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牛产肠毒素大肠杆菌毒力因子多重PCR检测方法的建立 总被引：6，自引：1，他引：6
通过多重PCR扩增产肠毒素大肠杆菌（enterotoxigentic E.coli，ETEC）的毒力因子F41菌毛、K99菌毛和STa肠毒素的编码基因来检测和鉴定ETEC。试验中对影响PCR扩增的dNTP、Mg^2＋、引物浓度以及退火温度等因素进行优化，在优化条件的基础上，确定多重PCR的特异性和灵敏性，以此建立同时检测ETEC多个毒力因子的多重PCR方法。用该方法对分离于犊牛腹泻和犊牛肠毒血症的7株大肠杆菌进行检测，结果2株为F41、K99和STa阳性，4株为F41、STa阳性，1株为K99STa阳性。这与玻片凝集试验检测菌毛的结果一致。试验表明，该方法特异性强、敏感性高、简便、快速，适用于临床鉴定和检测牛ETEC菌株。 相似文献
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为快速检测和鉴定产肠毒素大肠杆菌(ETEC)菌毛(K88和K99)和毒素(STa)基因,本研究设计合成了针对K88、K99和STa基因的3对特异性引物,对K88、K99和STa基因扩增条件进行优化,建立了检测K88、K99和STa的多重PCR方法.该方法对Kss、K99和STa基因的扩增产物分别为237 bp,314 bp和166 bp;此外,该方法具有良好的灵敏性和特异性.本实验建立的多重PCR方法为致幼畜腹泻ETEC的检测提供了快速准确方法.用所建立的多重PCR方法对实验室分离的23株大肠杆菌进行检测,结果2株为K99/STa阳性,1株为STa阳性. 相似文献
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为了对猪源产肠毒素性大肠杆菌（ETEC）的4种主要菌毛（K88、K99、F41和987p）进行快速检测和分型，建立了检测4种菌毛的多重PCR方法。首先设计合成4对4种菌毛特异性的引物，然后用4种菌毛参考ETEC菌株优化了多重PCR方法。该方法对K88、K99、F41和987p 4种菌毛的扩增产物分别为201，314，380和459bp。对4种扩增产物分别进行酶切鉴定，结果均得到与预期一致的2个片段。对各个参考菌株不同组合的检测结果为100％符合。结果表明，该多重PCR方法具有很好的特异性和敏感性，可用于ETEC性腹泻的辅助诊断及ETEC菌毛抗原分型检测。 相似文献
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《中国兽医杂志》2016,(4)
为建立一种简单、快速、灵敏、准确的产肠毒素大肠杆菌(ETEC)检测方法,根据产肠毒素大肠杆菌菌毛(K88)和毒素(STa和LT)基因分别设计合成了1对引物,对K88、STa和LT基因扩增条件进行优化,建立了检测K88、STa和LT的三重PCR方法。该方法对K88、STa和LT基因的扩增产物分别为499 bp,190 bp和373 bp;此外,该方法具有良好的灵敏性和特异性。本实验建立的三重PCR方法为致幼畜腹泻ETEC的检测提供了快速准确方法。用所建立的三重PCR方法对实验室从临床腹泻样品中分离的120株大肠杆菌进行检测,结果 9株为K88/LT/STa阳性,14株为K88/LT阳性,21株K88/STa阳性,13株LT/STa阳性,8株K88阳性,2株LT阳性,12株STa阳性。 相似文献
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《畜牧与兽医》2015,(1):86-90
在对大肠杆菌主要毒力因子基因序列分析的基础上,建立了用于快速检测毒素、菌毛和毒力岛(HPI)的PCR方法;用所建立的多重PCR或单重PCR方法,对江苏部分地区临床疑似大肠杆菌感染的新生仔猪腹泻样品110份和健康新生仔猪粪便35份进行了快速检测。结果表明:HPI已经成为致仔猪腹泻大肠杆菌最常见的毒力因子(58.18%),其次是肠毒素(LT1 10.91%、ST2 8.18%、ST1 2.73%),并揭示了至少46.36%的病例与HPI+大肠杆菌感染相关,11.82%的病例与HPI+大肠杆菌和产肠毒素大肠杆菌(ETEC)混合感染相关,仅有6.36%的病例与ETEC感染相关。菌毛仍然是ETEC的主要毒力因子,其中987P+菌株6.36%、K88+菌株3.64%、K99+菌株0.91%,未发现F41+菌株。此外,通过对健康新生仔猪样品的检测,发现48.57%的动物携带HPI+大肠杆菌,5.71%的动物携带ETEC(主要为ST2+),未发现所检测的其他毒力因子,这表明HPI+大肠杆菌很可能是条件性致病菌。 相似文献
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为了快速检测和鉴定产肠毒素大肠杆菌菌毛(K88和K99)基因,本研究设计合成了针对K88、K99的2对特异性引物,对扩增条件进行优化,建立了检测K88和K99的双重PCR方法。该方法对K88、K99基因的扩增产物大小分别为237和314 bp;最终确定dNTP终浓度0.4 mmol/L,K88、K99的引物终浓度均为25 μmol/L,退火温度为52℃。试验结果表明,该方法具有良好的灵敏性和特异性。用所建立的双重PCR方法对实验室分离的23株大肠杆菌进行检测,结果显示,K88单重PCR阳性2株,K99单重PCR阳性3株,K88和K99双重PCR阳性5株。本研究建立的双重PCR检测方法为致幼畜腹泻产肠毒素大肠杆菌的快速准确检测提供了方法。 相似文献
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产肠毒素大肠杆菌菌毛的DNA疫苗研究进展 总被引：4，自引：2，他引：2
肠毒素大肠杆菌是导致婴幼儿及旅游者急性腹泻,仔猪腹泻和水肿的主要病原菌之一。菌毛定居因子是该病原菌主要的致病因素。DNA疫苗既能激发机体的细胞免疫,也能诱导特异性的体液免疫。目前用于预防ETEC腹泻的DNA疫苗的研究已取得一定进展,作者从重组质粒的构建、免疫应答、疫苗的接种系统以及基因佐剂4个方面简单的概述了产肠毒素大肠杆菌菌毛的DNA疫苗的研究现状。 相似文献
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肠毒素大肠杆菌(Enterotoxigenic Escherichia coli,ETEC)是引起仔猪腹泻的重要病原之一,其致病性主要与菌毛粘附素及其产生的肠毒素有关。ETEC依靠菌毛粘附素与小肠粘膜上皮细胞上的受体特异性结合,这种粘附作用可以使ETEC抵抗肠道蠕动的冲刷作用而定植,并大量繁殖。ETEC在生长过程中,产生大量的肠毒素并不断释放到肠道内,造成肠道内水和电解质比例失衡,从而引起腹泻。不同肠毒性大肠杆菌(ETEC)菌株的菌毛抗原类型不同,主要有K88、K99、987P及F41,其中以K88的流行最为普遍,因而亦尤为重要。1K88粘附素菌毛,又称纤毛或柔毛,由菌… 相似文献
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J Osek 《Journal of veterinary diagnostic investigation》2001,13(4):308-311
A multiplex polymerase chain reaction (PCR) system was developed for identification of enterotoxigenic Escherichia coli (ETEC) strains and to differentiate them from other gram negative enteric bacteria. This test simultaneously amplifies heat-labile (LTI) and heat-stable (STI and STII) toxin sequences and the E. coli-specific universal stress protein (uspA). The specificity of the method was validated by single PCR tests performed with the reference E. coli and non-E. coli strains and with bacteria isolated from pig feces. The multiplex PCR allowed the rapid and specific identification of enterotoxin-positive E. coli and may be used as a method for direct determination of ETEC and to differentiate them from other E. coli and gram-negative enteric isolates. 相似文献
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本研究旨在建立一种快速鉴定致猪水肿病大肠埃希菌的多重PCR检测方法.分别针对大肠埃希菌16S rDNA、志贺毒素Stx2e A亚基和菌毛F18ab A亚基保守序列设计合成3对特异性引物,优化多重PCR反应条件,并进行特异性和敏感性检测.结果显示,阳性对照菌株扩增产物大小分别为1 062、733和313 bp.特异性和灵敏性检测结果表明,与肠炎沙门菌、多杀性巴氏杆菌、胸膜肺炎放线杆菌、副猪嗜血杆菌、支气管败血波氏杆菌和猪链球菌等猪常见致病菌均无交叉反应;菌体直接扩增法最低检出量为1 875 CFU.利用建立的多重PCR检测方法对分离收集的128株大肠埃希菌进行鉴定,得到36株致猪水肿病大肠埃希菌,其中30株既有菌毛F18ab又产志贺毒素Stx2e,另外6株仅产志贺毒素Stx2e.结果表明,本试验所建立的多重PCR检测方法对致猪水肿病大肠埃希菌的快速诊断和流行病学调查具有一定的应用价值. 相似文献
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根据NCBI上已收录的链球菌ef-tu基因、金黄色葡萄球菌nuc基因、沙门菌hut基因和大肠杆菌23SrRNA基因的序列,设计并合成4对特异性引物,通过优化多重PCR的反应条件,建立了能够同时检测4种细菌混合感染的多重PCR诊断方法。特异性分析结果表明,应用该方法可以从链球菌、金黄色葡萄球菌、沙门菌和大肠杆菌以及4种细菌的混合物中扩增出4条大小分别为197、278、495和652bp的特异性条带,其他对照组的检测结果均为阴性;敏感性分析表明,该方法对4种病原菌基因组DNA的检出量分别为链球菌25.6pg、金黄色葡萄球菌33.2pg、沙门菌35.7pg、大肠杆菌52.1pg;人工模拟感染样本检测表明,该方法能从混合感染的病料中特异地检测出4种病原菌。本试验建立的多重PCR方法具有特异性强,敏感度高,稳定性好的特点,可以有效的检测链球菌、金黄色葡萄球菌、沙门菌和大肠杆菌的混合感染。 相似文献
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Osek J Gallien P Protz D Truszczynski M 《Berliner und Münchener tier?rztliche Wochenschrift》2000,113(7-8):265-270
Enterotoxigenic Escherichia coli (ETEC) may produce heat-labile (LT) and heat-stable (STI or STII) enterotoxins. Differentiation between ETEC and other pathogenic and non-pathogenic E. coli as well as other Gram-negative bacteria responsible for induction of diarrhoea, requires isolation, biochemical identification and determination of toxins (or their genes--elt, estI, estII). A multiplex polymerase chain reaction (PCR) system for the rapid and specific detection of enterotoxin-gene-positive E. coli was developed. The primers described by other authors, specific for the universal stress protein A (UspA) of E. coli and enterotoxin genes were used and allowed a simultaneous amplification of the E. coli-specific uspA and the respective toxin genes. The specificity of this multiplex PCR system was confirmed by testing ETEC, non-ETEC and other non-E. coli bacteria. The specific 884 bp uspA gene and 280 bp (eltI), 166 bp (estI) or 278 bp (estII) amplification products were generated with the respective ETEC strains whereas no amplification was detected with non-E. coli bacteria. The multiplex PCR developed allowed the rapid and specific identification of enterotoxin-producing E. coli colonies directly grown from faecal samples of pigs with diarrhoea. The test may be used as a method for the determination of ETEC among other pathogenic groups of E. coli and other Gram-negative enteric isolates. 相似文献
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Ok M Güler L Turgut K Ok U Sen I Gündüz IK Birdane MF Güzelbekteş H 《Zoonoses and public health》2009,56(2):94-101
The purpose of this study was to determine aetiological agents of diarrhoea in neonatal calves and to investigate virulence gene markers of Escherichia coli strains isolated from calves by multiplex polymerase chain reaction (PCR). Eighty-two diarrhoeic calves and 18 healthy calves were used as subjects. Faeces were taken from the rectums of all the calves and were subjected to bacterial culture. Antigen enzyme-linked immunosorbent assay (ELISA) was performed to detect rotavirus, coronavirus and E. coli K99 in faeces of all the calves. A multiplex PCR was used to characterize E. coli strains in all the calves. Escherichia coli was isolated from 37 faeces samples, Enterococcus ssp. was isolated from 22 faeces samples and Salmonella was isolated from one faeces sample in diarrhoeic calves. Furthermore, only E. coli was isolated from all 18 faeces samples of healthy calves. Of the 37 E. coli isolated from diarrhoeic calves, K99 (18.9%), F41 (18.9%), heat-stable enterotoxin a (STa) (18.9%), Shiga toxin 1 (Stx1; 13.5%) and Shiga toxin 2 (Stx2; 5.4%) and intimin (8.1%) genes were identified by multiplex PCR. Of the 18 E. coli isolated from healthy calves, K99 (16.6%) and intimin (55.5%) genes were identified by PCR. A total of 15 rotavirus, 11 coronavirus and 11 E. coli K99 were detected in diarrhoeic calves by the antigen ELISA. As a result, this study shows that rotavirus, coronavirus, E. coli and Enterococcus ssp. were determined to play a role in the aetiology of diarrhoea in the neonatal calves. K99, F41, STa, Stx1 and Stx2 were found as the most common virulence gene markers of E. coli strains isolated from calves with diarrhoea. Multiplex PCR may be useful for characterization of E. coli isolated from calves. 相似文献
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A total of 720 Escherichia coli strains isolated from diarrheic piglets on 756 swine farms were screened for the presence of the enteroaggregative E. coli heat-stable enterotoxin 1 (EAST1) gene by polymerase chain reaction (PCR). Escherichia coli strains that carried EAST1 genes were also tested by PCR for the presence of 4 fimbriae (F4, F5, F6, F41), 2 heat-stable enterotoxins (STa and STb), and 1 heat-labile enterotoxin (LT) gene. One hundred sixty-four (22.7%) of the 720 E. coli isolates carried genes for EAST1. Of these 164 isolates, 62 (37.8%) carried EAST1 genes only, 11 (6.7%) carried genes for at least 1 of the fimbrial adhesins, 51 (31.1%) carried genes for at least 1 of the enterotoxins, and 40 (23.8%) carried genes for at least 1 of the fimbrial adhesins and enterotoxins. Forty-six percent of strains that carried EAST1 genes carried STa genes, and 16% of strains that carried EAST1 genes carried F4. The isolation rate of enterotoxigenic E. coli strains carrying genes for EAST1 gene was 63%. The 6 major genotypes observed in this study (in decreasing order) were EAST1+, EAST1+STa+, EAST1+STa+STb+, EAST1+STa+F5+, EAST1+STa+F4+, and EAST1+STb+F4+. EAST1 is widely prevalent among diarrheagenic strains of E. coli and may represent an important virulence determinant in the pathogenesis of enteric colibacillosis of preweaned pigs. 相似文献
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Bussalleu E Pinart E Yeste M Briz M Sancho S Torner E Bonet S 《Research in veterinary science》2012,93(1):31-33
In semen, bacteria's isolation from a pure culture is complex, laborious and easily alterable by the presence of antibiotics and inhibitors. We developed a PCR technique to detect the presence of the enterotoxigenic (ETEC) and verotoxigenic Escherichia coli (VTEC) (strains with high prevalence in the swine industry) in semen by adapting the protocols developed by Zhang et al. (2007) and Yilmaz et al. (2006). We artificially inoculated extended semen samples at different infective concentrations of bacteria (from 10(2) to 10(8) bacteria ml(-1)) with two enterotoxigenic and verotoxigenic strains, and performed two multiplex and one conventional PCR. This technique proved to be a quick, useful and reliable tool to detect the presence of ETEC and VTEC up to an infective dose of 10(5) bacteria ml(-1) in semen. 相似文献
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The prevalence of enterotoxigenic Escherichia coli in the feces of calves with diarrhea. 总被引：6，自引：1，他引：5 
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下载免费PDF全文 A study was undertaken to determine the prevalence of enterotoxigenicity among Escherichia coli isolated from calves with diarrhea and from a control group of normal calves. The test organisms consisted of 200 E. coli recovered from scouring calves less than two weeks of age and 100 E. coli from normal calves. The enterotoxigenicity of the cultures was evaluated by three methods, namely, injection of ligated segments of piglet intestine, injection of ligated segments of calf intestine and oral inoculation of suckling mice. Live cutures of all the test organisms were used for the ligated intestine studies whereas sterile broth culture supernatants were used in the suckling mouse tests. Of the isolates from scouring calves, 36% were enterotoxigenic in the piglet intestine and 28% in the calf intestine. Amongst the isolates from normal calves, none was enterotoxigenic in the piglet intestine and one was enterotoxigenic in the calf test system. The ligated piglet intestine was considered unsuitable for determining the enterotoxigenicity of bovine E. coli, whereas the ligated calf intestine test was satisfactory and correlated completely with the suckling mouse test. The enterotoxigenic E. coli of bovine origin produced an enterotoxin that resembled the heat stable enterotoxin of typical porcine enteropathogenic E. coli. 相似文献