为了鉴定异性双胎牛性器官发育紊乱疾病——牛自由马丁(freemartinism)综合征,试验选择7头中国荷斯坦异性孪生双胎的母牛进行微滴数字PCR(droplet digital PCR,ddPCR)检测,并根据异性孪生母犊不孕的个体染色体为XX/XY嵌合、正常母牛染...为了鉴定异性双胎牛性器官发育紊乱疾病——牛自由马丁(freemartinism)综合征,试验选择7头中国荷斯坦异性孪生双胎的母牛进行微滴数字PCR(droplet digital PCR,ddPCR)检测,并根据异性孪生母犊不孕的个体染色体为XX/XY嵌合、正常母牛染色体为XX来判定是否为嵌合体。结果表明:此7个样本均存在XX/XY染色体嵌合,均为异性孪生不孕母犊。说明ddPCR方法用于鉴定牛自由马丁现象是有效的。展开更多
Background Tuberous sclerosis complex(TSC),an inherited neurocutaneous disorder,is caused by variants in the TSC1 or TSC2 genes.The mosaic variants of TSC1 and TSC2 are scarcely detectable using the conventional nextg...Background Tuberous sclerosis complex(TSC),an inherited neurocutaneous disorder,is caused by variants in the TSC1 or TSC2 genes.The mosaic variants of TSC1 and TSC2 are scarcely detectable using the conventional nextgeneration sequencing(NGS).Therefore,this study aims to explore the detection and distribution of mosaic variants within affected families.Methods Through whole-exome sequencing(WES)or the TSC1/TSC2 panel to detect the variants of the TSC1 and TSC2 genes,the reaction system of droplet digital PCR(ddPCR)was designed to detect the mosaicism of these variants in affected families.Results Genetic testing was carried out on 29 TSC patients via WES or the TSC1/TSC2 panel.The results showed that 27 patients had positive results in the TSC gene variant tests.Fourteen cases were confirmed as de novo variants,and the asymptomatic fathers or mothers of 4 patients were identified as somatic mosaics by ddPCR,with mosaic proportions of 0.8%,24.18%,8.02%,and 0.33%respectively.Conclusions The ddPCR holds the potential to improve diagnostic accuracy,genetic risk assessment,and clinical diagnosis rates.Consequently,it could potentially be adopted as one of the modalities for prompt clinical diagnosis.展开更多
Carassius auratus herpesvirus(CaHV)is a pathogen isolated from crucian carp(Carassius auratus)associated with high mortality.A diagnosis method that can detect the virus at an early stage,specifically and accurately,i...Carassius auratus herpesvirus(CaHV)is a pathogen isolated from crucian carp(Carassius auratus)associated with high mortality.A diagnosis method that can detect the virus at an early stage,specifically and accurately,is an urgent requirement for the prevention of CaHV transmission.In the present study,a droplet digital PCR(ddPCR)method based on the tumor necrosis factor receptor(TNFR)gene was established to detect and quantify CaHV DNA with high specificity and no cross-reactions with other aquatic viruses.Skin mucus samples were collected from infected crucian carp from Day 1–8 after infection,and positive amplification was detected on the first day by ddPCR(0.54 copies/μL),whereas the presence of CaHV was not detected by routine PCR until Day 6.Tissue DNA was then collected from the head kidney of 20 fishes which were injected with CaHV and died during the experiment.The five negative samples checked by routine PCR were detected by ddPCR and real-time PCR(qPCR),respectively.The results showed that the positive detection rate of ddPCR(100%)was higher than that of qPCR(40%).The detection limit of the ddPCR was found to be 0.52 copies/μL,which was much lower than the 50.12 copies/μL determined by qPCR.Overall,ddPCR offers a highly promising diagnosis method for the absolute quantification of CaHV in carrier fish and samples from the skin mucus and head kidney with low viral concentrations.展开更多
文摘为了鉴定异性双胎牛性器官发育紊乱疾病——牛自由马丁(freemartinism)综合征,试验选择7头中国荷斯坦异性孪生双胎的母牛进行微滴数字PCR(droplet digital PCR,ddPCR)检测,并根据异性孪生母犊不孕的个体染色体为XX/XY嵌合、正常母牛染色体为XX来判定是否为嵌合体。结果表明:此7个样本均存在XX/XY染色体嵌合,均为异性孪生不孕母犊。说明ddPCR方法用于鉴定牛自由马丁现象是有效的。
基金supported by Wuhan Knowledge innovation special project(2023020201020554)the Key research and development program of Hubei Province(2023BCB136)+3 种基金the Construction of the Clinical Research Laboratory of Wuhan Children’s Hospital(2022 FEYJS001)Construction Project of Hubei Provincial Clinical Medical Research Center for Childhood Neurodevelopmental Disorders(No.HST2020-19)Nature Science Foundation of Hubei Province(2023 AFB360)Research Project Grant of Wuhan Children’s Hospital(2023 FEBSJJ002).
文摘Background Tuberous sclerosis complex(TSC),an inherited neurocutaneous disorder,is caused by variants in the TSC1 or TSC2 genes.The mosaic variants of TSC1 and TSC2 are scarcely detectable using the conventional nextgeneration sequencing(NGS).Therefore,this study aims to explore the detection and distribution of mosaic variants within affected families.Methods Through whole-exome sequencing(WES)or the TSC1/TSC2 panel to detect the variants of the TSC1 and TSC2 genes,the reaction system of droplet digital PCR(ddPCR)was designed to detect the mosaicism of these variants in affected families.Results Genetic testing was carried out on 29 TSC patients via WES or the TSC1/TSC2 panel.The results showed that 27 patients had positive results in the TSC gene variant tests.Fourteen cases were confirmed as de novo variants,and the asymptomatic fathers or mothers of 4 patients were identified as somatic mosaics by ddPCR,with mosaic proportions of 0.8%,24.18%,8.02%,and 0.33%respectively.Conclusions The ddPCR holds the potential to improve diagnostic accuracy,genetic risk assessment,and clinical diagnosis rates.Consequently,it could potentially be adopted as one of the modalities for prompt clinical diagnosis.
基金financially supported by the Science and Technology Commission of Shanghai Municipality(21ZR1427200).
文摘Carassius auratus herpesvirus(CaHV)is a pathogen isolated from crucian carp(Carassius auratus)associated with high mortality.A diagnosis method that can detect the virus at an early stage,specifically and accurately,is an urgent requirement for the prevention of CaHV transmission.In the present study,a droplet digital PCR(ddPCR)method based on the tumor necrosis factor receptor(TNFR)gene was established to detect and quantify CaHV DNA with high specificity and no cross-reactions with other aquatic viruses.Skin mucus samples were collected from infected crucian carp from Day 1–8 after infection,and positive amplification was detected on the first day by ddPCR(0.54 copies/μL),whereas the presence of CaHV was not detected by routine PCR until Day 6.Tissue DNA was then collected from the head kidney of 20 fishes which were injected with CaHV and died during the experiment.The five negative samples checked by routine PCR were detected by ddPCR and real-time PCR(qPCR),respectively.The results showed that the positive detection rate of ddPCR(100%)was higher than that of qPCR(40%).The detection limit of the ddPCR was found to be 0.52 copies/μL,which was much lower than the 50.12 copies/μL determined by qPCR.Overall,ddPCR offers a highly promising diagnosis method for the absolute quantification of CaHV in carrier fish and samples from the skin mucus and head kidney with low viral concentrations.
