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1.
肠毒素大肠杆菌((Ent erot oxi geni c E.col i,ETEC)是引起犊牛腹泻的主要病原之一。本试验建立了多重PCR检测ETEC毒力因子F41菌毛、K99菌毛和STa、LT肠毒素相关基因的技术方法。试验对影响PCR扩增的dNTP、引物浓度以及退火温度等因素进行优化,确定了多重PCR的特异性和灵敏性。结果表明:所建立的多重PCR方法快速、特异、灵敏,在2.5h-3h内就可以完成,为致犊牛腹泻肠毒素大肠杆菌的快速准确检测提供另一种选择。 相似文献
2.
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
3.
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
4.
《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
5.
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
6.
《畜牧与兽医》2014,(8):94-97
对上海地区分离的199株猪源大肠杆菌分离株进行血清型鉴定和毒力因子检测,使用大肠杆菌标准抗O因子血清进行玻片凝集试验;采用普通和多重PCR方法,对分离株进行STa、STb、LT、Stx2e等4种肠毒素和K88、K99、987P、F18、F41等5种黏附素检测。结果:199株猪源大肠杆菌分离株有110株鉴定出血清型,占检测菌株的55.28%,覆盖了30种血清型,优势血清型为O8、O45、O64、O78、O138、O157、O163、O7+O8,占定型菌株的60%。共有32株大肠杆菌检测到毒力因子,阳性率为16.08%,检测出9种毒力因子。其中有21株单独检测到肠毒素,占65.63%,STa和STb+LT阳性的菌株分别为14和4;6株单独检测到黏附素,3株菌株K99阳性;5株同时检测到肠毒素和黏附素。在32株毒力基因阳性的大肠杆菌中,有15株血清型定型,覆盖7种血清型,其中O138、O7+O8与肠毒素STa相关,O8、O45与STb+LT相关。研究表明,上海地区猪源大肠杆菌分离株优势血清型至少覆盖了包括O8、O45、O64、O78、O138、O163等在内的8种血清型;大肠杆菌分离株以产肠毒素为主,主要毒力因子包括黏附素K99和肠毒素STa、STb+LT等。 相似文献
7.
《黑龙江畜牧兽医》2017,(3)
为了进一步了解黑龙江省部分牛场致犊牛腹泻大肠杆菌毒力因子的流行情况,试验采用常规分离和PCR检测方法对2014—2015年黑龙江省部分牛场采集的72份犊牛病料进行检测,鉴定得到63株大肠杆菌,并应用PCR技术对CS31A、K88、K99、F17、F41、irp2、eae A、sta、lt、bfp A、EAST1等毒力因子进行检测。结果表明:有46株大肠杆菌含有毒力因子,通常含1~2种毒力因子,其中有18株携带(占39.13%)CS31A基因,说明CS31A基因为优势基因;通过毒力因子对大肠杆菌进行分型,共鉴定出产肠毒素性大肠杆菌(ETEC)35株(占76.09%),为犊牛腹泻的主要致病型。 相似文献
8.
《畜牧与兽医》2016,(7):89-92
为确定新疆石河子、沙湾、阿克苏、博乐4个地区6个规模化牛场初生犊牛腹泻死亡与大肠杆菌的相关性,采集6个规模化牛场因腹泻死亡犊牛的肠系膜淋巴结等病料进行细菌分离鉴定。结果:从60份样品中分离并经生化鉴定获得53株呈β溶血的大肠杆菌;49株携带K99、F41菌毛基因及STa肠毒素基因;10株不同地区来源代表株均能迅速致死小鼠;多数分离株对氨苄西林、阿莫西林、阿莫西林/克拉维酸、头孢噻吩、头孢拉啶、庆大霉素、环丙沙星等耐药,对阿米卡星、头孢西丁等敏感。研究表明,携带K99、F41菌毛及STa的产毒性大肠杆菌是引起这6个牛场初生犊牛腹泻并造成死亡的主要病原之一。 相似文献
9.
为建立一种可以同时扩增大肠杆菌(E.coli)F4、F5、F6、F41和F18菌毛基因保守序列的多重PCR检测方法,本研究设计合成5对分别针对F4、F5、F6、F41和F18菌毛基因的特异性引物,以具有相应菌毛基因的E.coli参考菌株DNA为模板,通过对多重PCR反应条件的优化,建立了检测F4、F5、F6、F41和F18菌毛基因的多重PCR方法。所建立的多重PCR方法能够特异性扩增F4、F5、F6、F41、F18菌毛基因的目的片段,大小分别为770 bp、533 bp、422 bp、643 bp和1140 bp,该方法对沙门氏菌、猪丹毒杆菌、巴氏杆菌以及无菌毛基因的E.coli等参考菌株均无特异性扩增片段,检出F4、F5、F6、F41和F18菌毛基因的最低活菌浓度分别为5.3×10^5cfu/mL、3.7×10^6cfu/mL、3.1×10^5cfu/mL、3.7×10^7cfu/mL、6.9×10^5cfu/mL。用不同批次的引物和试剂进行3次多重PCR检测均能扩增出目的条带,表明建立的多重PCR方法有很好的批内和批间重复性。对90株大肠杆菌临床分离菌株菌毛基因进行检测,F4阳性率为3.33%,F5阳性率为2.22%,未检测到F6、F41和F18阳性菌株,其检测结果与常规单一PCR的检测结果一致。研究表明:建立的E.coli菌毛基因多重PCR分型方法具有很好的特异性、敏感性和重复性,可用于E.coli分离菌株菌毛基因型的快速鉴定,同时提高了检测效率。 相似文献
10.
《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
11.
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
12.
J G Mainil F Bex E Jacquemin P Pohl M Couturier A Kaeckenbeeck 《American journal of veterinary research》1990,51(2):187-190
Colony hybridizations with DNA probes for 3 heat-stable (STaP, STaH, and STb) enterotoxins and 1 heat-labile (LT) enterotoxin and for 4 adhesins (K99, F41, K88, 987P) were performed on 870 Escherichia coli isolates to determine pathotypes prevalent among enterotoxigenic E coli (ETEC) isolated from cattle in Belgium. One hundred thirty-two E coli isolates (15.2%) hybridized with probes STaP, K99, and/or F41. The 5 other probes were not hybridized by E coli isolates. Therefore, only STaP enterotoxin and K99 and F41 adhesins were virulence factors of ETEC isolated from cattle. Two major pathotypes accounted for 95% of the ETEC: STaP+K99+F41+ (67.4%) and STaP+K99+ (27.3%). The last 5% of probe-positive isolates had STaP+, STaP+F41+, or K99+F41+ minor pathotypes. Of 12 American ETEC isolates also assayed, 7 were positive with STb and/or 987P probes (pathotypes STaP+STb+, STaP+ 987P+, or STaP+STb+987P+) and may be porcine- rather than bovine-specific enteropathogens. The remaining 5 American ETEC isolates belonged to 3 minor pathotypes (STaP+, STaP+F41+, and K99+F41+) also found among Belgian E coli isolates. Such isolates may be derivatives of STaP+K99+F41+ or STaP+K99+ ETEC after in vivo or in vitro loss of virulence genes and/or non-ETEC isolates, which have acquired virulence genes by in vivo transfer. 相似文献
13.
14.
Escherichia coli isolates from calves were investigated by multiplex PCR assays for the presence of genes encoding K99, F41, F17-related fimbriae, heat-stabile enterotoxin a (STa), intimin (eae) and Shiga toxins (stx1 and stx2). A total of 120 E. coli isolates, 75 isolated from diarrhoeic or septicemic calves and 45 from clinically healthy calves aged between 1 day and 2 months were tested. Each isolate was obtained from different calves in different herds. Among the isolates from diseased animals, 12 (16%) isolates from 1- to 7-day-old diarrhoeic calves were detected as enterotoxigenic E. coli which possessed K99, F41 and STa in combination; F17-related fimbriae genes were detected in 33 (44%) isolates and they were found in combination with K99 + F41 + STa in two isolates. Of 120 isolates, 16 carried eae, eight stx1 and five stx2 genes alone or in combination. None of the eae- or stx-positive strains was identified as O157:H7. However, results indicate that calves may be carrier of Shiga toxin-producing E. coli which have potential as a human pathogen. Antimicrobial susceptibility of 75 isolates from diseased calves was determined by agar disk diffusion method for 14 antimicrobial agents. In 77.3% of the isolates, multiresistance was detected. Higher resistance rates were detected for cephalothin (72%), tetracycline (69.3%), kanamycin (69.3%), ampicillin (65.3%), nalidixic acid (53.3%), trimethoprim-sulphamethoxazole (52%) and enrofloxacin (41.3%), respectively. No resistance was found for ceftiofur and cefoxitin. 相似文献
15.
Rapid detection of Escherichia coli virulence factor genes using multiplex real-time TaqMan PCR assays 总被引：1，自引：0，他引：1
West DM Sprigings KA Cassar C Wakeley PR Sawyer J Davies RH 《Veterinary microbiology》2007,122(3-4):323-331
Three multiplex real-time TaqMan PCR assays were developed for the detection of Escherichia coli virulence factor genes in veterinary samples. Target virulence factors chosen were the fimbriae K88 (F4), K99 (F5), F41, F17, F18 and 987p (F6) and the toxins LT, STa and CDT IV. Detection of genes coding GAD were included in each assay as an internal control. These assays allow rapid identification of virulence factor genes using identical cycling conditions on an Mx3000Ptrade mark real-time PCR machine with the capacity to test up to 20 strains for 9 virulence genes in 1h. 相似文献
16.
Enterotoxigenic Escherichia coli (ETEC) in farm animals. 总被引：11，自引：0，他引：11
Animal diseases due to enterotoxigenic Escherichia coli (ETEC) typically appear as severe watery diarrhoea during the first few days of life (also a few days after weaning in pigs). ETEC adhere to the small intestinal microvilli without inducing morphological lesions and produce enterotoxins acting locally on enterocytes. This action results in the hypersecretion (of water and electrolytes) and reduced absorption. Adhesins and toxins are the two prominent virulence attributes of ETEC and the level of knowledge of these factors determines the chances for successful prevention and therapy of the disease. For animal ETEC the most common adhesins are the fimbriae (pili) on the surface: F4(K88), F5(K99), F6(987P), F41, F42, F165, F17 and F18. Enterotoxins (extracellular proteins or peptides) of animal ETEC are classified as heat-labile (LT) and heat-stable (ST) enterotoxins with further subdivisions (LTh-I, LTp-I, LTIIa, LTIIb, STaH, STaP, STb) according to antigenic and biological differences. Fimbriae and LT enterotoxins are made up of large molecular weight proteins which facilitate their utilisation in vaccines and their detection using immunodiagnostic systems. The adhesive fimbriae and enterotoxins of animal ETEC are plasmid determined (except F41 and F17). Virulence gene probes (DNA hybridisation, PCR) are specific and sensitive diagnostic and epidemiologic tools for the detection of ETEC. Based on genetic typing, the ETEC, in spite of limited serogroups, seem to represent a population of E. coli with a diverse genetic background. 相似文献
17.
Genotypic prevalence of the adhesin involved in diffuse adherence in Escherichia coli isolates in pre-weaned pigs with diarrhoea in Korea 总被引：1，自引：0，他引：1
Ha SK Choi C Jung K Kim J Han DU Ha Y Lee SD Kim SH Chae C 《Journal of veterinary medicine. B, Infectious diseases and veterinary public health》2004,51(4):166-168
A total of 1002 Escherichia coli strains isolated from pre-weaned pigs with diarrhoea on 1114 swine farms were screened for the presence of the adhesin involved in diffuse adherence (AIDA) gene by polymerase chain reaction (PCR). Escherichia coli isolates that carried AIDA genes were also tested by PCR for the detection of five fimbriae (F4, F5, F6, F18 and F41), heat-stable (STa, STb) and heat-labile (LT) enterotoxin, enteroaggregative E. coli heat-stable enterotoxin 1 (EAST1), and Shiga toxin 2 oedema disease (Stx2e) genes. Twenty-three (2.3%) of the 1002 E. coli isolates carried the gene for AIDA. Among 23 isolates shown to carry genes for AIDA, three carried the AIDA gene as the only shown virulence factor. Other isolates carried other virulence factor genes in addition to AIDA. Four isolates carried genes for at least one of the fimbrial adhesins and enterotoxins. Sixteen isolates carried genes for enterotoxins only. The AIDA may represent an additional virulence determinant in pre-weaned pigs with diarrhoea. 相似文献
18.
Osek J 《Veterinary microbiology》2003,91(1):65-72
The presence of the astA gene responsible for production of enteroaggregative Escherichia coli heat-stable enterotoxin 1 (EAST1) was examined in E. coli strains isolated from pigs with postweaning diarrhoea. Two hundred and seven isolates were tested using PCR for the astA marker and for heat-labile I (LTI), heat-stable I (STI), and heat-stable II (STII) enterotoxin genes. Moreover, the isolates were also analysed for their serotypes (O and K antigens) as well as for fimbrial adhesins using agglutination methods. It was shown that 96 (46.4%) of the isolates possessed the astA genetic determinant. The most common EAST1-positive E. coli serotype was O149:K91 and these strains were mostly LTI/STII-positive. A close correlation between the presence of F4 fimbriae and the EAST1 gene was also observed: 88 of 96 (91.7%) astA(+) isolates tested possessed the F4 antigen. Thus, EAST1 enterotoxin may represent an additional virulence determinant playing a role in the pathogenesis of porcine colibacillosis. 相似文献