目的比较DNATyperTM15和IdentifilerTM试剂盒的遗传学调查应用结果,以评价DNATyperTM15试剂盒的指标和性能。方法用DNATyperTM15和IdentifilerTM试剂盒同时对290份北方汉族群体的血样本进行扩增检测,比较遗传学统计数据。结果DNATyperT...目的比较DNATyperTM15和IdentifilerTM试剂盒的遗传学调查应用结果,以评价DNATyperTM15试剂盒的指标和性能。方法用DNATyperTM15和IdentifilerTM试剂盒同时对290份北方汉族群体的血样本进行扩增检测,比较遗传学统计数据。结果DNATyperTM15和IdentifilerTM荧光检测试剂盒中各基因座在汉族群体中的基因型分布均符合Hardy-W e inberg平衡,累积个人识别率和累计非父排除率:DNATyperTM15试剂盒分别为2.66×10-18和0.9999997;IdentifilerTM试剂盒分别为1.28×10-17和0.9999984。且两个试剂盒对相同基因座的频率调查数据一致。结论DNATyperTM15具有较高的个体识别和亲权鉴定能力,对法医学检案和DNA数据库建设具有应用价值。展开更多
The DNATyper^(TM)21 kit has good species specificity,typing accuracy,and locus amplification balance,which can be used for forensic genetic analysis such as individual identification and paternity testing.To improve t...The DNATyper^(TM)21 kit has good species specificity,typing accuracy,and locus amplification balance,which can be used for forensic genetic analysis such as individual identification and paternity testing.To improve the anti-inhibitor ability of this kit,the addition of bovine serum albumin(BSA),bovine thrombin(BT),FastStart Taq DNA Polymerase,TaKaRa Ex Taq Hot Start Version(Ex Taq HS),MyFiTM DNA Polymerase,and Klentaq DNA Polymerase(Klentaq)as PCR enhancers to the PCR reaction system was explored in the presence of different concentrations of inhibitors,such as indigo,humic acid(HA),hemoglobin,heme,and melanin.The results revealed that BSA exhibited high efficiency in improving the detection rate of STR loci;BT only helped to overcome the typing inhibition caused by indigo,melanin,HA,and heme;Four types of DNA polymerase had different anti-inhibitor effects on different inhibitors,among which Ex Taq HS was more effective than the other three DNA polymerases,but displayed comparatively lower resistance to hemoglobin than BSA and BT.This study provides basic data for further optimization of the kit through comprehensive analysis and facilitates its application in daily forensic investigations.展开更多
We describe the optimization and validation of the DNATyper^(TM)15 multiplex polymerase chain reaction(PCR)genotyping system for autosomal short tandem repeat(STR)amplification at 14 autosomal loci(D6S1043,D21S11,D7S8...We describe the optimization and validation of the DNATyper^(TM)15 multiplex polymerase chain reaction(PCR)genotyping system for autosomal short tandem repeat(STR)amplification at 14 autosomal loci(D6S1043,D21S11,D7S820,CSF1PO,D2S1338,D3S1358,D13S317,D8S1179,D16S539,Penta E,D5S818,vWA,D18S51,and FGA)and amelogenin,a sex‑determining locus.Several DNATyper^(TM)15 assay variables were optimized,including hot start Taq polymerase concentration,Taq polymerase activation time,magnesium concentration,primer concentration,annealing temperature,reaction volume,and cycle number.The performance of the assay was validated with respect to species specificity,sensitivity to template concentration,stability,accuracy,influence of the DNA extraction methods,and the ability to genotype the mixture samples.The performance of the DNATyper^(TM)15 system on casework samples was compared with that of two widely used STR amplification kits,Identifiler^(TM)(Applied Biosystems,Carlsbad,CA,USA)and PowerPlex 16®(Promega,Madison,WI,USA).The conditions for PCR‑based DNATyper^(TM)15 genotyping were optimized.Contamination from forensically relevant nonhuman DNA was not found to impact genotyping results,and full profiles were generated for all the reactions containing≥0.125 ng of DNA template.No significant difference in performance was observed even after the DNATyper^(TM)15 assay components were subjected to 20 freeze‑thaw cycles.The performances of DNATyper^(TM)15,Identifiler^(TM),and PowerPlex 16®were comparable in terms of sensitivity and the ability to genotype the mixed samples and case‑type samples,with the assays giving the same genotyping results for all the shared loci.The DNA extraction methods did not affect the performance of any of the systems.Our results demonstrate that the DNATyper^(TM)15 system is suitable for genotyping in both forensic DNA database work and case‑type samples.展开更多
Haemoglobinopathies are very serious clinical conditions caused by genetic mutations. They belong to autosomal recessive disorders and the most frequent genetic inherited diseases seen, specifically and above all amon...Haemoglobinopathies are very serious clinical conditions caused by genetic mutations. They belong to autosomal recessive disorders and the most frequent genetic inherited diseases seen, specifically and above all among Mediterranean countries. Thalassaemia syndromes (included Beta Thalassaemia and Sickle Cell Disease) have been the first diagnosed diseases since in intrauterine life using reccombinant DNA techniques. So, the better understanding of their pathophysiology has given a spectacolar improvement and a considerable impact on these conditional managements. Every year there are nearly 300,000 children born with haemoglobinopathies globally, and there are 60,000 - 70,000 children with Beta Thalassemia among them. Nowadays in Albania like everywhere, there is a significant increase of survival in these patients. As a result of life longevity and improvement of patients life quality, we can see that these patients may suffer from other concomitant illnesses. In our country, there are registered approximately 500 patients with haemoglobinopathies. We studied 50 pediatric patients at random ranging from age 2 until 18 years old. We excluded other pathologies among them. We found high values of biochemical indicators in blood (Ca 15-3 was found elevated in 75% of our patients, Lactate Dehydrogenases was found elevated in 70% of cases, Indirect Bilirubin was found elevated in 66% of cases). All three parameters are indicators of hemolysis. We found a correlation between high values of Ca 15-3 marker and high levels of LDH, Indirect Bilirubin and low level of hemoglobin (p < 0.05). Ca 15-3 is much elevated among patients non regularly transfused and in those who take an unsufficient amount of blood. Continuous monitoring of these biochemical parameters is going to help in the more effective follow up of patients with haemoglobinopathies.展开更多
文摘目的比较DNATyperTM15和IdentifilerTM试剂盒的遗传学调查应用结果,以评价DNATyperTM15试剂盒的指标和性能。方法用DNATyperTM15和IdentifilerTM试剂盒同时对290份北方汉族群体的血样本进行扩增检测,比较遗传学统计数据。结果DNATyperTM15和IdentifilerTM荧光检测试剂盒中各基因座在汉族群体中的基因型分布均符合Hardy-W e inberg平衡,累积个人识别率和累计非父排除率:DNATyperTM15试剂盒分别为2.66×10-18和0.9999997;IdentifilerTM试剂盒分别为1.28×10-17和0.9999984。且两个试剂盒对相同基因座的频率调查数据一致。结论DNATyperTM15具有较高的个体识别和亲权鉴定能力,对法医学检案和DNA数据库建设具有应用价值。
文摘The DNATyper^(TM)21 kit has good species specificity,typing accuracy,and locus amplification balance,which can be used for forensic genetic analysis such as individual identification and paternity testing.To improve the anti-inhibitor ability of this kit,the addition of bovine serum albumin(BSA),bovine thrombin(BT),FastStart Taq DNA Polymerase,TaKaRa Ex Taq Hot Start Version(Ex Taq HS),MyFiTM DNA Polymerase,and Klentaq DNA Polymerase(Klentaq)as PCR enhancers to the PCR reaction system was explored in the presence of different concentrations of inhibitors,such as indigo,humic acid(HA),hemoglobin,heme,and melanin.The results revealed that BSA exhibited high efficiency in improving the detection rate of STR loci;BT only helped to overcome the typing inhibition caused by indigo,melanin,HA,and heme;Four types of DNA polymerase had different anti-inhibitor effects on different inhibitors,among which Ex Taq HS was more effective than the other three DNA polymerases,but displayed comparatively lower resistance to hemoglobin than BSA and BT.This study provides basic data for further optimization of the kit through comprehensive analysis and facilitates its application in daily forensic investigations.
基金the National Science and Technology Supporting Program during the 10th 5‑year plan period(2001BA801B02)。
文摘We describe the optimization and validation of the DNATyper^(TM)15 multiplex polymerase chain reaction(PCR)genotyping system for autosomal short tandem repeat(STR)amplification at 14 autosomal loci(D6S1043,D21S11,D7S820,CSF1PO,D2S1338,D3S1358,D13S317,D8S1179,D16S539,Penta E,D5S818,vWA,D18S51,and FGA)and amelogenin,a sex‑determining locus.Several DNATyper^(TM)15 assay variables were optimized,including hot start Taq polymerase concentration,Taq polymerase activation time,magnesium concentration,primer concentration,annealing temperature,reaction volume,and cycle number.The performance of the assay was validated with respect to species specificity,sensitivity to template concentration,stability,accuracy,influence of the DNA extraction methods,and the ability to genotype the mixture samples.The performance of the DNATyper^(TM)15 system on casework samples was compared with that of two widely used STR amplification kits,Identifiler^(TM)(Applied Biosystems,Carlsbad,CA,USA)and PowerPlex 16®(Promega,Madison,WI,USA).The conditions for PCR‑based DNATyper^(TM)15 genotyping were optimized.Contamination from forensically relevant nonhuman DNA was not found to impact genotyping results,and full profiles were generated for all the reactions containing≥0.125 ng of DNA template.No significant difference in performance was observed even after the DNATyper^(TM)15 assay components were subjected to 20 freeze‑thaw cycles.The performances of DNATyper^(TM)15,Identifiler^(TM),and PowerPlex 16®were comparable in terms of sensitivity and the ability to genotype the mixed samples and case‑type samples,with the assays giving the same genotyping results for all the shared loci.The DNA extraction methods did not affect the performance of any of the systems.Our results demonstrate that the DNATyper^(TM)15 system is suitable for genotyping in both forensic DNA database work and case‑type samples.
文摘Haemoglobinopathies are very serious clinical conditions caused by genetic mutations. They belong to autosomal recessive disorders and the most frequent genetic inherited diseases seen, specifically and above all among Mediterranean countries. Thalassaemia syndromes (included Beta Thalassaemia and Sickle Cell Disease) have been the first diagnosed diseases since in intrauterine life using reccombinant DNA techniques. So, the better understanding of their pathophysiology has given a spectacolar improvement and a considerable impact on these conditional managements. Every year there are nearly 300,000 children born with haemoglobinopathies globally, and there are 60,000 - 70,000 children with Beta Thalassemia among them. Nowadays in Albania like everywhere, there is a significant increase of survival in these patients. As a result of life longevity and improvement of patients life quality, we can see that these patients may suffer from other concomitant illnesses. In our country, there are registered approximately 500 patients with haemoglobinopathies. We studied 50 pediatric patients at random ranging from age 2 until 18 years old. We excluded other pathologies among them. We found high values of biochemical indicators in blood (Ca 15-3 was found elevated in 75% of our patients, Lactate Dehydrogenases was found elevated in 70% of cases, Indirect Bilirubin was found elevated in 66% of cases). All three parameters are indicators of hemolysis. We found a correlation between high values of Ca 15-3 marker and high levels of LDH, Indirect Bilirubin and low level of hemoglobin (p < 0.05). Ca 15-3 is much elevated among patients non regularly transfused and in those who take an unsufficient amount of blood. Continuous monitoring of these biochemical parameters is going to help in the more effective follow up of patients with haemoglobinopathies.
