摘要
建立多重PCR诊断方法。以复活羊败血性链球菌病活疫苗作为研究对象,根据细菌16S rRNA基因通用引物和羊败血性链球菌种属特异性sodA基因,参考文献设计合成引物并进行PCR扩增。结果显示:(1)疫苗株接种BHI增菌培养,细菌生长缓慢,24 h后BHI培养基变浑浊;在血平板上形成有光泽、透明湿润、黏稠,表面光滑、边缘整齐,露滴状细小、灰白色的菌落,菌落周围有明显的β溶血。(2)羊败血性链球菌病疫苗株细菌镜检结果显示,镜检可见呈革兰氏染色阳性,常成对或3~5个短链排列的球形或椭圆形球菌,有荚膜。(3)羊败血性链球菌病疫苗株核酸提取结果显示,提取核酸稳定性良好,可用于后续实验研究。(4)PCR扩增结果显示,该方法特异性和敏感性好,可为羊败血性链球菌病的实验室诊断检测和分子流行病学调查提供技术支撑。研究认为,多重PCR检测方法简便、快捷,敏感性和特异性良好,技术易掌握,普遍适合基层单位使用,可快速获得诊断结果,确定病原,为临床合理用药提供科学依据。
A multiplex PCR diagnostic method was established.The live vaccine of resurrected ovine Streptococcus septicus disease was used as the research object.According to the universal primers of bacterial 16S rRNA gene and the species-specific sodA gene of ovine streptococcus septicus,the primers were designed and synthesized and amplified by PCR.The results showed as follows:(1)The vaccine were inoculated with BHI medium,the growth of bacteria was slow,and the BHI medium became cloudy after 24 h;On the blood plate,shiny,transparent,moist,viscous,smooth surface,neat edges,small dew drops,gray and white colonies were formed,and there was obvious beta hemolysis around the colonies.(2)The microscopic examination results of the bacteria of the ovine septicemia streptococci vaccine strain showed that the microscopic examination showed gram staining positive,often in pairs or 3 to 5 short chain arranged spherical or oval coccus,with capsules.(3)The results of nucleic acid extraction showed that the extracted nucleic acid was stable and could be used for subsequent experimental studies.(4)PCR amplification results showed that the method had good specificity and sensitivity,and could provide technical support for laboratory diagnostic detection and molecular epidemiological investigation of sheep septicemia streptococcus disease.The study concluded that the multiplex PCR detection method is simple,rapid,sensitive and specific,easy to master,and generally suitable for grass-roots units to use,can quickly obtain diagnostic results,identify the pathogen,and provide scientific basis for clinical rational drug use.
作者
魏梦君
郑思思
游潇倩
孙生祯
辛莉
阚威
WEI Mengjun;ZHENG Sisi;YOU Xiaoqian;SUN Shengzhen;XIN Li;KAN Wei(Qinghai Provincial Center for Animal Disease Control and Prevention,Xining,Qinghai 81oooo,China)
出处
《畜牧兽医杂志》
2024年第5期80-82,共3页
Journal of Animal Science and Veterinary Medicine
