摘要
为建立一种鰤鱼诺卡氏菌(Nocardia seriolae)多重PCR的分型方法,对已公布的 20 个鰤鱼诺卡氏菌全基因组数据进行对比分析,筛选鰤鱼诺卡氏菌种内差异基因片段并设计PCR引物,经单重PCR验证确认后进行引物组合、引物配比优化,构建鰤鱼诺卡氏菌的多重PCR菌株分型方法,并对该菌株分型方法进行灵敏度分析和病例应用.结果表明:在试验中构建的多重PCR菌株分型方法可用于鰤鱼诺卡氏菌培养物和感染鱼样分型,并确认鰤鱼诺卡氏菌ArsC、AraC、ISOPREN、ALAT、IclR基因片段具有种内差异;其中多重PCR引物组合Ⅰ(ISOPREN-F/R、ALAT-F/R、IclR-F/R)引物最优配比为ISOPREN-F/R∶ALAT-F/R∶IclR-F/R=2∶1∶2,灵敏度达 125 pg/μL,组合Ⅱ(AraC-F/R、ALAT-F/R、IclR-F/R)引物最佳配比为AraC-F/R∶ALAT-F/R∶IclR-F/R=1∶1∶1,灵敏度达 500 pg/μL,组合Ⅲ(ArsC-F/R、ALAT-F/R、IclR-F/R)引物最佳配比为ArsC-F/R∶ALAT-F/R∶IclR-F/R=1∶2∶2,灵敏度达 500 pg/μL.研究表明,本研究中建立的鰤鱼诺卡氏菌的多重PCR菌株分型方法,可在 1～2 h内完成鰤鱼诺卡氏菌的菌株分型鉴定,为水产实践中鰤鱼诺卡氏菌感染病例的菌株分型提供了快速、准确、实用的鉴定方法,可助力鱼类诺卡氏菌病的防控.
Abstract
To establish a multiplex PCR typing method for pathogenic bacterium(Nocardia seriolae)strain,the whole genome data of 20 published N.seriolae strains were compared and analyzed to screen the differential gene frag-ments and to design PCR primers.After confirmation by simplex PCR,the primer combinations and primer ratios were optimized to construct a multiplex PCR N.seriolae strains typing method,then sensitivity analysis and case application of the strain typing method were conducted on.The results showed that multiplex PCR strains typing method construc-ted here was used to type N.seriolae cultures and infecte fish,with confirmation of the intraspecific difference of N.se-riolae ArsC,AraC,ISOPREN,ALAT,and IclR gene fragment.The optimal primer ratio in primers combination Ⅰ(ISOPREN-F/R,ALAT-F/R,IclR-F/R),the ratio was shown to be ISOPREN-F/R:ALAT-F/R:IclR-F/R=2:1:2,with the sensitivity of 125 pg/μL for the strain typing method using multiplex PCR.For primers combina-tion Ⅱ(AraC-F/R,ALAT-F/R,IclR-F/R),the optimal primer ratio was found to be AraC-F/R:ALAT-F/R:IclR-F/R=1:1:1,with the sensitivity of 500 pg/μL.For primers combinationⅢ(ArsC-F/R,ALAT-F/R,IclR-F/R),the optimal primer ratio was ArsC-F/R:ALAT-F/R:IclR-F/R=1:2:2,with the detection sensitivity of 500 pg/μL.In the present research,N.seriolae multiplex PCR strains typing method was established,and completed the typing identification of N.seriolae in 1 to 2 hours.The findings provide a rapid,accurate and practical identifica-tion method for strains typing of N.seriolae infection cases in aquaculture practice.
维普
万方数据