共查询到10条相似文献，搜索用时 593 毫秒
1.
采用一种新型的引物设计方法——双启动寡核苷酸引物（dual-priming oligonucleotide,DPO）,以霍乱弧菌mdh基因为靶序列,建立了霍乱弧菌DPO-PCR特异性检测方法,分析了DPO引物退火温度不敏感性、检测灵敏度及特异性,并对检测方法进行了初步应用。灵敏度结果显示,DPO-PCR方法对霍乱弧菌的最低检出限为1.07×10^2 CFU/mL。退火温度不敏感性试验中,与常规PCR引物相比,DPO引物在45～65℃退火温度均能够高效扩增出靶基因片段。特异性结果显示,DPO-PCR方法的特异性比常规PCR方法强,不产生任何非特异性扩增。利用建立的霍乱弧菌DPO-PCR检测方法对采集的550份样本进行检测,检出43份霍乱弧菌阳性样本,经行业标准法（SN/T2425-2010）复检,检测结果相同,表明所建立的DPO-PCR检测方法具有良好的实用性,为霍乱弧菌的快速准确检测提供了新途径。 相似文献
2.
为建立一种基于铜绿假单胞菌flgE基因的PCR检测方法,本试验根据GenBank中已发表的铜绿假单胞菌flgE基因序列,设计合成了1对特异性引物,由铜绿假单胞菌基因组DNA中扩增获得了目的基因。从退火温度、循环次数、Mg2+浓度、dNTPs浓度和引物浓度5个方面优化了反应条件,并检测了该方法的特异性和敏感性。结果成功扩增出了铜绿假单胞菌1 400 bp的flgE特异性基因片段,最佳退火温度、循环次数、Mg2+浓度、dNTPs浓度和引物浓度分别为56℃、30个循环、1.6 mmol/L、0.2 mmol/L和0.3μmol/L。特异性试验表明,仅铜绿假单胞菌中可扩增出目的片段,而多杀性巴氏杆菌、鼠伤寒沙门氏菌、志贺氏菌、致病性大肠杆菌和金黄色葡萄球菌中均未扩增出相应片段。敏感性试验结果表明,本方法可检测到最低10 pg/μL的铜绿假单胞菌基因组DNA以及100 CFU/mL的病原菌。 相似文献
3.
非洲猪瘟病毒实时荧光定量PCR检测方法的建立及应用 总被引：4，自引：1，他引：3
本研究为了建立一套检测非洲猪瘟病毒(African swine fever virus,ASFV)的实时荧光定量PCR检测方法,根据GenBank公布的23株编码ASFV结构蛋白p72的基因序列,设计引物和探针,优化退火温度、Mg2＋浓度和引物、探针浓度,生成标准曲线,进行重复性、敏感性、特异性试验,并检测样品。结果显示,优化的退火温度为60 ℃,Mg2＋终浓度为4 mmol/L,引物、探针终浓度分别为0.8、0.3 μmol/L。重复性试验变异系数均小于1.3%,敏感性试验最低能够检测到10拷贝/μL的质粒,以其他5种猪病病毒和ASFV质粒为模板进行特异性试验,只有ASFV质粒出现扩增曲线。结果表明,建立的实时荧光定量PCR方法是快速、灵敏、特异的检测ASFV的方法。 相似文献
4.
The aim of this study was to establish a simultaneous triple PCR detection method for Staphylococcus aureus (S.aureus),Pseudomonas aeruginosa (P.aeruginosa) and Klebsiella pneumoniae (K.pneumoniae).Three pairs of specific primers had been designed according to nuc gene of S.aureus,toxR gene of P.aeruginosa and PhoE gene of K.pneumoniae.The triple PCR reaction conditions were optimized on the basis of single PCR methods.At the same time,specificity,sensitivity and repeatability tests of the triple PCR method were studied,and the results of bacteria isolation and culture and the triple PCR method were compared.The results showed that the amplification product sizes were 484,278 and 368 bp,respectively.The optimal annealing temperature was 56 to 59 ℃,the concentrations of primers were all 0.2 μmol/L,dNTP concentration was 200 μmol/L,Mg2+ concentration was 2.5 mmol/L.The specificity test showed that there was no cross reaction between these three bacteria templates and other eight kinds of common bacteria templates,such as Bordetella bronchiseptica.The minimum of simultaneous detection of three bacteria genomic DNA was 10-5 ng/μL.The results of three repeatability tests of triple PCR were the same which indicated the repeatability was good.30 samples from mice were detected by bacteria isolation and culture and the triple PCR method,the detection rate of triple PCR was slightly higher than the bacteria isolation and culture.The positive samples detected by bacteria isolation and culture were also positive detected by triple PCR.The results showed that a specific,sensitive and efficient triple PCR system had been established and could provide the technical support for bacteria detection and epidemiological investigation of laboratory animals. 相似文献
5.
LEI Chenghong CHEN Kaiyun XU Xinfeng HUDUSI·Aierken XU Jun BAI Meihua SHI Qian XIAO Yuanyuan WANG Keke 《中国畜牧兽医》2020,47(5):1299-1306
The purpose of this study was to establish a highly sensitive 3D digital PCR (3D-dPCR) method for the detection of equine herpesvirus 1 (EHV-1),which could accurately and quantitatively detect the samples with low EHV-1 content and realize the early diagnosis and prevention of equine rhinopneumonia.According to the conserved region of EHV-1 glycoprotein B gene,we designed specific primers and probes,optimized the concentration and annealing temperature of primers in the 3D-dPCR reaction system,analyzed the sensitivity,specificity and repeatability of this method,and established the 3D-dPCR method of EHV-1.In this study,the best concentration of primer and probe of 3D-dPCR was 0.4 and 0.4 μmol/L respectively,the best annealing temperature was 60 ℃,R2 of the absolute quantitative curve of the method was 0.998,the linear relationship was good,the sensitivity was about 10 times higher than that of Real-time PCR,and the minimum detection limit was 5.83 copies/μL.There was no cross reaction with EHV-4,Theileria equi and the nucleic acid of equine arteritis.The results showed that the positive rate of 3D-dPCR was 66.7%,which was higher than that of Real-time PCR for EHV-1 in OIE (64.2%).The results of 3D-dPCR were consistent with those of Real-time PCR,and the sensitivity of 3D-dPCR to the samples with low virus content was higher,which could effectively detect suspicious samples.The results showed that the established 3D-dPCR method was more sensitive,specific and reproducible for the detection of clinical samples with low copy number,and could be used for the accurate and quantitative detection of EHV-1. 相似文献
6.
本研究旨在建立一种能同时检测金黄色葡萄球菌(Staphylococcus aureus,S.aureus)、绿脓杆菌(Pseudomonas aeruginosa,P.aeruginosa)、肺炎克雷伯杆菌(Klebsiella pneumoniae,K.pneumoniae)的三重PCR检测方法.根据金黄色葡萄球菌nuc基因、绿脓杆菌toxR基因、肺炎克雷伯杆菌PhoE基因设计并合成引物,在单一PCR条件基础上优化建立三重PCR反应条件,并进行特异性、敏感性和重复性分析及与细菌分离培养的比对试验.结果显示,3对引物均能特异性扩增出目的条带,大小分别为484、278和368 bp.最佳退火温度在56~59 ℃之间,引物浓度均为0.2 μmol/L,dNTP浓度为200 μmol/L,Mg2+浓度为2.5 mmol/L.3种细菌间无交叉反应.对支气管鲍特杆菌等其他8种实验动物常见致病菌均无交叉反应.最低能同时检测到10-5 ng/μL的细菌基因组DNA.3次重复结果一致,表明建立的三重PCR方法重复性好.同时采用细菌分离培养法和三重PCR方法对30份实验小鼠样本进行检测,对比结果显示三重PCR方法检出率略高于细菌分离培养法,细菌分离培养法呈阳性的样品,三重PCR方法均能检出.结果表明,本试验建立的三重PCR检测方法具有特异、敏感、高效等优点,为实验动物细菌快速检测和流行病学调查提供了技术支持. 相似文献
7.
试验旨在建立一种快速检测猪流行性腹泻病毒（PEDV）的环介导等温扩增方法（LAMP）,为诊断PEDV提供简便、敏感、准确可靠的工具。参考GenBank中PEDV基因序列（登录号：KT799997）,针对PEDVN基因设计了6条引物,对所建立的LAMP反应体系、反应温度进行优化,建立可特异性扩增PEDV的LAMP方法。结果显示,本试验成功建立了PEDV LAMP检测方法,在60℃恒温下反应60 min,能特异性地检测PEDV,检测限量为91拷贝/μL,比常规PCR方法的敏感性高100倍。对比75份临床样本的LAMP和常规RT-PCR法检测结果,显示两种方法符合率为97.3%。综上所述,本试验建立的LAMP方法具有特异性强、敏感性高,操作简单,设备要求低的特点,适用于PEDV临床样本的快速检测。 相似文献
8.
1种检测新城疫病毒的微滴式数字PCR方法 总被引：1，自引：1，他引：0
本研究旨在构建一种新城疫病毒（Newcastle disease virus，NDV）的微滴式数字PCR方法（droplet digital PCR，ddPCR）。以NDV F基因为靶基因，选取保守区域设计引物和探针，构建了NDV ddPCR。对ddPCR反应引物和探针浓度、退火温度进行了优化，并对该方法的灵敏度、特异性和重复性进行测定。试验结果表明，ddPCR的最佳引物和探针浓度分别为900和250 nmol·L-1，最佳的退火温度为55℃。ddPCR的线性良好，最低检测下限为1.8 copies·μL-1，与禽传染性支气管炎病毒等其他6种病毒无交叉反应，样本重复的变异系数为2.4%。结果提示，本研究建立的ddPCR方法灵敏度高、特异性强，可对新城疫病毒感染的临床样品进行定量检测。 相似文献
9.
LUO Ya-kun LIANG Lin WANG Jing LIU Cun LIU Qi LIU Chang LIN Wen-cheng CUI Shang-jin 《中国畜牧兽医》2017,44(2):344-349
This study was aimed to establish a loop-mediated isothermal amplification (LAMP) assay for detection of porcine epidemic diarrhea virus (PEDV), and provide a simple, sensitive, accurate and reliable tool for diagnosis of PEDV.The conservative PEDV N gene (GenBank accession number: KT799997) of PEDV was selected as a target to design six specific primers.The reaction system and temperature of LAMP were optimized, and the LAMP method for specific amplification of PEDV was established. Results showed that the PEDV LAMP detection method was established successfully, and it could detect PEDV specifically at 60℃ for 60 min,and the detection limit was 91 copies/μL, which was one hundred-fold higher than conventional RT-PCR method.75 clinical samples were detected by LAMP and PCR, respectively, the coincidence of LAMP and PCR was about 97.3%. All the data suggested that the LAMP assay had strong specificity, high sensitivity, simple operation, low equipment requirement, and was suitable for rapid detection of PEDV clinical samples. 相似文献
10.
MEI Li WANG Yingchao WU Di LI Yue SONG Yanjun WEI Haitao WANG Lin GAO Xiaolong FENG Xiaoyu 《畜牧兽医学报》1956,51(12):3187-3192
This experiment was conducted to establish a droplet digital PCR (ddPCR) method for the detection of Newcastle disease virus (NDV). A ddPCR method was developed, which the primers and probes were designed based on the conservative regions of F gene of NDV. The concentration of primer and probe, the annealing temperature in ddPCR reaction were optimized. The sensitivity, specificity and reproducibility of ddPCR method were evaluated. In results, the optimal primer concentration and the probe concentration were 900 and 250 nmol·L-1, the optimum annealing temperature was 55℃. The detection limit of ddPCR method was 1.8 copies·μL-1 with a good linear response, it had no cross reaction with other six viruses (include IBV), the coefficient of variation of sample repetition was 2.4%. All the results showed that NDV ddPCR was sensitive and specific, it was suitable for quantitative detection of clinical samples infected with NDV. 相似文献