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1.
为建立一种可准确、快速鉴定畜禽临床病例中3种常见革兰氏阴性杆菌的多重PCR检测方法,本试验针对大肠埃希菌23S rRNA基因、巴氏杆菌KMT基因和沙门氏菌invA基因分别设计合成了1对特异性引物,构建可同时快速鉴别大肠埃希菌、巴氏杆菌和沙门氏菌的多重PCR反应体系,并进行反应条件优化及方法性能评估。结果显示,所建立的方法最佳引物浓度分别为1.0、1.5和1.0 μmol/L,最佳退火温度为56 ℃。性能评价结果显示,所建立的多重PCR检测方法具有较好的特异性和敏感性,其中对沙门氏菌检测敏感性最高,为1.44 pg/μL。对70株革兰氏阴性临床分离细菌进行检测,结果表明,所建立的多重PCR检测方法能实现3种临床分离细菌的快速准确鉴定。本方法的建立为相关临床病例快速诊断及流行病学调查提供了有效的技术支持。 相似文献
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本研究旨在建立一种能够同时鉴别包括大肠杆菌O157：H7、沙门菌、空肠弯曲菌、单核细胞增生李斯特菌、霍乱弧菌、副溶血弧菌和金黄色葡萄球菌7种常见食源性致病菌的GeXP多重PCR检测方法。根据这些致病菌在GenBank上公布的保守基因序列,设计合成了7对特异性GeXP引物。用单一或混合细菌样品的DNA模板优化反应条件,设置对照组,构建重组质粒,随机组合不同浓度的样品,验证所建立的GeXP方法的特异性、敏感性和准确性。最后用该方法检测120份临床样品,进一步验证所建立的GeXP检测方法的准确性和可靠性。结果显示,单一或混合模板的GeXP检测均能特异性出现相应清晰峰值,可在103拷贝·μL-1水平上同时特异地检测出7种细菌病原体,不同浓度模板混合时,本试验所建立的方法依然可检测出对应病原体。检测120份临床样品,GeXP多重PCR阳性率为2.50%（3/120）~15.83%（19/120）,普通PCR阳性率为2.50%（3/120）~15.00%（18/120）,GeXP多重PCR多检出8份阳性,表明GeXP方法更为敏感与准确。本研究建立的同时鉴别7种食源性致病菌的GeXP多重PCR检测方法,具有高通量、特异性强和敏感性高的特点,为食源性常见致病菌感染或混合感染提供了快速分子诊断方法。 相似文献
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ZHANG Yanfang XIE Zhixun ZENG Tingting LIU Jiabo XIE Liji DENG Xianwen XIE Zhiqin LUO Sisi HUANG Jiaoling WANG Sheng 《畜牧兽医学报》1956,51(10):2536-2546
This experiment was developed to simultaneously detect the seven common foodborne pathogenic bacterias include E. coli O157:H7, Salmonella, V. cholera, L. monocytogenes, C. jejuni, V. Parahemolyticus and S. aureus. Seven pairs of specific primers were designed according to the conserved sequences of the genes from each pathogen available in the GenBank database. Single and mixed pathogen DNA templates were used to evaluate the specificity of the GeXP-multiplex assay. Control group was set up, recombinant plasmids were constructed, and samples of different DNA concentrations were randomly combined to verify the sensitivity, specificity, accuracy and anti-interference of the established GeXP method. To certify the accuracy and reliability of the GeXP assay, it was evaluated using 120 clinical specimens that were compared with the single PCR. The obtained results showed that the corresponding specific fragments of genes were amplified by the single and the multiplex GeXP PCR assay. The detection limit of GeXP was 103 copies·μL-1 when all of seven bacterial pathogens were detected. The results of the interference assay showed the presence of specific amplification peaks when different templates. The detection rate of the GeXP multiplex PCR method was 2.50% (3/120)-15.83% (19/120) while the conventional PCR was 2.50% (3/120)-15.00% (18/120), and GeXP multiple PCR detected 8 more positive cases, which means that, the GeXP was more sensitive and accurate in the detection of the clinical samples. In conclusion, this GeXP-based multiplex PCR is a high-throughput, specific and sensitive test to detect seven common foodborne pathogenic bacterias. This assay provides a method in rapid molecular diagnosis for mix clinical seven common foodborne pathogenic bacterias. 相似文献
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张耀东 朱红 AFAYIBO Doss&# h Jean Ap&# tre 王瑶 易正飞 信素华 陶程琳 李涛 祁晶晶 田明星 丁铲 于圣青 王少辉 《畜牧兽医学报》2020,51(12):3193-3198
本研究旨在建立β-内酰胺类、四环素类、氨基苷类、酰胺醇类、磺胺类抗菌药物耐药基因的多重PCR检测方法，用于耐药基因的快速检测。根据GenBank公布的上述5类抗菌药物的耐药基因序列，设计17对特异性引物。通过优化PCR体系和反应程序，建立4组耐药基因（cat+floR+tetB+tetC；dfrA12+sul2+sul1+blaCTX-M+balTEM-1；aac3+aph3+aadA1+strB；tetA+cmlA+strA+sul3）的多重PCR反应体系。然后，检测多重PCR方法的特异性和敏感性。利用建立的多重PCR方法检测42株禽致病性大肠杆菌的耐药基因，同时，检测其耐药性，比较分析耐药基因和耐药表型之间的相关性。结果显示，建立的4组多重PCR体系可有效扩增出17个耐药基因片段，测序结果表明特异性较好。敏感性结果表明，4组多重PCR体系的菌液敏感性分别为103、104、104和105CFU。禽致病性大肠杆菌分离株的耐药基因多重PCR和单重PCR检测结果一致，其携带的耐药基因和耐药表型的符合率为92.86%。本研究建立的耐药基因多重PCR方法能简便、快速地检测常见的耐药基因，可用于耐药基因的传播、流行调查。 相似文献
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ZHANG Yaodong ZHU Hong AFAYIBO Doss&# h Jean Ap&# tre WANG Yao YI Zhengfei XIN Suhua TAO Chenglin LI Tao QI Jingjing TIAN Mingxing DING Chan YU Shengqing WANG Shaohui 《畜牧兽医学报》1956,51(12):3193-3198
To detect the drug resistance genes, the multiplex PCR method for drug resistance genes of β-lactams, tetracyclines, aminoglycosides, amphenicols, and sulfonamides were developed. Based on the sequences of drug resistance genes from GenBank, 17 pairs of specific primers were designed. Then, 4 multiple PCR assays were established through the optimization of PCR reaction conditions and primers concentrations (cat+floR+tetB+tetC; dfrA12+sul2+sul1+blaCTX-M+balTEM-1; aac3+aph3+aadA1+strB; tetA+cmlA+strA+sul3). The sensitivity and specificity of these assays were determined. The multiplex PCR assays were used to detect the drug resistance genes of 42 avian pathogenic Escherichia coli (APEC). The drug sensitivity of these APEC strains were also determined, which were compared to the distributions of drug resistance genes in these strains. The results showed that the 17 drug resistance genes were effectively and specifically amplified in these 4 optimized multiplex PCR assays. The detection limits of the 4 multiplex PCR were 103, 104, 104 and 105CFU of bacteria, respectively. The established multiplex PCR assays are specific and rapid for the detection of drug resistance genes in APEC strains, which showed 92.86% coincident with the drug resistance for these strains. The developed 4 multiplex PCR are simple and rapid assays for drug resistance genes detection, which can be used for the epidemiologic study for drug resistance genes. 相似文献
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Boes J Alban L Bagger J Møgelmose V Baggesen DL Olsen JE 《Preventive veterinary medicine》2005,69(3-4):213-228
A pilot study was carried out on a Danish swine farm infected with multi-resistant Salmonella Typhimurium DT104 (MRDT104). We aimed to (1) investigate to which degree the decline of Escherichia coli and Salmonella in swine slurry applied to farmland depended on the application method; (2) estimate the survival times of E. coli and Salmonella in the soil surface following deposition of naturally contaminated pig slurry; and (3) simulate survival of Salmonella in different infection levels using E. coli data as input estimates. Slurry was deposited by four different methods: (1) hose applicator on black soil followed by ploughing and harrowing; (2) hose applicator on black soil followed only by harrowing; (3) hose applicator on a field with winter-wheat seedlings without further soil treatment; (4) slurry injector on a field with winter-wheat seedlings without further soil treatment. E. coli and Salmonella could not be detected at all in soil following treatment 1. Following the other treatments, E. coli was not detected in soil samples after day 21 and Salmonella was no longer detected after day 7. Simulation results showed that clinical (4 log CFU g−1) and sub-clinical Salmonella levels (2500 CFU g−1) would fall below the detection limit within 10 or 5 days, respectively. Analysis of samples from 62 Danish MRDT104-infected swineherds showed that nearly 75% of these herds had low levels of MRDT104 (<10 CFU g−1) in their slurry. Our results show that ploughing and harrowing of soil amended with contaminated pig slurry was an effective means to reduce environmental exposure to E. coli and Salmonella on this clay-soil farm. 相似文献
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牛诺如病毒实时荧光定量PCR检测方法的建立及应用 总被引：1，自引：1，他引：0
牛诺如病毒（BNoV）是国内新发的犊牛腹泻病原,笔者拟建立检测BNoV的Real-time PCR方法。根据BNoV流行株的RNA聚合酶（RdRp）基因序列设计引物,通过优化反应条件和体系,成功建立基于EvaGreen检测BNoV的Real-time PCR方法。该检测方法的Ct值与标准品模板在2.24×102~2.24×108拷贝·μL-1线性关系良好,相关系数R2=0.997,扩增效率为98.44%;该方法可特异性检出BNoV,对其他犊牛腹泻相关病原呈阴性;最低检测限为22.4拷贝·μL-1;批间和批内的变异系数均小于2%,重复性好。对2017年9月-2019年5月采自河南省的221份犊牛腹泻样本中BNoV的检出率为11.31%（25/221）,采样场阳性率为92.86%（13/14）。本试验所建方法灵敏度高、特异性强、稳定性好,为BNoV的检测和流行病学调查提供了有力手段。 相似文献
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本研究旨在构建1种布鲁氏菌（Brucella）微滴式数字PCR方法（droplet digital PCR，ddPCR）。以布鲁氏菌BCSP31基因为靶基因，选取保守区域设计引物和探针，构建了布鲁氏菌微滴式数字PCR方法。然后优化了ddPCR反应中的引物、探针浓度及退火温度，并进行方法的灵敏度、特异性和重复性试验。结果表明，ddPCR的最佳引物和探针浓度分别为900 nmol·L-1和250 nmol·L-1，最佳的退火温度为58℃。ddPCR的线性良好，最低检测下限为1.12 copies·μL-1，与大肠杆菌O：157菌株等其他4种细菌无交叉反应，而且批内重复性和批间重复性都较好。综上表明，本研究建立的ddPCR方法灵敏度高、特异性强，可对布鲁氏菌感染的临床样品进行定量检测。 相似文献
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ZHANG Xun WU Peng LI Shi-zhen FU Yin-feng XU Zhui LIANG Tian YANG Su-fang CHEN Xin-kai QI Ya-yin SHENG Jin-liang 《中国畜牧兽医》2015,42(10):2612-2618
The aim of this study was to establish a multiplex PCR method for simultaneously detecting porcine reproductive and respiratory syndrome virus (PRRSV),porcine circovirus type 2 (PCV2),porcine pseudorabies virus (PRV) and Mycoplasma hyopneumoniae (Mh).Four pairs of primers were synthesized according to the reference.The multiplex PCR method was developed by optimizing the reaction condition,specificity and sensitivity detection.Four different amplicons with size of 424,490,298 and 360 bp for PRRSV,PCV2,PRV and Mh,respectively,were yielded.The sensitivity of multiplex PCR indicated that the detection limit was 3 copies by using Multiplex PCR Master Mix,and other common pathogens were not amplified.A total of 60 specimens from piglets were tested by multiplex PCR method.The positive accordance rate between simple and multiplex PCR was 100%.This study indicated that multiplex PCR might be a useful tool for rapid and sensitive etiological diagnosis and provided an effective technical support for pathogenic molecular epidemiology investigation. 相似文献
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新疆地区4种猪病多重PCR检测方法的建立与初步应用 总被引：1，自引：0，他引：1
本试验旨在建立一种可同时检测猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)、猪圆环病毒2型(porcine circovirus type 2,PCV2)、猪伪狂犬病病毒(porcine pseudorabies virus,PRV)、猪肺炎支原体(Mycoplasma hyopneumoniae,Mh)的多重PCR检测方法。参考相关文献合成PRRSV、PCV2、PRV、Mh的特异性引物,通过对反应条件优化、特异性、敏感性测定,建立了可同时检测以上4种猪多发传染病的PCR诊断方法。扩增的片段长度分别为424 bp (PRRSV)、490 bp (PCV2)、298 bp (PRV)和360 bp (Mh),对其他常见猪病病原无特异性扩增,采用Multiplex PCR Master Mix对PRRSV、PCV2、PRV、Mh的核酸最低检出量为3个拷贝。采用多重PCR方法对60份临床样本反复检测,与单重PCR相比,4种病原符合率均为100%。结果表明,建立的多重PCR诊断方法可用于猪群中上述4种病原的单一或混合感染的快速鉴别诊断和流行病学调查。 相似文献
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本研究旨在调查山东地区鸡源沙门氏菌的流行情况及毒力基因分布。在山东地区规模化养鸡场采集病料,利用四硫磺酸钠煌绿增菌液（TTB）进行沙门氏菌选择增菌,并对分离菌株进行纯化培养、革兰氏染色、生化试验、培养基菌落形态鉴定,利用沙门氏菌属及血清型特异性引物对分离菌进行PCR扩增鉴定,并利用PCR方法检测分离菌中33种毒力基因分布。结果显示,分离菌在LB固体培养基和麦康凯培养基上呈无色半透明、光滑且边缘整齐的菌落,镜检为革兰氏阴性、两端钝圆的小杆菌。生化试验、培养基菌落形态鉴定、沙门氏菌属及血清型特异性引物PCR扩增结果显示,共分离到25株鸡源沙门氏菌,其中肠炎沙门氏菌9株,鸡白痢沙门氏菌和鼠伤寒沙门氏菌各8株。毒力基因检测结果显示,33种毒力基因在25株分离菌中均有检出,其中20种毒力岛基因（invJ、virK、sopA、mogA、hilA、hisJ、ssaB、ssaQ、ssiD、misL、mgtC、orf319、siiE、siiD、bcfA、pipC、sopB、araB、spoB和avrA基因）、肠毒素基因（stn基因）及菌毛毒力基因（fimA基因）检出率均为100%;2种毒力岛基因（sipA和sodC基因）及4种毒力质粒基因（spvA、spvC、spvD和spvR基因）检出率均为96%,spoE基因检出率为68%,fliC基因检出率为36%,gipA与spvB基因检出率均为32%,sseL基因检出率为4%;25株鸡源沙门氏菌中,分别携带31种（8株）、30种（1株）、29种（15株）和24种（1株）毒力基因;毒力基因gipA和spvB只在鼠伤寒沙门氏菌中检出,且在鼠伤寒沙门氏菌中检出率为100%。本研究结果表明,山东地区鸡源沙门氏菌主要为肠炎沙门氏菌、鸡白痢沙门氏菌和鼠伤寒沙门氏菌,分离菌皆携带多种毒力基因,其中gipA和spvB毒力基因只在鼠伤寒沙门氏菌中检出,可作为鼠伤寒沙门氏菌鉴定的备选基因之一。 相似文献
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研究旨在克隆鼠伤寒沙门菌cya基因并对其进行生物信息学分析。以鼠伤寒沙门菌SL1344株为模板PCR扩增并克隆了鼠伤寒沙门菌cya基因,构建原核表达质粒pET-32a-cya,转化大肠杆菌BL21（DE3）感受态细胞,用1 mmol/L IPTG对其进行诱导表达、纯化,同时利用相关的生物信息学软件对其进行生物信息学分析。结果显示,鼠伤寒沙门菌cya基因大小为1 185 bp。经SDS-PAGE和Western blotting分析显示,在原核表达系统中主要以包涵体形式存在,蛋白分子质量大小约为58 ku。生物信息学分析表明,Cya蛋白由394个氨基酸组成,理论分子质量为44.97 ku,分子式为C2026H3146N562O578S11,等电点（pI）为7.01,其中带负电荷残基总数（Asp+Glu）43个,带正电荷残基总数（Arg+Lys）42个。Cya蛋白无信号肽与跨膜区,为膜内蛋白,含有4个N-糖基化位点、1个O-糖基化位点、27个磷酸化位点、13个线性B细胞结合位点、11个T细胞结合位点。二级结构中α-螺旋、延伸链、β-转角、无规则卷曲所占比例分别为38.07%、19.80%、5.58%和36.55%。本试验结果为进一步研究鼠伤寒沙门菌Cya蛋白的功能及建立以Cya蛋白为抗原的免疫鉴别检测方法奠定了基础。 相似文献
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为建立适用于羊源多杀性巴氏杆菌的重组酶聚合酶扩增（RPA）诊断方法,本研究依据前期测序鉴定的羊源多杀性巴氏杆菌KMT1基因序列构建pET-28a （＋）-KMT1质粒标准品。设计基于RPA技术的特异性引物及RPA荧光探针,建立实时荧光RPA方法的最适反应体系。采用10倍梯度连续稀释的质粒标准品检测该RPA方法的敏感性并绘制相关性曲线;以10种不同菌株的基因组DNA为模板验证方法的特异性;用感染多杀性巴氏杆菌的山羊及小鼠组织样品对方法的可靠性进行验证。结果显示,本试验建立的实时荧光RPA方法最适反应温度为39 ℃,最佳引物为KMT1-Fe1,灵敏度达100拷贝/μL,检测下限为10拷贝/μL。与大肠杆菌、金黄色葡萄球菌、副伤寒沙门氏菌、副溶血弧菌、绿脓杆菌、产酸克雷伯菌、布鲁氏菌S2株和鲍曼不动杆菌均无交叉反应。对13个组织样本进行检测,阳性率为76.9%,与实时荧光定量PCR检测结果的符合率达92.3%。综上所述,本研究建立的羊源多杀性巴氏杆菌实时荧光RPA方法具有特异性强、灵敏度高、可靠、快速便捷等特点,适用于多杀性巴氏杆菌的临床分子诊断。 相似文献
15.
本试验旨在建立一种乳品中大肠杆菌PMA-qPCR活菌检测方法.优化qPCR检测方法,探究菌浓度为1×108 CFU/mL的大肠杆菌活菌悬液、热致死菌悬液细胞数来确定不同的PMA剂量、暗孵育时间、曝光时间对死菌抑制效果的影响,确定最佳PMA处理方案.结果表明,qPCR检测可特异性扩增大肠杆菌,1×108 CFU/mL的大肠杆菌经90 ℃水浴30 s全部致死后,采用10 μg/mL的 PMA暗孵育15 min后冰上曝光10 min为最佳处理方案,这种处理方案可最大程度抑制死细胞信号,而对活细胞几乎没有影响,样品中微生物初始浓度不低于1×108 CFU/mL时较稳定,得到标准曲线回归方程y=-3.356x+47.413,R2=0.9989,最低检测限为103 CFU/mL,加标样本检测结果与实际相符.该方法为利用PMA-qPCR检测食品中的活大肠杆菌杆菌奠定了基础. 相似文献
16.
GAI Dong-xue REN Hong-lin LU Shi-ying HU Pan MENG Xian-mei LIU Xi SONG De-gang JIN Wen ZHANG Song CHANG Jiang LIU Yan-yan LIU Zeng-shan 《中国畜牧兽医》2016,43(2):493-498
In order to detect viable E.coli in milk,a new PMA-qPCR method was established.The influences of different PMA concentration,dark incubation time,exposure time on dead bacteria inhibition effect were determined by detection of the cell numbers of viable and heat-killed E.coli suspensions at concentration of 1×108 CFU/mL through fluorescence quantitative PCR (qPCR) method.The results showed that qPCR assay could specifically detect E.coli,and the viable E.coli must be exposed to 90 ℃ for 30 s in water bath to be lethal.The best treatment was 10 μg/mL PMA with 15 min of dark incubation time and 10 min of exposure time.This treatment could inhibit dead cell signals to a largest extend,while had little impact on aviable cells.The stability of PMA-qPCR assay was kept while the concentration of bacteria was more than 1×108 CFU/mL.The regression equation of standard curve was y=-3.356x+47.413,R2=0.9989,the lowest detection limit was 103 CFU/mL.The result of adding assay was agreed with the actual situation.This study laid a foundation for using of PMA-qPCR to detect the viable E.coli in food. 相似文献
17.
为了检测猪伪狂犬病病毒(pseudorabies virus,PRV)的感染情况,并进行强弱毒株的鉴别诊断,本研究建立了快速、简便、灵敏度高、特异性强的鉴别猪PRV的双重PCR方法。针对PRVgE和gB基因序列,分别设计了2对特异性引物,通过对退火温度(50~60℃,按照1℃递增)、引物浓度(0.2~1.4μL,依次增加0.2μL)的优化,结果表明,双重PCR反应的最佳退火温度为56℃、最适引物添加量为1μL。特异性试验结果表明,该方法可以扩增出PRVgE(316bp)和gB(432bp)的目的片段,对PCV2、PTV、CSFV、PPV、PRRSV、大肠杆菌的DNA或cDNA均无扩增。敏感性试验结果表明,PRVgE和gB的最低核酸检出量分别为4.4×103和3.3×103拷贝/μL,与单一PCR方法的敏感性相近。应用该方法对广东、广西地区临床送检的56份组织样品进行检测,检测结果显示,强毒感染阳性率为53.6%(30/56),阴性率为46.4%(26/56),未发现有弱毒感染。 相似文献
18.
本研究旨在建立能同时检测猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)、伪狂犬病病毒(PRV)、猪细小病毒(PPV)、猪圆环病毒2型(PCV2)、坏死梭杆菌(Fn)和副猪嗜血杆菌(Hps)7种猪场常见高致死性流行病原的多重PCR检测方法。利用7种病原体的保守序列设计7对特异性引物,同时合成了Cy-5标记的通用引物。将通用引物分别连接到特异性引物的5′端形成7对特异性嵌合引物。优化反应条件,分别使用7组嵌合引物和通用引物混合,扩增7种病原的混合cDNA/DNA,验证其单重PCR的特异性。利用GeXP多重基因表达遗传分析系统,混合7种嵌合引物和通用引物,扩增单一病原的cDNA/DNA,验证其多重PCR特异性;将其他常见猪病病原的基因组作为干扰的阳性标本,利用7对混合嵌合引物和通用引物进行多重PCR分析,扩增加入了阳性标本的混合模板,验证其多重PCR的抗干扰能力。利用重组质粒和体外转录的RNA进行梯度稀释,确定GeXP多重检测体系的灵敏度。结果表明,7种不同引物分别进行GeXP单重及多重检测,均能检测出特异性目的片段的信号,无明显的干扰片段信号出现;GeXP多重检测抗干扰试验结果显示,在混入3种干扰病原模板后,依然可同时特异性检测出7种病原;GeXP多重检测灵敏度分析显示,在10~3拷贝/μL浓度条件下能检测到7种不同基因的特异性结果。本研究建立的同时检测7种猪场常见高致死性流行病原的GeXP检测方法具有高通量、高特异性和高灵敏度的特点,为快速诊断猪流行性疾病的交叉感染和混合感染提供了新型的检测方法。 相似文献
19.
【目的】试验旨在获得高效表达的鼠伤寒沙门菌SptP蛋白并进行生物信息学分析,为其功能研究和互作蛋白的筛选提供理论依据。【方法】通过PCR技术扩增SptP基因,并将该序列连接至pET-32a （+）载体,构建鼠伤寒沙门菌SptP基因原核表达载体pET32a-SptP。通过热激法将重组质粒导入大肠杆菌BL21（DE3）感受态细胞后经IPTG诱导表达、纯化重组蛋白,并经过SDS-PAGE和Western blotting验证;应用在线软件对SptP蛋白进行生物信息学分析。【结果】PCR成功扩增出大小为1 632 bp的SptP基因。SptP重组蛋白在大肠杆菌BL21（DE3）感受态细胞中成功诱导表达、纯化,得到分子质量为79.7 ku的蛋白。SptP蛋白分子式为C2625H4257N745O812S25,分子质量为60 047.68 u,无跨膜结构,无信号肽存在,理论等电点为8.75,有57个潜在的磷酸化位点,主要定位于细胞核、细胞质、高尔基体、细胞骨架、分泌系统的囊泡、质膜,占比分别为43.5%、34.8%、8.7%、4.3%、4.3%和4.3%。SptP蛋白二级结构由α-螺旋、延伸链、β-转角及无规则卷曲组成,占比分别为43.65%、14.92%、4.42%和37.02%。【结论】本研究构建了表达SptP蛋白的重组质粒pET32a-SptP,获得分子质量为79.7 ku的SptP重组蛋白,阐明了SptP蛋白的基本理化性质和生物学功能,为后续SptP蛋白与宿主细胞互作的作用机制及鼠伤寒沙门菌新疫苗的制备提供理论基础和试验依据。 相似文献
20.
In order to detect the procine pseudorabies virus (PRV) infection and differentiate virulent and avirulent PRV, a duplex PCR method was established with two pairs of specific primers designed based on the conserved sequences of gE and gB genes of PRV.Optimize the reaction conditions by adjusting concentration of primers from 0.2 to 1.4 μL, each time adding 0.2 μL and temperature of annealing from 56 to 60 ℃, added 1 ℃ each time, and so on.The results showed that the suitable annealing temperature was 56 ℃ and the suitable primer dose was 1 μL.Two special fragments of 316 bp (PRV gE) and 432 bp (PRV gB) were amplified by the optimized duplex PCR.The specific tests showed that no PCR products were detected for PCV2, PTV, CSFV, PPV, PRRSV and E.coli.The sensitivity test showed that DNA used for the duplex PCR might be as low as 4.4×103 copy/μL for PRV gE and 3.3×103 copy/μL for PRV gB.The sensitivity of the duplex PCR was close to the single PCR.Fifty-six clinical samples of sick pigs from different areas in Guangdong and Guangxi province were detected by the duplex PCR and the virulent detection rate was 53.6% (30/56) for PRV and the negative rate was 46.4%(26/56), and there was no attenuated PRV infection. 相似文献