Establishment and application of multiplex PCR typing method for pathogenic bacterium(Nocardia seriolae)
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.
万方数据
维普
