猫疱疹病毒I型(FHV-1)是威胁猫科动物健康的重要传染性疾病,本研究旨在研发一种灵敏、高效的FHV-1检测技术。依据GenBank上FHV-1 US6保守区域的基因序列,设计合成1对针对FHV-1 gD基因的引物,建立了一种SYBR Green I荧光定量PCR的检测方...猫疱疹病毒I型(FHV-1)是威胁猫科动物健康的重要传染性疾病,本研究旨在研发一种灵敏、高效的FHV-1检测技术。依据GenBank上FHV-1 US6保守区域的基因序列,设计合成1对针对FHV-1 gD基因的引物,建立了一种SYBR Green I荧光定量PCR的检测方法,构建标准曲线后分别验证该方法的特异性、敏感性、重复性,并将其进一步应用于人工感染猫产生的临床样本检测。结果:该特异性引物与猫杯状病毒(FCV)、猫细小病毒(FPV)和猫冠状病毒(FCoV)均未出现交叉反应,检测下限为14.78 copies/μL,组内和组间重复试验的变异系数均低于2%;该方法对临床样本的检出率比常规PCR高出25.46%;通过该方法检测人工感染FHV-1强毒后猫的每日排毒量,结果呈现上升趋势,与临床发病程度相符,猫的脏器病毒载量存在个体差异,但集中在心脏、肺脏、肠道和膀胱中检出。综上,该研究建立的SYBR Green I荧光定量PCR方法对FHV-1具有较好的特异性、灵敏度和重复性,为FHV-1感染的快速诊断以及疾病的防控提供方法支持。展开更多
DNA barcoding has been extensively used for species identification.However,species identification of mixed samples or degraded DNA is limited by current DNA barcoding methods.In this study,we use plant species in Jugl...DNA barcoding has been extensively used for species identification.However,species identification of mixed samples or degraded DNA is limited by current DNA barcoding methods.In this study,we use plant species in Juglandaceae to evaluate an assembly-free reads accurate identification(AFRAID)method of species identification,a novel approach for precise species identification in plants.Specifically,we determined(1)the accuracy of DNA barcoding approaches in delimiting species in Juglandaceae,(2)the minimum size of chloroplast dataset for species discrimination,and(3)minimum amount of next generation sequencing(NGS)data required for species identification.We found that species identification rates were highest when whole chloroplast genomes were used,followed by taxon-specific DNA barcodes,and then universal DNA barcodes.Species identification of 100%was achieved when chloroplast genome sequence coverage reached 20%and the original sequencing data reached 500,000 reads.AFRAID accurately identified species for all samples tested after 500,000 clean reads,with far less computing time than common approaches.These results provide a new approach to accurately identify species,overcoming limitations of traditional DNA barcodes.Our method,which uses next generation sequencing to generate partial chloroplast genomes,reveals that DNA barcode regions are not necessarily fixed,accelerating the process of species identification.展开更多
The East China Sea(ECS)off the Coast of Zhoushan Archipelago,Zhejiang(ECS-CZA)is home to abundant fishery resources and an important spawning,feeding,and nursing ground for a variety of fish species.Due to long-term o...The East China Sea(ECS)off the Coast of Zhoushan Archipelago,Zhejiang(ECS-CZA)is home to abundant fishery resources and an important spawning,feeding,and nursing ground for a variety of fish species.Due to long-term overfishing,the ichthyoplankton structure has been dramatically altered.Understanding the species composition and distribution of fish eggs and larvae is one of the most essential tasks to accurately regulate fishery resources and formulate effective management policies;however,little is known about the ichthyoplankton in this region.In this study,an integrated strategy of morphology identification(MI)and mitochondrial COI DNA barcoding was used to identify species of fish eggs and larvae collected from the ECSCZA.MI revealed 15 fish egg species belonging to 12 families and 12 fish larva species belonging to 12 families;in contrast,DNA barcoding altogether identified 30 species,including 18 fish egg species and 13 fish larva species.One species was shared between the egg and larva samples.Our study offers useful tools and critical scientific information for further understanding the diversity,distribution,and conservation management of various ichthyoplankton species in the marine environment.展开更多
文摘猫疱疹病毒I型(FHV-1)是威胁猫科动物健康的重要传染性疾病,本研究旨在研发一种灵敏、高效的FHV-1检测技术。依据GenBank上FHV-1 US6保守区域的基因序列,设计合成1对针对FHV-1 gD基因的引物,建立了一种SYBR Green I荧光定量PCR的检测方法,构建标准曲线后分别验证该方法的特异性、敏感性、重复性,并将其进一步应用于人工感染猫产生的临床样本检测。结果:该特异性引物与猫杯状病毒(FCV)、猫细小病毒(FPV)和猫冠状病毒(FCoV)均未出现交叉反应,检测下限为14.78 copies/μL,组内和组间重复试验的变异系数均低于2%;该方法对临床样本的检出率比常规PCR高出25.46%;通过该方法检测人工感染FHV-1强毒后猫的每日排毒量,结果呈现上升趋势,与临床发病程度相符,猫的脏器病毒载量存在个体差异,但集中在心脏、肺脏、肠道和膀胱中检出。综上,该研究建立的SYBR Green I荧光定量PCR方法对FHV-1具有较好的特异性、灵敏度和重复性,为FHV-1感染的快速诊断以及疾病的防控提供方法支持。
基金supported by the funds from Natural Science Foundation of Hebei Province(C2022402017).
文摘DNA barcoding has been extensively used for species identification.However,species identification of mixed samples or degraded DNA is limited by current DNA barcoding methods.In this study,we use plant species in Juglandaceae to evaluate an assembly-free reads accurate identification(AFRAID)method of species identification,a novel approach for precise species identification in plants.Specifically,we determined(1)the accuracy of DNA barcoding approaches in delimiting species in Juglandaceae,(2)the minimum size of chloroplast dataset for species discrimination,and(3)minimum amount of next generation sequencing(NGS)data required for species identification.We found that species identification rates were highest when whole chloroplast genomes were used,followed by taxon-specific DNA barcodes,and then universal DNA barcodes.Species identification of 100%was achieved when chloroplast genome sequence coverage reached 20%and the original sequencing data reached 500,000 reads.AFRAID accurately identified species for all samples tested after 500,000 clean reads,with far less computing time than common approaches.These results provide a new approach to accurately identify species,overcoming limitations of traditional DNA barcodes.Our method,which uses next generation sequencing to generate partial chloroplast genomes,reveals that DNA barcode regions are not necessarily fixed,accelerating the process of species identification.
文摘The East China Sea(ECS)off the Coast of Zhoushan Archipelago,Zhejiang(ECS-CZA)is home to abundant fishery resources and an important spawning,feeding,and nursing ground for a variety of fish species.Due to long-term overfishing,the ichthyoplankton structure has been dramatically altered.Understanding the species composition and distribution of fish eggs and larvae is one of the most essential tasks to accurately regulate fishery resources and formulate effective management policies;however,little is known about the ichthyoplankton in this region.In this study,an integrated strategy of morphology identification(MI)and mitochondrial COI DNA barcoding was used to identify species of fish eggs and larvae collected from the ECSCZA.MI revealed 15 fish egg species belonging to 12 families and 12 fish larva species belonging to 12 families;in contrast,DNA barcoding altogether identified 30 species,including 18 fish egg species and 13 fish larva species.One species was shared between the egg and larva samples.Our study offers useful tools and critical scientific information for further understanding the diversity,distribution,and conservation management of various ichthyoplankton species in the marine environment.
