摘要
This study was to develop an efficient and simple method for the detection of duck Tembusu virus( DTMUV) by loop-mediated isothermal amplification( LAMP). Six pairs of LAMP primers were designed according to the conserved region of the DTMUV E gene sequence in Gen Bank,which were then used for the optimization of various reaction components and reaction system of specific LAMP for DTMUV. Further the fluorescent reagent SYBR Green I and a certain proportion of calcium and manganese ion were used to determin the color development of products for visible analysis instead of agarose gel electrophoresis. The results showed that the sensitivity SYBR Green I as the fluorescent reagent was 10 copies viruses per μL,which is 100 times higher than normal PCR method,while the detection limit of combined use of calcium and manganese ion was 1 000 copies viruses per μL. Although the sensitivity of mixture of calcium and manganese ion is lower than SYBR Green I,it can avoid the aerosol contamination. The fluorogenic analysis-based LAMP system established in our study has a high sensitivity and avoid the cross contamination,which is of huge potential in research institutions,grass-roots laboratories and field testing and can provide effective means to completely curb the occurrence and spreading of DTMUV.
This study was to develop an efficient and simple method for the detection of duck Tembusu virus( DTMUV) by loop-mediated isothermal amplification( LAMP). Six pairs of LAMP primers were designed according to the conserved region of the DTMUV E gene sequence in Gen Bank,which were then used for the optimization of various reaction components and reaction system of specific LAMP for DTMUV. Further the fluorescent reagent SYBR Green I and a certain proportion of calcium and manganese ion were used to determin the color development of products for visible analysis instead of agarose gel electrophoresis. The results showed that the sensitivity SYBR Green I as the fluorescent reagent was 10 copies viruses per μL,which is 100 times higher than normal PCR method,while the detection limit of combined use of calcium and manganese ion was 1 000 copies viruses per μL. Although the sensitivity of mixture of calcium and manganese ion is lower than SYBR Green I,it can avoid the aerosol contamination. The fluorogenic analysis-based LAMP system established in our study has a high sensitivity and avoid the cross contamination,which is of huge potential in research institutions,grass-roots laboratories and field testing and can provide effective means to completely curb the occurrence and spreading of DTMUV.
基金
Supported by Special Fund for Agro-scientific Research in the Public Interest(201003012)
