鸭禽致病性大肠杆菌分离鉴定及多重PCR检测方法的建立

发布时间：2018-10-21 07:39

【摘要】：近年来,由禽致病性大肠杆菌(avian pathogenic Escherichia coli,APEC)引起的禽大肠杆菌病给全球养禽业造成了严重的经济损失。因此,快速准确的诊断是治疗和控制该病的重要前提。本研究自江苏省徐州市周边养鸭厂病死樱桃谷鸭肝脏分离到7株APEC菌株,研究其种系发生型、耐药性、毒力基因分布,并建立多重PCR检测方法,为该地区APEC的临床防控和进一步研究提供支持。主要研究结果如下:1.对2015年采集自江苏省徐州市周边养鸭场临床疑似大肠杆菌病的病死樱桃谷鸭肝脏进行病原菌的分离,根据形态特征、培养特性、生化试验、16S rRNA分子鉴定可确定分离到7株大肠杆菌。对分离菌株的种系发生型进行鉴定可确定7株大肠杆菌均属B2型。取编号为E17和E4的两株大肠杆菌进行动物致病性试验,表明分离到的两株病原菌皆有致病性。为了解分离株对药物敏感性的差异,采用kirby-Bauer纸片法,用29种药敏纸片对分离到的7株鸭源大肠杆菌分离株进行药敏实验,结果表明7株大肠杆菌都具有多重耐药性,最少的对5种药物耐药,最多的对21种药物耐药。亚胺培南、呋喃妥因和呋喃唑酮可作为高敏药物对本地区的鸭源大肠杆菌病防治选药提供一定的指导作用。对7株分离菌株的12个毒力基因进行检测,结果显示除菌株E14、E17、EB未检出stx基因外,其余的4株都含有全部的12种毒力基因。2.根据Genbank中已发表的APEC iss、cvaC、iucD、irp-2、iroN、tsh基因序列,用Primer 5.0软件设计6对特异引物,建立鸭源APEC的多重PCR检测方法。检测多重PCR方法的特异性和灵敏性,结果表明该方法具有特异性,扩增最小检出DNA核酸量为100 pg/μL,最小检出菌落数为105 CFU。利用该多重PCR检测25株APEC(均属于B2型)和79株非致病大肠杆菌(31.65%属于A群,50.63%属于B1群,1.27%属于B2群,16.46%属于D群)验证多重PCR方法的可行性。结果为,在分离菌株含有上述6个毒力基因中3个或3个以上时,即有92.0%的概率认定该分离株具致病性,误差率为8.0%。建立的多重PCR方法能够简便、快速地检测APEC。本研究将为江苏徐州地区APEC的进一步研究提供一定支持。
[Abstract]:In recent years, avian Escherichia coli caused by avian pathogenic Escherichia coli (avian pathogenic Escherichia coli,APEC) has caused serious economic losses to poultry industry worldwide. Therefore, rapid and accurate diagnosis is an important prerequisite for the treatment and control of the disease. In this study, 7 strains of APEC were isolated from the liver of dead Cherry Valley Duck from a duck farm in Xuzhou City, Jiangsu Province. The genotypes, drug resistance, virulence gene distribution and multiplex PCR detection were studied. To provide support for clinical prevention and control and further research of APEC in this area. The main results are as follows: 1. The pathogenic bacteria were isolated from the dead Cherry Valley duck liver collected from the duck farm around Xuzhou City, Jiangsu Province in 2015. According to the morphological characteristics and the culture characteristics, the pathogenic bacteria were isolated from the liver of the dead Cherry Valley duck, which was suspected of E. coli disease. Biochemical test, 16s rRNA molecular identification can confirm that 7 strains of Escherichia coli were isolated. Identification of the phylogenetic types of the isolated strains confirmed that all 7 strains of Escherichia coli belong to B2 type. Two strains of Escherichia coli named E17 and E4 were selected for animal pathogenicity test. The results showed that the two strains were pathogenicity. In order to understand the difference of drug sensitivity of 7 strains of duck Escherichia coli, the drug sensitivity of 7 strains of Escherichia coli isolated from duck was tested by kirby-Bauer disk method. The results showed that all 7 strains of Escherichia coli had multiple drug resistance. The least resistant to 5 drugs and the most resistant to 21 drugs. Imipenem, furantoin and furazolidone can be used as Gao Min drugs for the prevention and treatment of duck Escherichia coli. 12 virulence genes of 7 isolates were detected. The results showed that all the other 4 strains contained 12 virulence genes except the stx gene of E14 E17 EB. 2. According to the published APEC iss,cvaC,iucD,irp-2,iroN,tsh gene sequence in Genbank, six pairs of specific primers were designed with Primer 5.0 software to establish a multiplex PCR detection method for duck APEC. The specificity and sensitivity of the multiplex PCR method were tested. The results show that the method is specific, the minimum DNA nucleic acid amount detected by amplification is 100 pg/ 渭 L, and the minimal detected colony number is 105 CFU.. The multiplex PCR was used to detect 25 strains of APEC (all belong to B2 type) and 79 strains of non-pathogenic Escherichia coli (31.65% belong to group A, 50.63% belong to group B1, 1.27% belong to group B2 and 16.46% belong to group D). The results showed that when the isolates contained 3 or more of the above 6 virulence genes, the probability was 92.0% that the isolates were pathogenicity, and the error rate was 8.0%. The established multiplex PCR method can be used to detect APEC. easily and quickly. This study will provide some support for the further study of APEC in Xuzhou, Jiangsu Province.
【学位授予单位】：牡丹江师范学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.61

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