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 conse...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.展开更多
为明确鸭特异性寡聚脱氧核苷酸(Cp G ODN)体外对鸭坦布苏病毒(DTMUV)复制的抑制作用,研究在Cp G ODN最佳活性分子筛选的基础上,与DTMUV感染后的细胞进行共孵育,分别采用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐法(MTT法)和实时荧光定...为明确鸭特异性寡聚脱氧核苷酸(Cp G ODN)体外对鸭坦布苏病毒(DTMUV)复制的抑制作用,研究在Cp G ODN最佳活性分子筛选的基础上,与DTMUV感染后的细胞进行共孵育,分别采用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐法(MTT法)和实时荧光定量PCR(q RT-PCR)检测不同浓度Cp G ODN-D2对DTMUV复制的抑制作用。结果显示:对非洲绿猴肾(Vero)细胞来说,Cp G ODN-D2浓度在300μg/m L以内均为安全浓度;MTT法和q RT-PCR检测结果均表明,各浓度Cp G ODN-D2对DTMUV的复制均有一定的抑制作用,并随浓度升高呈现先上升后下降的趋势,其中30μg/m L的浓度效果最佳,达到84.74%。结果证实了Cp G ODN-D2作为新型免疫调节剂可有效抑制DTMUV的复制。展开更多
鸭坦布苏病毒(DTMUV)为新出现的病毒,主要引起鸭的产蛋量急剧下降。为评价真核表达DTMUV的E蛋白的免疫原性,本研究利用杆状病毒表面展示技术,构建表面展示DTMUV JSXZ株E蛋白的重组杆状病毒(r BV-DTMUV-E),将其感染昆虫细胞,从而将E蛋白...鸭坦布苏病毒(DTMUV)为新出现的病毒,主要引起鸭的产蛋量急剧下降。为评价真核表达DTMUV的E蛋白的免疫原性,本研究利用杆状病毒表面展示技术,构建表面展示DTMUV JSXZ株E蛋白的重组杆状病毒(r BV-DTMUV-E),将其感染昆虫细胞,从而将E蛋白展示在杆状病毒的表面,并对表达的重组E蛋白(r E)的免疫原性和保护效果进行研究。经western blot和间接免疫荧光试验证明r E能够在Sf9细胞中有效表达;并且呈可溶性表达。重组蛋白经镍柱纯化,并采用氢氧化铝乳化(0.1μg/m L),分别以0.2 m L、0.4 m L和0.6 m L的剂量免疫雏鸭,通过ELISA抗体检测结果证明,r E可以诱导产生高水平的Ig G血清抗体;MTT试验结果表明,r E能够刺激T淋巴细胞增殖;攻毒保护试验结果显示,其0.2 m L组免疫保护率为80%,0.4 m L和0.6 m L组保护率均为100%,而对照组雏鸭均表现为典型的DTMUV感染症状,其中4只死亡。以上研究结果为DTMUV亚单位疫苗的研究奠定了基础。展开更多
基金Supported by Special Fund for Agro-scientific Research in the Public Interest(201003012)
文摘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.
文摘为明确鸭特异性寡聚脱氧核苷酸(Cp G ODN)体外对鸭坦布苏病毒(DTMUV)复制的抑制作用,研究在Cp G ODN最佳活性分子筛选的基础上,与DTMUV感染后的细胞进行共孵育,分别采用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐法(MTT法)和实时荧光定量PCR(q RT-PCR)检测不同浓度Cp G ODN-D2对DTMUV复制的抑制作用。结果显示:对非洲绿猴肾(Vero)细胞来说,Cp G ODN-D2浓度在300μg/m L以内均为安全浓度;MTT法和q RT-PCR检测结果均表明,各浓度Cp G ODN-D2对DTMUV的复制均有一定的抑制作用,并随浓度升高呈现先上升后下降的趋势,其中30μg/m L的浓度效果最佳,达到84.74%。结果证实了Cp G ODN-D2作为新型免疫调节剂可有效抑制DTMUV的复制。
文摘鸭坦布苏病毒(DTMUV)为新出现的病毒,主要引起鸭的产蛋量急剧下降。为评价真核表达DTMUV的E蛋白的免疫原性,本研究利用杆状病毒表面展示技术,构建表面展示DTMUV JSXZ株E蛋白的重组杆状病毒(r BV-DTMUV-E),将其感染昆虫细胞,从而将E蛋白展示在杆状病毒的表面,并对表达的重组E蛋白(r E)的免疫原性和保护效果进行研究。经western blot和间接免疫荧光试验证明r E能够在Sf9细胞中有效表达;并且呈可溶性表达。重组蛋白经镍柱纯化,并采用氢氧化铝乳化(0.1μg/m L),分别以0.2 m L、0.4 m L和0.6 m L的剂量免疫雏鸭,通过ELISA抗体检测结果证明,r E可以诱导产生高水平的Ig G血清抗体;MTT试验结果表明,r E能够刺激T淋巴细胞增殖;攻毒保护试验结果显示,其0.2 m L组免疫保护率为80%,0.4 m L和0.6 m L组保护率均为100%,而对照组雏鸭均表现为典型的DTMUV感染症状,其中4只死亡。以上研究结果为DTMUV亚单位疫苗的研究奠定了基础。
