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为确定疑似禽霍乱病例病原种类及其病原基因型,本研究采用细菌分离技术对病原菌进行实验室分离培养,应用传统方法和分子生物学方法对分离细菌进行鉴定,并应用PCR扩增和基因序列分析对分离细菌进行基因分型。结果显示,分离菌具有多杀性巴氏杆菌（Pasteurella multocida,Pm）典型培养特征,菌落形态和菌体染色特征、生理生化特性均与多杀性巴氏杆菌相符;PCR扩增到457 bp的基因片段。采用5对分型引物对分离菌进行基因分型显示,仅有A型引物扩增到大小1 050 bp的目的基因片段,序列分析也显示分离菌荚膜基因与A型多杀性巴氏杆菌参考菌株荚膜特异性基因同源性高达97.6%~100.0%,系统进化与A型多杀性巴氏杆菌处于同一进化分支。结果表明,疑似禽霍乱病例病原为A型多杀性巴氏杆菌,本研究结果将为禽霍乱的防控提供参考资料。 相似文献
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为了克隆从患肺炎犊牛和健康犊牛上分离的多杀性巴氏杆菌血红素结合受体(heme acquisition system receptor,HasR)基因并进行序列分析,分别以致犊牛肺炎和健康犊牛携带的多杀性巴氏杆菌DNA为模板,通过PCR反应扩增出血红素结合受体基因的全部序列,PCR产物纯化后克隆到pMD19-T载体上,经菌液PCR和酶切鉴定后进行序列测定。各菌株HasR基因序列比对结果发现,Pm-BS-a、Pm142-x-4、Pm142-x-3、Pm149-x、Pm-BS-d的序列之间亲源关系较近,而Pm149-xby与参考菌株Pm70的同源性较高。健康牛源的多杀性巴氏杆菌与致犊牛肺炎多杀性巴氏杆菌之间HasR基因的同源性非常高,说明致犊牛肺炎多杀性巴氏杆菌是一种条件性致病菌,其引起的犊牛肺炎可能属内源性感染。 相似文献
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副猪嗜血杆菌、多杀性巴氏杆菌和胸膜肺炎放线杆菌多重PCR检测方法的建立及应用 总被引：1，自引：0，他引：1
在已建立的副猪嗜血杆菌(HPS)、多杀性巴氏杆菌(Pm)、胸膜肺炎放线杆菌(APP)的单重PCR检测方法的基础上,通过对扩增条件的优化,利用一次PCR反应可同时扩增出APP的256 bp、Pm的457 bp、HPS的822 bp的特异性片段,建立了HPS、Pm、APP的多重PCR实验室诊断方法。该复合PCR可检测60 pg的HPS、120 pg的Pm、50 pg的APP。利用该方法检测猪繁殖与呼吸综合征病毒(PRRSV)阳性猪体内分离到的39株细菌,结果显示HPS、Pm、APP分别为12、16、2株,对39份病料的检测与常规细菌分离鉴定结果一致,可见该方法适用于HPS、APP、Pm的临床快速检测。 相似文献
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从鲫鱼肠道中分离出1株细菌,暂时编号为SR-1,进行细菌形态学观察、理化特征、16S rDNA序列分析及药敏试验等研究,结果表明SR-1菌株为山梨醇发酵阳性的温和气单胞菌;PCR扩增SR-1菌株的16S rDNA序列为1509 bp;BLAST比对分析结果表明SR-1菌株与温和气单胞菌(Aeromonas sobria)亲缘关系最近,序列同源性为99.87%～100%;药敏试验结果显示SR-1菌株对阿奇霉素、氯霉素、四环素、诺氟沙星、头孢噻肟、头孢克肟等药物敏感。 相似文献
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The 16S r RNA gene of 49 streptococci of serological group B isolated from various origins was amplified by polymerase chain reaction (PCR) and subsequently digested with the restriction enzymes Rsa I and Msp I. The restriction profiles of all group B streptococci appeared to be identical indicating no intraspecies sequence variations of this gene. A fragment of the gene of two group B-streptococcal reference strains, including the hypervariable V2 region, could be amplified by PCR and sequenced. The sequence appeared to be identical and allowed the design of species-specific oligonucleotide primers. The primer pair used produced an amplicon with a size of 1250 bp and correctly identified all 49 group B-streptococci investigated but none of the control strains of various species and serogroups. This primer could be used in a multiplex PCR and allowed a rapid identification of bacteria of this species. 相似文献
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RT-PCR检测猪瘟病毒方法的建立与应用 总被引：7，自引：1，他引：6
建立RT PCR检测猪瘟病毒的方法。根据已发表的猪瘟病毒E2基因 (囊膜糖蛋白gP55基因 )序列 ,设计合成了一对特异性引物 ,扩增片段的大小为 50 7bp ,用RT PCR技术对石门系标准株和 1 0株分离株进行检测。结果这对引物对标准株和 1 0株分离株均能扩增出与预期大小相符 50 7bpRT PCR产物 ,而对其他 6种猪病病原核酸的扩增结果为阴性。该RT PCR可检出 1 0 0pg的猪瘟病毒RNA模板 ,对人工感染猪不同组织样品进行检测 ,结果对白细胞抽提的核酸样品检出率最高为 1 0 0 % (2 4 / 2 4 ) ,其次为扁桃体、脾、肾 ,检出率为 83 3 % (2 0 / 2 4 ) ,再者为淋巴结 ,检出率为66 7% (1 6/ 2 4 )。对送检的 1 9份疑似猪瘟的病死猪病料组织进行RT PCR检测 ,结果有 1 6份样品为猪瘟病毒阳性。兔体交叉反应试验结果RT PCR阳性的 1 6份病料中 ,有 1 4份样品被判为含有猪瘟病毒 ,其他病料兔体交叉反应试验结果全为阴性 相似文献
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鹅细小病毒VP3基因真核表达载体的构建及其在Vero细胞中表达 总被引：2，自引：0，他引：2
根据已发表的鹅细小病毒(GPV)B株核苷酸序列,设计并合成了1对引物,PCR扩增GPV吉林分离株主要结构蛋白VP3基因,获得大小为1605bp的核苷酸片段。将该片段纯化后克隆入pMD18-T载体,转化感受态大肠杆菌JM109,双酶切鉴定,筛选阳性重组质粒并测序。经过酶切和连接反应,将VP3基因克隆入真核表达载体pVAX1,转化感受态大肠杆菌DH5α,筛选阳性克隆,提取质粒,进行了PCR和酶切鉴定。通过脂质体法将pVAX1-VP3转染Vero细胞,RT-PCR和间接免疫荧光法检测。结果显示,VP3基因克隆成功,与GPVB株核苷酸序列同源性为96.2%;PCR和酶切鉴定结果证实,成功构建了含VP3基因的GPV真核表达载体pVAX1-VP3。提取转染该质粒的Vero细胞RNA,RT-PCR扩增,在1000～2000bp可见一明显DNA条带;间接荧光抗体染色转染细胞,在细胞表面可见特异荧光。 相似文献
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Identification and detection of Actinobacillus pleuropneumoniae by PCR based on the gene apxIVA 总被引：5，自引：0，他引：5
Schaller A Djordjevic SP Eamens GJ Forbes WA Kuhn R Kuhnert P Gottschalk M Nicolet J Frey J 《Veterinary microbiology》2001,79(1):47-62
The apxIVA gene, a recently discovered RTX determinant of Actinobacillus pleuropneumoniae, was shown to be species-specific. DNA hybridization experiments using probes for various regions of apxIVA revealed that the 3'-terminus of this gene was present in all 14 serotypes of A. pleuropneumoniae but absent from phylogenetically related species. A primer pair spanning this region specifically amplified a 422bp fragment in PCR experiments with DNA from the reference strains of the 14 serotypes and 194 field strains isolated from various geographic locations worldwide. DNA sequence analysis of PCR products derived from all serotypes were identical except in serotypes 3, 8, and 10, which showed minor differences. The PCR did not amplify any product when DNA from 17 different bacterial species closely related to A. pleuropneumoniae was used as template. In addition, the PCR was negative with DNA of several Actinobacillus sp. which were initially characterized as A. pleuropneumoniae using routine phenotypic and serological analyses but which were subsequently shown by 16S rRNA sequence analysis to belong to yet undefined Actinobacillus species. The sensitivity of the PCR was determined to be 10pg of A. pleuropneumoniae DNA. A set of nested primers amplified a 377bp fragment specifically with A. pleuropneumoniae DNA. DNA titration experiments using the flanking and nested primer pairs showed an improved level of sensitivity to approximately 10fg of genomic DNA. The nested PCR was used to monitor the spread of A. pleuropneumoniae in pigs experimentally infected with a virulent serotype 1 strain and housed in a controlled environment facility. A. pleuropneumoniae DNA could be detected by nested PCR in nasal swab samples of infected pigs receiving either a high dose (5x10(5)) or a low dose (1x10(4)) challenge and in unchallenged cohorts that were contact-infected by the inoculated animals. Furthermore, PCR confirmed the presence of A. pleuropneumoniae in 16/17 homogenates from necrotic lung lesions, while the bacterium was successfully recovered from 13 of these lesions by culture. 相似文献
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为鉴定临床疑似鸭多杀性巴氏杆菌感染肉鸭的病原菌，本试验通过细菌分离培养、菌体形态观察、细菌生化鉴定、16S rRNA基因测序分析、细菌种特异性鉴定、荚膜分型鉴定和动物回归试验进行鉴定，并通过药敏试验和耐药基因检测进行耐药性分析。结果显示，从患病鸭肝脏组织分离到的细菌在鲜血琼脂培养基中呈现表面光滑凸起、灰白色菌落，为革兰氏阴性短小杆菌，瑞氏染色呈两极浓染；生化鉴定结果显示，分离菌能发酵葡萄糖、蔗糖和甘露醇，硫化氢、氧化酶和吲哚等试验阳性；16S rRNA基因序列系统进化树分析显示，该分离菌与多杀性巴氏杆菌聚为一支，同源性 > 99%；细菌种特异性鉴定结果与多杀性巴氏杆菌相符；荚膜分型鉴定结果仅扩增到约为1 050 bp的目的基因片段，与荚膜血清A型相符；动物回归试验显示，该分离菌有较强的致病性；药敏试验结果显示，该分离菌对羧苄西林、氨苄西林、复方新诺明和四环素等12种药物耐药；经耐药基因PCR检测显示，该分离菌携带Sul1、Sul3、tet（X）和Intl1 4种耐药基因，与药敏表型相符。本试验成功分离到1株鸭源荚膜血清A型多杀性巴氏杆菌，为鸭多杀性巴氏杆菌病的防治提供参考依据。 相似文献
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为研究牛源多杀性巴氏杆菌（Pasteurella multocida，Pm）Oma87基因的分子特征及同源性，本研究对荚膜血清A型（capsular serotype A，Pm8）Oma87基因进行扩增、克隆和测序，利用在线软件对其进行分子特征、抗原性及遗传进化分析。结果显示，Oma87基因全长为2 376 bp，编码791个氨基酸；氨基酸序列分析发现，该蛋白为酸性的亲水性蛋白，稳定性高，具有信号肽结构但不存在跨膜区域；二、三级结构分析发现，该蛋白为无规则卷曲及多段延伸链所形成的"N"字型三维立体结构。遗传进化分析显示，新疆分离株Pm8 Oma87基因与其他不同地域的牛源A型Pm和部分猪源A型Pm的同源性较近，与其他型Pm的同源性较远；通过VaxiJen软件分析发现，Oma87蛋白的保护性抗原的总体预测值为0.5478，高于正常阈值0.4，证明Oma87蛋白可作为免疫原性蛋白，为进一步研究Oma87蛋白奠定了理论基础。 相似文献
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XU Li-pu WANG Jing-bo ZHANG Wen WANG Xiao-liang CAO Huan WANG Shu PAN Yong JIANG Yu-lin 《中国畜牧兽医》2015,42(6):1592-1596
A chronic and infective disease occurred on the farmed Chinook salmon (Oncorhynchus tshaivytscha) in a farm in Huairou district of Beijing and it caused high accumulative mortality.The typical clinical symptoms indicated that it might be bacterial kidney disease (BKD).The purpose of this study was to confirm its pathogen.The typical clinical symptoms of sick fish,morphology observation of bacteria in kidney tissue smears and sequence analysis of bacteria DNA were conducted.A number of gram positive brevi bacteria were observed in kidney tissue print.16S rDNA sequence of K1 strain was more than 99% homology with that of Renibacterium salmoninarum and they were in the same cluster.383 and 320 bp fragment were amplified by PCR and nested PCR.The fragments were sequenced and analyzed.The results showed the fragment was shared 100% nucleotide identity with P57 surface protein gene of Renibacterium salmoninarum. It was indicated that these diseased fish were suffering from BKD caused by Renibacterium salmoninarum.This was the first report about Renibacterium salmoninarum detected in farmed fish in China. 相似文献
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北京怀柔某养殖场的大鳞大麻哈鱼发生一种慢性,但累计死亡率很高的流行病,为研究大鳞大麻哈鱼感染病原菌的种类,本试验根据病鱼临床症状观察判断疑似感染鲑肾杆菌.试验对病鱼肾脏组织进行印片染色观察细菌形态特征,抽提病鱼的肝脏、肾脏组织DNA,扩增16S rDNA和鲑肾杆菌P57表面蛋白基因片段,测序并运用BLAST进行同源性比对.结果显示,病鱼肾脏组织印片在显微镜下可见大量形态一致的革兰氏阳性短杆菌;采用病鱼肝脏、肾脏组织扩增的16S rDNA基因构建的系统发育树显示与鲑肾杆菌聚为一支;通过PCR扩增出鲑肾杆菌P57表面蛋白基因DNA片段,测序结果与鲑肾杆菌标准株ATCC 33209完全一致,符合率为100%.结果表明病鱼感染鲑肾杆菌并引发细菌性肾病(BKD).这是中国首次在养殖鱼体内检出鲑肾杆菌. 相似文献
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9株鸡毒支原体29 Ku多肽基因的克隆与序列分析 总被引：4，自引：0，他引：4
根据已发表的鸡毒支原体(MG)S6株29Ku多肽基因序列设计了1对引物,以9株(广西分离株5株、标准株4株)DNA为模板进行PCR扩增,均得到802bp的特异性片段,将9株MG PCR产物纯化后克隆到pMD18-T载体上,得到重组质粒.重组质粒经PCR法和EcorⅠ、SalⅠ双酶切等方法鉴定后,测定了9株29 Ku多肽基因序列,并在基因库中S6标准株的29 Ku多肽基因序列进行分析比较.结果表明,5株分离株与5株标准株29Ku多肽基因核苷酸序列同源性分别为94.4%～99.9%,推导的氨基酸同源性分别为89.7%～99.2%.从各毒株的进化分析表明,5个分离株与标准强毒株S6、A5969、K1501和PG31强毒株间遗传距离较近,而5个分离株与标准株F疫苗株间遗传距离则较远. 相似文献