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《中国兽医杂志》2019,(8)
分析不同血清型/生物型沙门菌全基因组序列筛选出鸡白痢和鸡伤寒沙门菌特异性基因组序列,设计两对引物,建立双重PCR方法鉴别检测鸡白痢和鸡伤寒沙门菌不同生物型,并进行初步的临床应用。双重PCR方法结果显示,鸡白痢沙门菌显示417 bp条带,鸡伤寒沙门菌显示417 bp和636 bp两个条带,而阴性对照未出现条带,与预期设计相符。双重PCR体系对56株不同血清型沙门菌鉴定结果与细菌学分离的血清型鉴定结果完全一致,说明本试验建立的双重PCR体系特异性良好,应用上述方法检测鸡场疑似20份临床样本,结果发现8株鸡白痢沙门菌阳性,1株鸡伤寒沙门菌阳性。上述结果表明,已建立双重PCR方法特异性检测鸡白痢和鸡伤寒沙门菌。本试验为鸡白痢和鸡伤寒不同生物型沙门菌的检测提供了一种简洁、敏感、特异的新方法。 相似文献
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参照GenBank公布的鸡白痢沙门菌与鸡伤寒沙门菌fimH基因序列,根据两者在第37和第544位碱基的不同,设计了一对等位基因特异性引物,建立了一种鸡白痢沙门菌等位基因特异性PCR(AS-PCR)检测方法,并应用该方法对鸡白痢沙门菌临床样品进行了检测.结果表明,该AS-PCR的扩增产物大小为543 bp,核酸检出限为12.6 pg/μL,菌液检出限为2.3×104 cfu/mL,适用于鸡泄殖腔拭子、鲜蛋、饲料和饮水中鸡白痢沙门菌的检测.该AS-PCR检测方法具有简便、快速、特异性强、敏感度高等特点,可应用于鸡白痢沙门菌的快速检测. 相似文献
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本研究旨在建立和优化禽沙门菌分离鉴定的方法。选择山东省某地方品种鸡疑似发生沙门菌病的未出壳死胚样品24份,通过4组不同的培养基组合比较沙门菌的分离情况,选择沙门菌通用引物两对和鸡白痢特异性引物一对进行分离株的PCR检测,比较其特异性,然后进行血清分型和MLST分型,确定沙门菌类型并研究其相关性。结果表明,该批样品中TTB比SC增菌效果好,XLD和XLT4选择培养基对沙门菌分离效果相同,最终通过PCR方法鉴定出沙门菌阳性率为58.3%;发现两对通用引物特异性一致,表明可任选一对引物进行PCR鉴定;用鸡白痢沙门菌特异性引物完成的PCR鉴定结果显示,14株沙门菌中有7株为鸡白痢沙门菌;血清型鉴定结果表明,14株沙门菌中有7株鸡白痢沙门菌、7株肠炎沙门菌,共两种血清型;MLST分子分型结果表明,14株沙门菌分为3个ST型,分别是7株ST11、3株ST92和4株ST2151,其中ST11为肠炎沙门菌,ST92和ST2151为鸡白痢沙门菌;本研究表明,疑似沙门菌感染样品经BPW和TTB增菌、XLD或XLT4选择培养基分离培养,最后通过PCR方法可准确完成沙门菌的分离鉴定;同时,应用该方法可完成对鸡白痢沙门菌的鉴定,并与血清分型和MLST分子分型结果一致。 相似文献
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《中国兽医杂志》2017,(12)
为了有效防控雏鸡白痢沙门菌病,试验自保定某蛋种鸡场疑似为鸡沙门菌病雏鸡采取肝脏病料进行细菌的分离培养、生化鉴定、鸡白痢沙门菌多价血清学鉴定及沙门菌inv A基因PCR鉴定,并对分离菌进行致病性检测及对7种抗菌药物的耐药性检测。结果表明,10株分离菌的形态特征、生化特性、血清学鉴定结果符合鸡白痢沙门菌的特征,说明从病死雏鸡组织中分离出的细菌为鸡白痢沙门菌。沙门菌inv A基因PCR扩增出373 bp目的片段,进一步证实分离菌为鸡沙门菌。用分离菌株接种雏鸡,其死亡率高达90%,表明该菌株有很高的致病性。10株分离菌株对阿莫西林和青霉素耐药率达100%,对阿齐霉素的耐药率达90%,而对阿米卡星、头孢唑林和庆大霉素耐药性较弱、对氧氟沙星较为敏感。3耐菌株多重耐药比例为42.86%,4耐菌株多重耐药比例为57.14%,5耐菌株多重耐药比例为71.43%,说明雏鸡沙门菌分离株对所试抗菌药物均表现出不同程度的耐药性,且表现出多重耐药现象。本试验为鸡白痢沙门菌病的防治提供参考。 相似文献
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《中国家禽》2019,(8)
为了解广东某黄羽肉种鸡场鸡白痢沙门菌的感染及耐药情况,随机采集该场孵化至21日胚龄时的117个死亡胚进行鸡白痢沙门菌分离,通过生化鉴定、PCR、血清分型及rfbS基因序列测定等鉴定鸡白痢沙门菌分离株,并采用琼脂稀释法测定分离株对16种抗菌药物的最小抑菌浓度(MIC)。结果显示:共分离鉴定出18株鸡白痢沙门菌,分离率为15.38%;18个鸡白痢沙门菌分离株对头孢噻肟、头孢吡肟、亚胺培南、多黏菌素B、庆大霉素、阿米卡星、氟苯尼考、链霉素均敏感,而对磺胺异吡恶唑(100%)、萘啶酸(72.2%)、氨苄西林(55.6%)、四环素(44.4%)耐药率较高,且分离株均有多重耐药性。研究表明该黄羽肉种鸡场已受到鸡白痢沙门菌感染的困扰,有必要尽快启动鸡白痢沙门菌防控与净化。 相似文献
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《中国预防兽医学报》2017,(3)
为建立鸡白痢沙门氏菌与其他常见致病性沙门氏菌的血清型特异性快速PCR检测方法,本研究通过对Gen Bank中鸡白痢沙门氏菌、鸡伤寒沙门氏菌和肠炎沙门氏菌全基因组进行生物信息学分析,确定SEEP17495基因为鸡白痢沙门氏菌的检测靶基因,设计特异性引物,建立了一种能够直接从粪便样品中检测鸡白痢沙门氏菌的PCR检测方法。结果显示:该方法对于两株鸡白痢沙门氏菌均可以特异性地扩增出356 bp的目的片段,但对于7株常见非沙门氏菌致病菌和29株其他常见致病性沙门氏菌血清型的扩增结果均为阴性。该方法无论检测鸡白痢沙门氏菌纯培养菌,还是检测模拟鸡粪样品中的鸡白痢沙门氏菌,检测的灵敏度均为103cfu/m L。本研究建立的PCR检测方法具有良好的特异性和灵敏性,并且能够快速检测出粪便样品中的鸡白痢沙门氏菌,为鸡白痢沙门氏菌的检测提供了一种快速、有效的方法。 相似文献
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通过培养特性、生化反应、血清学试验及PCR检测,对2010年10月～2012年6月江苏、安徽两省送检的禽源病料进行鸡白痢沙门菌的分离鉴定,并对分离株进行药敏试验.结果从1 420份样品中分离鉴定出55株鸡白痢沙门菌;药敏试验结果表明,分离株对萘啶酸、羧苄青霉素、链霉素、磺胺异恶唑和氨苄西林等抗菌药物具有较高的耐药性,而对庆大霉素、头孢曲松、氯霉素和卡那霉素具有较强敏感性.上述鸡白痢沙门菌分离株中,有49株(89.09％)为多重耐药菌株,其中以四耐菌株比例最高(25.45％).本研究对于指导临床合理用药及控制鸡白痢沙门菌发生与流行具有重要的现实意义. 相似文献
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为了建立一种快速、高效和简便的鸡白痢沙门菌套式PCR检测方法,根据沙门菌侵袭蛋白基因invA的高度保守区域,利用Primmer 5.0软件设计2对特异性的套式引物,以提取的沙门菌DNA为模板克隆invA基因,将其连入T载体并计算拷贝数作为套式PCR的模板,在对反应条件进行优化的基础上,建立最佳的套式PCR反应条件,确定其上、下游引物用量分别为0.3μL,套式PCR的第1次退火温度为57.4℃,第2次为53.5℃。应用该方法对已构建好的不同浓度的标准阳性质粒作为模板进行检测,其灵敏度为102拷贝数/20μL,远高于常规PCR的106拷贝数/20μL。用建立的套式PCR方法对从新疆石河子某鸡场采集的25样品进行检测,与传统的诊断方法比较符合率达95.00%。建立的鸡白痢沙门菌套式PCR检测方法具有快速、可行、特异性强、准确率高等特点,为鸡沙门菌病的早期诊断和研究提供了技术支撑。 相似文献
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An allele-specific PCR assay for the rapid and serotype-specific detection of Salmonella pullorum 总被引：2，自引：0，他引：2
Desai AR Shah DH Shringi S Lee MJ Li YH Cho MR Park JH Eo SK Lee JH Chae JS 《Avian diseases》2005,49(4):558-561
Salmonella serovar Pullorum is a causative agent of pullorum disease (PD) in poultry and is responsible for severe economic losses to the poultry industry in many parts of the world. A definitive detection of Pullorum requires culture followed by serotyping and biochemical identification, a process that is tedious and takes several weeks to accomplish. We have developed a rapid allele-specific polymerase chain reaction (PCR) method based on the nucleotide polymorphism in rfbS gene sequence for the serotype-specific detection of Pullorum and its differentiation from the closely related Gallinarum. The specificity of this PCR assay was tested using DNA samples from Pullorum (n = 13), Salmonella serotypes other than Pullorum (n = 19), and closely related non-Salmonella organisms (n = 5). The PCR assay was highly serotype-specific as the PCR amplicon of 147 base pairs was observed only in the case of Pullorum, while all the other DNA samples tested PCR negative. A definitive identification of Pullorum cultures was possible in less than 3 hr. As little as 100 pg of SP DNA was detected. This allele-specific PCR method is highly specific as well as sensitive and may be an effective molecular tool in the rapid and serotype-specific detection of Pullorum and differentiation from other Salmonella species. 相似文献
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Salmonella pullorum is the cause of pullorum disease, which is characterized by white diarrhea and a high mortality rate in poultry. During the 1990s, the serologic "pullorum" test has occasionally failed to detect infected birds during the early stage of disease. To determine if any recent genetic changes have taken place in S. pullorum to account for poor seroconversion sometimes observed in infected flocks, S. pullorum from 1990s outbreaks and strains isolated prior to the 1980s were typed by random amplified polymorphic DNA (RAPD). Of 40 S. pullorum isolates typed by this method, eight distinct DNA patterns were identified with one of three RAPD polymerase chain reaction primers. Sixty-two percent of S. pullorum isolates shared the same RAPD DNA pattern, and a major proportion of these strains were from recent flock infections. The RAPD patterns for S. pullorum were clearly distinct from the avian Salmonella group B isolates included in this analysis. The distribution of Salmonella virulence genes among avian Salmonella isolates was also examined. Eighty-five percent of the S. pullorum isolates had both the virulence plasmid gene, spvB, and the invasion gene, invA, with the same percentage positive for the Salmonella enteriditis fimbrial gene, sef. However, significant variability was observed among S. pullorum in their ability to invade avian epithelial cells, despite the presence of the Salmonella invasion gene in these isolates. 相似文献
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Ceelen LM Haesebrouck F Favoreel H Ducatelle R Decostere A 《Veterinary microbiology》2006,113(1-2):45-53
Helicobacter pullorum has been associated with diarrhoea, gastroenteritis and liver disease in humans and with hepatitis and enteritis in poultry. The purpose of the present study was to examine whether cytolethal distending toxin was present among 10 poultry and three human H. pullorum isolates and whether a different level of cytolethal distending toxin activity was noted. A PCR assay was performed to detect the cdtB gene. In addition, epithelial Hep-2 cells inoculated with sonicate from all strains were observed microscopically and DNA analysis of these cells was done by flow cytometry. All H. pullorum isolates harboured the cdtB gene, but functional cytolethal distending toxin activity was only demonstrated in the human H. pullorum strain CCUG 33839. A significant number of cells treated with sonicate from this strain were enlarged. The nuclei were distended proportionally. Giant cells and multinucleated cells were observed as well. In addition, stress fibers accumulated. DNA analysis by flow cytometry revealed 31.0% of these cells at the S/G2 stage of the cell cycle. The tested poultry and human H. pullorum isolates all possess the cdtB gene, but under the circumstances adopted in this study only the human strain CCUG 33839 seems to show biological activity typically for CDT in vitro. 相似文献
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OBJECTIVE: To identify swimming motility in Salmonella pullorum isolates and to characterize the flagellar proteins produced by motile isolates. SAMPLE POPULATION: 30 S pullorum isolates and isolates of 7 other Salmonella sp. PROCEDURE: Salmonella pullorum isolates were inoculated into high motility medium to evaluate swimming motility. Putative flagellar proteins were purified from the organisms and analyzed by means of gel electrophoresis and western blotting procedures, using various antisera specific for flagellar proteins. Antisera shown to be reactive with putative flagellar proteins were incorporated into the growth medium to examine their effects on motility of the isolates. RESULTS: All S pullorum isolates had evidence of swimming motility. Two putative flagellar proteins were purified from 2 of the S pullorum isolates: a 60 to 62 kd protein shown to react with antiserum specific for type y flagellar protein, and a 58 to 59 kd protein shown to react with antiserum specific for type d flagellar protein and with antibody reactive to a highly conserved flagellar epitope found on various Enterobacteriaceae. Antiserum specific for type d flagellar protein inhibited swimming motility of S pullorum isolates, but antiserum specific for type y flagellar protein did not. CONCLUSIONS: Results suggest that S pullorum isolates can be induced to manifest swimming motility when grown on medium with a low agar concentration and possess a 58 to 59 kd protein of d serotype and a second protein of 60 to 62 kd that also may be a flagellar protein. 相似文献
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Three-day-old specific pathogen-free chickens (n = 24) located in isolators were inoculated orally with Helicobacter pullorum. One group (n = 12) was infected with a H. pullorum field isolate from human origin, another one (n = 12) with the American Type Culture Collection H. pullorum reference isolate 51801 originating from chickens. Both isolates were positive for cytolethal distending toxin, investigated using a polymerase chain reaction (PCR). A third group (n = 4) was kept as a negative control. Starting on day 7 of life, birds from each group were euthanatized at different time points up to 35 days. Various organ samples were taken aseptically and processed by culture and a H. pullorum-specific PCR. In the group infected with the human isolate the nucleic acid of H. pullorum was detected in the caecal tonsils and caeca of 12 and 11 birds, respectively. Live bacteria were cultivated from the caecal tonsils and caeca of five birds 24 and 31 days postinfection. Live bacteria were also isolated from the heart of one bird, whereas PCR had to be used to detect the nucleic acid of H. pullorum in the gallbladder of four birds. No live bacteria were reisolated at any time from birds infected with the avian isolate, but bacterial nucleic acid was detected in the caeca of five birds and in the gallbladder of one. In both groups neither live H. pullorum nor its nucleic acid were detected in the liver, spleen, and duodenum. Compared to the avian H. pullorum isolate the human isolate proved to be more invasive. No obvious clinical symptoms or disease was seen in the chickens during the entire experiment. The reisolation of live bacteria at the end of the experiments indicates that H. pullorum could enter the food chain even after early infection in birds. Furthermore, PCR was demonstrated to be helpful in tracing these fastidious bacteria. 相似文献
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Detection and identification of Mycoplasma conjunctivae in infectious keratoconjunctivitis by PCR based on the 16S rRNA gene. 总被引：2，自引：0，他引：2
M Giacometti J Nicolet K E Johansson T Naglic M P Degiorgis J Frey 《Zentralblatt für Veterin?rmedizin. Reihe B. Journal of veterinary medicine. Series B》1999,46(3):173-180
A specific PCR assay based on unique sequences of the rrs genes (16S rRNA) of Mycoplasma conjunctivae was developed for direct detection and identification of this pathogen from clinical material. DNA from eye swabs was amplified after a simple lysis step by either a single PCR with the M. conjunctivae specific primer pair McoR1 and McoF1, or by a nested PCR with the Mycoplasma genus specific primer pair MOLIGEN1-L and 16UNI-R in the first step and McoR1 and McoF1 in the second step. The specificity of the primer pair McoR1 and McoF1 was verified with purified DNA from the type strain, from 17 field isolates of M. conjunctivae and from several Mollicutes which are phylogenetically related to M. conjunctivae or which can be isolated from the same host animals. This method identified mycoplasma isolates from goat, sheep, ibex and chamois originating from different countries as M. conjunctivae. No cross amplifications with other mycoplasmas which are related to M. conjunctivae were observed. Eye swab samples containing known numbers of M. conjunctivae cells were analysed after direct lysis of the material. The detection level was estimated to be 20 cells per swab when the nested PCR procedure was used and 2 x 10(5) by the single PCR method. In an experimental infection model of sheep, the nested PCR method for detection of M. conjunctivae gave results which were comparable to mycoplasmal culture. These are the implications for diagnostic purposes: M. conjunctivae isolates can be identified by the one-step PCR method, whereas for detection and identification of M. conjunctivae in clinical material the two-step method should be used (higher sensitivity). 相似文献
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Specific detection by PCR of Streptococcus agalactiae in milk. 总被引：3，自引：0，他引：3
The aim of this study was to develop a simple and specific method for direct detection of Streptococcus agalactiae from cow's milk. The method was based on polymerase chain reaction (PCR) using species-specific and universal primers derived from the 16S rRNA gene. The amplification product was verified by restriction endonuclease digest and sequencing. Specific identification was proven on a collection of 147 S. agalactiae isolates of bovine and human origin. In addition, 17 strains belonging to different bacterial species that potentially can be found in milk samples also tested negative. The PCR developed was used for direct detection of S. agalactiae in milk, using for the first time with gram-positive bacteria the nucleic acid-binding properties of diatomaceous earth. The test, which has high specificity, high sensitivity (100 cfu/mL), and can be carried out in less than 24 h, represents an innovative diagnostic tool for the detection of S. agalactiae in milk. 相似文献