鸡胚胎性病原茵多重PCR检测方法的建立

为建立同时检测禽波氏杆菌(Bordetella avium)、沙门氏茵(Salmonella)、大肠杆菌(Escherichia coli)和绿脓杆菌(Pseudomonas aeruginosa)4种导致鸡胚死亡病原菌的多重PCR方法,本研究根据B.aviun的ompA基因、Salmonella的invA基因、E.coli的phoA基因和P.aeruginosa的toxR基因序列,各设计一对特异性引物进行多重PCR反应,并对反应体系和条件进行优化.结果显示,4对引物分别扩增出597bp、724bp、372bp和278bp的目的条带;并且特异性强不与其他非目的茵发生反应.经优化B.aviun、P.aeruginosa和E.coli多重PCR检测灵敏度达到104cfu/mL,而Salmonella为103cfu/mL.本研究建立的多重PCR方法为相关病原茵的快速检测提供方法.     

牛产肠毒素大肠杆菌毒力因子多重PCR检测方法的建立

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。     

猪源肠毒素性大肠杆菌菌毛基因多重PCR检测方法的建立

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。     

产气荚膜梭菌菌落多重PCR方法的建立及初步应用

根据GenBank中已发布的产气荚膜梭菌α、β、ε、τ毒素基因序列,分别设计并合成针对4种毒素基因的特异引物,通过优化多重PCR反应条件,建立1种简单的产气荚膜梭菌定型菌落多重PCR方法。结果显示：A、B、C、D、E5型产气荚膜梭菌参考菌株均扩增出了相应的预期目的条带,而大肠杆菌、巴氏杆菌和芽孢杆菌则均未能扩增出相应条带;将单个菌落稀释100倍,仍能扩增出相应的目的片段,该方法对B型和E型参考菌株最低检测量分别为2．6×10^4cfu／mL、1．2×10^4cfu／mL。应用该多重PCR方法从106份样品中检测到30株产气荚膜梭菌且均为A型,其中病死鸡的盲肠内容物分离率为36．5％（19／52）,健康鸡群新鲜粪便样品分离率为20．4％（11／54）。本研究建立的多重PCR方法特异性强,敏感度高,重复性好,可以有效进行产气荚膜梭菌的快速检测及5种血清型的鉴别,对产气荚膜梭菌的感染及食品安全问题的研究均具有重要意义。     

致猪水肿病大肠杆菌鄂E株菌毛F18基因FedF亚基的克隆、表达及其生物学特性

以致猪水肿病大肠杆菌鄂E株为模板,通过PCR的方法扩增大肠杆菌菌毛F18主要亚基FedF的全长及去信号肽亚基因FedFs,扩增片段分别为1000、900 bp。将纯化的扩增产物克隆到pMD18-T中,通过酶切鉴定和序列分析表明,含SacⅠ及HindⅢ酶切位点的基因全长为967 bp,鄂E株FedF基因编码区全长903 bp,编码301个氨基酸,碱基序列同标准株107/86的同源性为100%。与菌毛AF/R1相比,编码的蛋白质没有同源性。利用pGEX-KG分别构建表达载体,SDS-PAGE检测表明FedFc/pGEX-KG无表达带,FedFs/pGEX-KG则有约58 000的特异性表达带;West-ern blotting检测表明重组蛋白具有免疫原性。利用重组蛋白GST-FedF免疫新西兰兔所制备的抗血清,能抑制Ee株与刷状缘细胞的粘附。利用表达的蛋白建立了F18的ELISA检测方法,并检测790份临床送检血样,其中536份呈阳性,血清学阳性率为67.8%。结果表明,FedF是猪大肠杆菌病新型疫苗及F18^＋E.coli感染鉴别诊断的良好候选抗原,通过本试验建立的ELISA方法揭示国内的F18^＋E.coli感染严重,血清学阳性率高达67.8%。     

检测987p^＋肠毒素性大肠埃希氏菌PCR方法的建立

以987p菌毛结构基因保守序列为靶序列，设计合成了1对可扩增459bp目的片段的引物，建立了检测肠毒素性大肠埃希氏菌987p菌毛基因的PCR方法。该方法对K88^ ( 为菌毛阳性）、K99^ 、F41^ 参考菌株和链球菌、葡萄球菌、巴氏杆菌的检测结果均为阴性；该方法的敏感度可达10^2CFU，对20株腹泻仔猪粪例分离物进行检测，有1株为阳性，与血清学检测的结果一致。结果表明，此方法特异性和敏感性都很高，可用于临床987p肠毒素性大肠埃希氏菌病的快速诊断和流行病学调查。     

Percoll分离法与多重PCR快速检测肉品中沙门氏菌、金黄色葡萄球菌和志贺氏菌

为从食品中有效分离致病菌,去除PCR反应的抑制因子,提高多重PCR检测灵敏度,本研究利用Percoll密度梯度离心的前处理法从鲜肉样品中高效分离沙门氏菌、金黄色葡萄球菌和志贺氏菌,以沙门氏菌invA基因、金黄色葡萄球菌nuc基因和志贺氏菌ipaH基因为靶基因,经过PCR分别扩增出255 bp、506 bp、620 bp的DNA片段,DNA测序证实这些片段为目的扩增产物。该体系检测猪肉匀浆液中3种致病菌的灵敏度为6.2×103 cfu/mL、1.2×103 cfu/mL和2.4×103 cfu/mL,检测时间约6 h。Percoll前处理法与多重PCR检测肉中沙门氏菌、金黄色葡萄球菌和志贺氏菌为同时检测肉类样品中多种致病菌提供了有效的检测方法。     

奶牛乳腺炎5种病原菌多重PCR快速检测方法的建立与评价

为建立一种能同时快速检测多种奶牛乳腺炎致病菌的多重PCR检测方法,本研究以化脓隐秘杆菌ISR基因、无乳链球菌cfb基因、大肠杆菌phoA基因、副乳房链球菌23S rRNA基因和金黄色葡萄球菌nuc基因为靶基因,设计并合成了5对特异性引物,通过方阵法对多重PCR反应体系及反应条件优化,建立了一种能快速检测5种病原菌的多重PCR方法。对各菌种随机组合后利用该多重PCR方法检测,结果显示,该方法能同时快速检测奶牛乳腺炎乳样中5种主要病原菌,而其他病原菌则呈阴性结果,具有较强特异性。分别将化脓隐秘杆菌、无乳链球菌、大肠杆菌、副乳房链球菌、金黄色葡萄球菌菌液10倍倍比稀释后,利用建立的多重PCR方法进行检测,确定该方法敏感性,结果显示,该方法对5种病原菌最低检测浓度分别为:金黄色葡萄球菌6.25×10~4cfu/mL,大肠杆菌2.95×10~6cfu/mL,化脓隐秘杆菌2.95×10~5cfu/mL,副乳房链球菌4.3×10~5cfu/mL,无乳链球菌3.15×10~5cfu/mL。对临床送检及人工模拟的乳样检测结果显示该方法具有较好符合率及灵敏度。本研究首次建立了对包括副乳房链球菌在内的奶牛乳腺炎致病菌多重PCR检测方法,为快速检测奶牛乳腺炎病原菌提供了可行的技术手段。     

奶牛乳房炎病原菌多重PCR检测方法的建立

为了同时检测奶牛乳房炎多种病原菌,试验采用金黄色葡萄球菌(S.aureus)nuc基因、牛支原体(M.bovis)oppD/F基因、大肠杆菌(E.coli)16S-23S rRNA基因内转录间隔区作为靶标设计3对特异性引物,在优化单菌退火温度的基础上确定多重PCR的退火温度,建立S.aureus、M.bovis和E.coli的多重PCR检测方法,验证多重PCR的重复性、特异性、敏感性及实用性。结果表明:基于单一PCR三者共同的最佳退火温度(55.3℃、56.5℃、57.8℃)优化多重PCR退火温度,最终确定57.8℃为三者最佳的退火温度。该方法能扩增出长度为237 bp(S.aureus)、333 bp(M.bovis)、634 bp(E.coli)的基因片段;靶标菌特异性扩增而非靶标菌无扩增条带,最低检测浓度分别为S.aureus 1 pg/μL、M.bovis 1 pg/μL、E.coli 100 fg/μL;正常乳样和临床乳房炎乳样均检出S.aureus(25.9%、53.8%)、M.bovis(10.3%、17.9%)和E.coli(32.8%、71.8%),但乳房炎乳样的阳性检出率高于正常乳样。说明本研究建立的多重PCR方法特异性和敏感性强、稳定性和实用性高,具有良好的推广应用前景。     

新型凝集技术快速检测产肠毒素性大肠杆菌菌毛的研究

为建立以彩色二氧化硅微球(CSN)为载体,快速检测产肠毒素性大肠杆菌(ETEC)菌毛K88ab、K99、987p和F41的新型凝集技术,本研究采用反相微乳液法制备二氧化硅微球,将其与菌毛单因子血清偶联制成免疫彩色二氧化硅微球(ICSN),通过优化反应条件建立了快速检测ETEC菌毛的方法。特异性试验结果显示,ICSN只与携带其对应菌毛的菌株发生反应,与携带其它菌毛的菌株无交叉反应;敏感性试验结果显示菌液的最小检出浓度为1.0×10~6cfu/m L~5.1×10~6cfu/m L;稳定性试验显示4℃保存30 d,ICSN特异性、敏感性无明显变化;双盲样品检测准确率为100%。本研究有助于ETEC菌毛的快速检测和幼畜大肠杆菌性腹泻的诊断。     

Prevalence of enterotoxigenic <Emphasis Type="Italic">Escherichia coli</Emphasis> virulence genes from scouring piglets in Zimbabwe

World-wide, enterotoxigenic Escherichia coli (ETEC) and verotoxigenic E. coli (VTEC)-induced diarrhea are economically important for porcine producers. Our aim was to investigate the prevalence of toxin and fimbrial genes among E. coli isolated from diarrheic piglets from randomly selected piggeries in Zimbabwe.We used multiplex PCR for screening STa, STb, LT, and Stx-2e toxins. Subsequently F4, F5, F6, F18 and F41 fimbriae genes were screened in toxin positive isolates. Toxin positive strains lacking tested fimbriae genes were characterized using transmission electron microscopy, agglutination and agglutination inhibition tests. Approximately 32% of the 1,984 isolates tested positive for STa, STb, LT or Stx-2e genes. Of these, approximately 81% had F4, F5, F6, F18 or F41 fimbriae genes. The remaining toxin positive strains lacked tested fimbriae genes and appeared to either express F1-like fimbriae, or lacked fimbriae. The data constitute an important framework for implementation of prevention measures, such as using relevant fimbriae-based vaccines against ETEC induced diarrhea or VTEC-induced edema.     

The use of polymerase chain reaction for determination of virulence factors of Escherichia coli strains isolated from pigs in Poland.

E. coli strains isolated from pigs with postweaning diarrhea or edema disease were tested by phenotypic and genotypic methods for the presence of virulence antigens and genes, respectively. The slide agglutination and ELISA analyses were used for determination of F4, F5, F6, F17, and F41 fimbriae whereas the prevalence of fimbrial fedA and toxin eltI, estI, estII, stx1, stx2 and stx2e genes were recorded by the means of PCR. Only F4 antigen (ac variant) was found in strains of the serogroup O149:K91 isolated from pigs with diarrhea. PCR analyses showed that the fedA gene encoding F18 fimbriae was present in 61.9% of strains isolated from pigs with diarrhea and in 84.2% of strains isolated from pigs with edema disease. The eltI genes encoding heat-labile toxin I (LTI) were present only in 9 out of 21 strains recovered from pigs with diarrhea. Shiga toxin 2 variant (stx2e) genes were found in six isolates from edema disease and also in one strain from diarrhea. The PCR test used in the study was a sensitive and valuable method for determination of virulence factors of E. coli strains.     

致猪水肿病大肠埃希菌多重PCR检测方法的建立及应用

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.     

Rapid detection of Escherichia coli virulence factor genes using multiplex real-time TaqMan PCR assays

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.     

Analysis of the AIDA-I gene sequence and prevalence in Escherichia coli isolates from pigs with post-weaning diarrhoea and oedema disease

Three hundred and twenty-four strains of Escherichia coli isolated from weaned pigs with diarrhoea or oedema disease in Eastern China were screened by multiplex PCR for the presence of the gene encoding adhesin involved in diffuse adhesion I (AIDA-I). Two AIDA-I positive strains were subjected to analysis of the nucleotide sequence of the complete orfA and orfB of the AIDA gene. The AIDA-I positive E. coli isolates were also assessed for five fimbriae (F4, F5, F6, F18 and F41) by monoclonal antibodies and for toxin genes (STa, STb, LT, EAST1, Stx2e) by PCR. Twenty-one (6.5%) of the isolates possessed AIDA-I genes. Of these isolates, two carried AIDA-I genes as the only demonstrated virulence factors, and the remaining isolates carried other virulence factor genes. Comparing the AIDA-I sequence from porcine and human sources, a high homology of orfA both in porcine E. coli and human E. coli was observed. However, each orfB of the two porcine E. coli isolates was 3864 nucleotides long compared with 3861 for the E. coli 2787 orfB, and showed 96.5% homology to E. coli 2787. The data indicated (1) that AIDA-I may be an occasional virulence factor in post-weaning diarrhoea and oedema disease in pigs, (2) that it has the potential to transfer between porcine and human E. coli, and (3) that there is a genetic diversity in orfB between human and porcine E. coli.     

Prevalence of the enteroaggregative Escherichia coli heat-stable enterotoxin 1 (EAST1) gene in isolates in weaned pigs with diarrhea and/or edema disease

A total of 476 Escherichia coli isolated from weaned pigs with diarrhea and/or edema disease were screened for the presence of the enteroaggregative E. coli heat-stable enterotoxin 1 (EAST1) gene by polymerase chain reaction (PCR). E. coli strains that carried EAST1 genes were also tested by PCR for the presence of genes for five fimbriae (F4, F5, F6, F18 and F41), two heat-stable (STa and STb) and one heat-labile (LT) enterotoxin, and Shiga toxin 2e (Stx2e). One hundred and forty nine (31.3%) of the 476 E. coli isolates carried the gene for EAST1. Of these 149 isolates, 66 (44.3%) carried the east1 gene only and 83 (55.7%) carried genes for the fimbrial adhesins or enterotoxins. E. coli which carried east1 gene also possessed genes for STa or F4 frequently. EAST1 may represent an additional determinant in the pathogenesis of E. coli diarrhea in weaned pigs.     

Prevalence of serogroups and virulence factors of Escherichia coli strains isolated from pigs with postweaning diarrhoea in eastern China

This study was undertaken to determine the present distribution of serogroups, hemolytic activity and virulence factors among Escherichia coli strains isolated from pigs with postweaning diarrhoea from eight provinces in eastern China. Two hundred and fifteen E. coli isolates were serogrouped with O-antisera, investigated for hemolytic activity, assessed for F4, F5, F6, F18 and F41 fimbrial antigens by monoclonal antibodies and detected for genes of enterotoxins and shiga-toxin-two-variant (Stx2e) by a multiplex polymerase chain reaction (PCR). Among these E. coli isolates, 140 were determined to be placed in serogroups, 52 were unable to be serogrouped and the rest 23 auto-agglutinated. These isolates distributed in 45 serogroups and 64.3% (90/140) belonged to 12 O serogroups: O8, O9, O11, O20, O32, O91, O93, O101, O107, O115, O116 and O131. Hemolytic activity was detected in 11.6% (25/215) of all isolates. Several uncommon O serogroups were discovered in this study. Agglutination tests showed that 50.2% (108/215) of these isolates were positive for one or more of the five fimbrial antigens. Seventy-two E. coli strains expressed single fimbria and 36 strains expressed two or more fimbriae. Among these 215 E. coli isolates, strains expressing F18, F4, F6, F6 + F18 or F5 + F41 occurred more frequently. PCR analysis showed that 60.5% (130/215) of the isolates only harboured the gene of estI (STI) while 6.0% (13/215) strains possessed the genes of stx2e, estI and estII and 5.6% (12/215) of strains had the genes of estI/estII. Of all these isolates, 107 (49.8%) were negative for the fimbrial antigens examined. The fimbria-negative isolates usually possessed genetic determinant of estI (78, 72.9%).     

Prevalence of the enteroaggregative Escherichia coli heat-stable enterotoxin 1 gene and its relationship with fimbrial and enterotoxin genes in E. coli isolated from diarrheic piglets.

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.