降低，更适于为数众多的中小型企业的使用。  相似文献   

非洲猪瘟病毒环介导恒温扩增快速检测技术的建立及应用     

杨吉飞  关贵全  刘志杰  汪月凤  李有全  马米玲  刘爱红  任巧云  苟惠天  杜鹏飞  罗建勋  殷宏 《中国兽医寄生虫病》2011,19(4):7-12

本研究基于非洲猪瘟病毒（African swine fever virus, ASFV）vp72基因设计引物,建立了能够快速检测非洲猪瘟病毒的环介导恒温扩增技术（loop-mediated isothermal amplification, LAMP）。将LAMP与OIE参考的PCR检测方法进行比较,并且应用LAMP对非洲猪瘟参考实验室提供的非洲猪瘟病毒17个毒株的基因组以及国内收集的50份猪的基因组、30份蜱的基因组进行检测。结果显示,本研究设计的引物具有良好的特异性和敏感性,所建立的LAMP能够成功扩增非洲猪瘟病毒17个毒株的基因组,而野外收集的猪和蜱的基因组检测均为阴性。因此,本研究所建立的方法能够用于非洲猪瘟的快速诊断以及防控。  相似文献   

LAMP法鉴定奶牛早期胚胎性别的效果研究     

黄金明  熊杰  柳尧波  游伟 《家畜生态》2005,(1)

本试验利用Lamp法对28枚鲜胚进行了性别鉴定,将其中13枚雌性胚和3枚雄性胚胎给受体牛移植,妊娠率达45.5%,性别鉴定的准确率为100%。说明Lamp法用于奶牛早期胚胎性别鉴定是切实可行的。  相似文献   

LAMP技术诊断猪支原体肺炎的应用研究     

夏伟  李鹏 《黑龙江八一农垦大学学报》2012,24(4):21-24

应用环介导等温扩增技术(LAMP)对猪支原体肺炎进行诊断应用研究,对来自黑龙江不同地区的患猪呼吸道疾病的肺脏病料64份进行检测,54份诊断为阳性,阳性率84.3%。健康猪鼻拭子共176份,经LAMP检测,157份为阳性,阳性率为89.2%。其中健康猪拭子带菌率较高,结果表明,LAMP技术可应用于猪支原体肺炎的临床诊断。  相似文献