鲤春病毒血症病毒(spring viremia of carp virus,SVCV)是一种高致病性病原,对鲤科鱼类造成巨大危害,被世界动物卫生组织列为必须通报疫病。2002年,鲤春病毒血症(spring viremia of carp,SVC)在中国首次检出后,迅速在全国蔓延,给我国鲤...鲤春病毒血症病毒(spring viremia of carp virus,SVCV)是一种高致病性病原,对鲤科鱼类造成巨大危害,被世界动物卫生组织列为必须通报疫病。2002年,鲤春病毒血症(spring viremia of carp,SVC)在中国首次检出后,迅速在全国蔓延,给我国鲤鱼养殖业造成巨大威胁。因此,我国农业农村部将其列为二类动物疫病。迄今为止,早期、快速、准确的诊断仍然是控制其传播和流行的重要手段。本研究基于CRISPR/Cas13a系统结合重组酶介导等温核酸扩增技术(recombinase-aided amplification,RAA),通过对GenBank上登录的SVCV全基因序列比对,针对SVCV聚合酶L基因高度保守的区域设计了一组RAA扩增引物和相应crRNA;建立了一种可用于SVCV现场快速检测的RAA-CRISPR/Cas13a的方法,可以覆盖SVCV的4种不同基因型(Ia、Ib、Ic和Id)。按照世界动物卫生组织推荐的检测方法验证程序,经实验验证该检测方法有较好的重复性,最低检出限为115 copies/μL,与其他病原之间不发生交叉反应。通过对实验室分离保存的60份来自进出境水生动物疫病监测、国内重大水生动物疫病监测以及攻毒实验的样本进行检测,RAA-CRISPR/Cas13a检测结果与实时荧光定量PCR、套式RT-PCR和病毒分离培养结果完全一致,诊断灵敏度和诊断特异性均达到100%。采用本研究建立的方法在4个不同实验室开展比对,在相同条件下对20份样本进行检测,4个实验室的检测结果一致。结果表明,本研究建立的方法重现性良好,为首次采用CRISPR-Cas13a系统进行SVCV检测的研究,对SVCV快速诊断和防控具有重要现实意义。展开更多
The advancement of Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)gene editing technology has revolutionized the comprehension of human genome,propelling molecular and cellular biology research into ...The advancement of Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)gene editing technology has revolutionized the comprehension of human genome,propelling molecular and cellular biology research into unexplored realms and accelerating progress in life sciences and medicine.CRISPR-based gene screening,recognized for its efficiency and practicality,is widely utilized across diverse biological fields.Aging is a multifaceted process governed by a myriad of genetic and epigenetic factors.Unraveling the genes regulating aging holds promise for understanding this intricate phenomenon and devising strategies for its assessment and intervention.This review provides a comprehensive overview of the progress in CRISPR screening and its applications in aging research,while also offering insights into future directions.CRISPR-based genetic-manipulation tools are positioned as indispensable instruments for mitigating aging and managing age-related diseases.展开更多
文摘鲤春病毒血症病毒(spring viremia of carp virus,SVCV)是一种高致病性病原,对鲤科鱼类造成巨大危害,被世界动物卫生组织列为必须通报疫病。2002年,鲤春病毒血症(spring viremia of carp,SVC)在中国首次检出后,迅速在全国蔓延,给我国鲤鱼养殖业造成巨大威胁。因此,我国农业农村部将其列为二类动物疫病。迄今为止,早期、快速、准确的诊断仍然是控制其传播和流行的重要手段。本研究基于CRISPR/Cas13a系统结合重组酶介导等温核酸扩增技术(recombinase-aided amplification,RAA),通过对GenBank上登录的SVCV全基因序列比对,针对SVCV聚合酶L基因高度保守的区域设计了一组RAA扩增引物和相应crRNA;建立了一种可用于SVCV现场快速检测的RAA-CRISPR/Cas13a的方法,可以覆盖SVCV的4种不同基因型(Ia、Ib、Ic和Id)。按照世界动物卫生组织推荐的检测方法验证程序,经实验验证该检测方法有较好的重复性,最低检出限为115 copies/μL,与其他病原之间不发生交叉反应。通过对实验室分离保存的60份来自进出境水生动物疫病监测、国内重大水生动物疫病监测以及攻毒实验的样本进行检测,RAA-CRISPR/Cas13a检测结果与实时荧光定量PCR、套式RT-PCR和病毒分离培养结果完全一致,诊断灵敏度和诊断特异性均达到100%。采用本研究建立的方法在4个不同实验室开展比对,在相同条件下对20份样本进行检测,4个实验室的检测结果一致。结果表明,本研究建立的方法重现性良好,为首次采用CRISPR-Cas13a系统进行SVCV检测的研究,对SVCV快速诊断和防控具有重要现实意义。
文摘The advancement of Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)gene editing technology has revolutionized the comprehension of human genome,propelling molecular and cellular biology research into unexplored realms and accelerating progress in life sciences and medicine.CRISPR-based gene screening,recognized for its efficiency and practicality,is widely utilized across diverse biological fields.Aging is a multifaceted process governed by a myriad of genetic and epigenetic factors.Unraveling the genes regulating aging holds promise for understanding this intricate phenomenon and devising strategies for its assessment and intervention.This review provides a comprehensive overview of the progress in CRISPR screening and its applications in aging research,while also offering insights into future directions.CRISPR-based genetic-manipulation tools are positioned as indispensable instruments for mitigating aging and managing age-related diseases.
