鱼源藤黄微球菌三重PCR检测法的建立及耐药性分析

为鉴定感染黄颡鱼菌株fsznc-CL、建立一种快速检测的方法和探讨其耐药特性。采用形态学、理化特性及16S rDNA序列分析方法进行菌株鉴定,人工回归感染研究其致病性。通过对三对特异性引物的反应体系和条件进行优化,建立一种三重PCR检测法并应用到人工感染黄颡鱼的检测中。运用PCR方法扩增耐药基因,用琼脂纸片扩散法检测该菌的药敏特性。结果表明,分离菌株fsznc-CL为藤黄微球菌(Micrococcus luteus),该菌对黄颡鱼具有一定的致病性。三重PCR检测法可准确扩增出藤黄微球菌CBS、Sig和Pol三对目的基因,而其他菌株未扩增出,检测该菌的最低模板浓度为5.822×10^-3 ng/μL,该方法检测带菌样品的阳性率约为83.33%。该菌携带了TEM、aph(3')-Ⅱa、aac(6')-Ⅰ、Sul1和Sul2耐药基因。菌株fsznc-CL对诺氟沙星、苯唑西林、红霉素和呋喃唑酮耐药。本试验建立的三重PCR检测法具有较高的特异性和灵敏度,目前藤黄微球菌对少部分抗生素耐药。

Development of a Triple PCR Detection Method and Antibiotic Resistance Analysis for Micrococcus luteus Isolated from Fish

To identify strain fsznc-CL isolated from Pelteobagrus fulvidraco,establish a rapid detection method and discuss the drug sensitivity. The strain was identified by the morphological,physical and chemical characteristics and 16S rDNA sequence. Through the optimization of reaction system and conditions for three pairs of specific primers,a triple PCR detection method was established. This method was applied to the detection of thesamples infected Pelteobagrus fulvidraco. The drug resistance genes were amplified by PCR and the drug sensitivity of was detected by disk agar diffusion method. The results showed that strain fsznc-CL was Micrococcus luteus. Strain fsznc-CL was lethal to Pelteobagrus fulvidraco. Three pairs of target genes of Micrococcus luteus CBS,Sig and Pol could be accurately amplified by triple-PCR detection,while the other strains could not be amplified. The minimum template concentration for the detection of Micrococcus luteus was 5.822×10-3 ng/μL. The positive rate of this method in detected sampleswas about 83.33%.This strain has drug resistance genes of TEM,aph(3')-Ⅱa,aac(6')-I,Sul1 and Sul2.Strain fsznc-CL was resistant to norfloxacin,oxacillin,erythromycin and furazolidone. This triple PCR method had high specificity and sensitivity. Micrococcus luteus was resistant to a few antibiotics at present.