目的建立自动化工作站同步提取不同种类涉案法医生物检材DNA的新方法。方法选用TECAN Freedom EVO 100-4、75-2型自动化提取、加样工作站,采用磁珠法及Chelex-100法对各类涉案生物检材进行DNA提取、PCR扩增、毛细管电泳检测其STR分型,...目的建立自动化工作站同步提取不同种类涉案法医生物检材DNA的新方法。方法选用TECAN Freedom EVO 100-4、75-2型自动化提取、加样工作站,采用磁珠法及Chelex-100法对各类涉案生物检材进行DNA提取、PCR扩增、毛细管电泳检测其STR分型,进行比较测试。在"全国公安机关DNA数据库应用系统"中建立并应用实验室信息管理系统(LIMS)模拟实施规范化DNA检案。结果1552份各类检材,采用工作站-磁珠法提取DNA效果最佳,STR检测成功率为95%,工作站-Chelex法为88%;二者分别与其手工提取法比较,成功率无明显差异。92个样本同期检测,自动化工作站较手工操作DNA检案时间可缩减1.25倍。结论工作站磁珠法提取涉案检材DNA,可获得满意的STR分型结果。应用LIMS管控,可有效防控污染,明显提高检案效率及鉴定质量。展开更多
Bones and teeth often represent the only sources of DNA available for identifying human remains.DNA in bones and teeth is generally better preserved than that in soft tissues because of the presence of hard connective...Bones and teeth often represent the only sources of DNA available for identifying human remains.DNA in bones and teeth is generally better preserved than that in soft tissues because of the presence of hard connective tissue with a high level of calcium.Because of the extensive mineralisation,the choice of an efficient DNA extraction procedure is important to minimise the sampling of a high level of minerals and to remove polymerase chain reaction(PCR)inhibitors.Some protocols are available for DNA extraction from bones and teeth as part of the Qiagen EZ1 DNA Investigator Kit using the EZ1 Advanced XL automated purification platform.To improve the efficiency of DNA extraction from skeletal remains,the present study focuses on a modification to these already available protocols.In this study,different bones and teeth collected between 1 and 50 years after death were subjected to DNA extraction using the standard EZ1 protocol,a supplementary protocol,and a modified protocol.The modified approach included a decalcification step,whereas the Qiagen protocols worked directly on non-decalcified powder.In all three procedures,150 mg samples were used for DNA extraction.We evaluated the quantity of DNA recovered from samples,the presence of any PCR inhibitors co-extracted,the level of DNA degradation,the quality of short tandem repeat(STR)profiles,and the reproducibility of the modified procedure.When compared with the other protocols,the modified protocol resulted in the best recovery of DNA that was free of PCR inhibitors.Additionally,the STR profiles were reliable and of high quality.In our opinion,the decalcification step increases DNA recovery by softening tissues,which allows lysis solutions to act more effectively.Furthermore,the use of two lysis solutions and the variation added to the EZ1 purification step allow for DNA recovery with quality and quantity superior to those of the previously available Qiagen-based protocols.These findings may be helpful solutions to the problems commonly encountered when dealing with difficult samples,such as bones and teeth.展开更多
Sexual assault evidence often contains sperm cells,which are typically separated from nonsperm cells using manual differential lysis procedures.The goal of this study was to evaluate the automated QIAGEN QIAcube for t...Sexual assault evidence often contains sperm cells,which are typically separated from nonsperm cells using manual differential lysis procedures.The goal of this study was to evaluate the automated QIAGEN QIAcube for this purpose and to compare it to manual QIAGEN and manual organic differential methods using DNA yields and STR profile data for assessment.DNA yields were determined by qPCR,followed by multiplex STR amplification,CE analysis,and mixture interpretation.The automated method was capable of effective cell separation,producing DNA yields sufficient for STR amplification.Further,sperm fraction human:male DNA ratios from the QIAcube samples were consistently closer to the desired 1:1 and STR profiles were less likely to result in mixtures,with 6–8fewer female alleles detected(median 1.5 alleles).Ultimately,using the QIAcube for automated differential processing of semen-containing mixtures reduces the need for downstream mixture interpretation and improves STR profile quality with substantially less hands-on time.展开更多
文摘目的建立自动化工作站同步提取不同种类涉案法医生物检材DNA的新方法。方法选用TECAN Freedom EVO 100-4、75-2型自动化提取、加样工作站,采用磁珠法及Chelex-100法对各类涉案生物检材进行DNA提取、PCR扩增、毛细管电泳检测其STR分型,进行比较测试。在"全国公安机关DNA数据库应用系统"中建立并应用实验室信息管理系统(LIMS)模拟实施规范化DNA检案。结果1552份各类检材,采用工作站-磁珠法提取DNA效果最佳,STR检测成功率为95%,工作站-Chelex法为88%;二者分别与其手工提取法比较,成功率无明显差异。92个样本同期检测,自动化工作站较手工操作DNA检案时间可缩减1.25倍。结论工作站磁珠法提取涉案检材DNA,可获得满意的STR分型结果。应用LIMS管控,可有效防控污染,明显提高检案效率及鉴定质量。
文摘Bones and teeth often represent the only sources of DNA available for identifying human remains.DNA in bones and teeth is generally better preserved than that in soft tissues because of the presence of hard connective tissue with a high level of calcium.Because of the extensive mineralisation,the choice of an efficient DNA extraction procedure is important to minimise the sampling of a high level of minerals and to remove polymerase chain reaction(PCR)inhibitors.Some protocols are available for DNA extraction from bones and teeth as part of the Qiagen EZ1 DNA Investigator Kit using the EZ1 Advanced XL automated purification platform.To improve the efficiency of DNA extraction from skeletal remains,the present study focuses on a modification to these already available protocols.In this study,different bones and teeth collected between 1 and 50 years after death were subjected to DNA extraction using the standard EZ1 protocol,a supplementary protocol,and a modified protocol.The modified approach included a decalcification step,whereas the Qiagen protocols worked directly on non-decalcified powder.In all three procedures,150 mg samples were used for DNA extraction.We evaluated the quantity of DNA recovered from samples,the presence of any PCR inhibitors co-extracted,the level of DNA degradation,the quality of short tandem repeat(STR)profiles,and the reproducibility of the modified procedure.When compared with the other protocols,the modified protocol resulted in the best recovery of DNA that was free of PCR inhibitors.Additionally,the STR profiles were reliable and of high quality.In our opinion,the decalcification step increases DNA recovery by softening tissues,which allows lysis solutions to act more effectively.Furthermore,the use of two lysis solutions and the variation added to the EZ1 purification step allow for DNA recovery with quality and quantity superior to those of the previously available Qiagen-based protocols.These findings may be helpful solutions to the problems commonly encountered when dealing with difficult samples,such as bones and teeth.
文摘Sexual assault evidence often contains sperm cells,which are typically separated from nonsperm cells using manual differential lysis procedures.The goal of this study was to evaluate the automated QIAGEN QIAcube for this purpose and to compare it to manual QIAGEN and manual organic differential methods using DNA yields and STR profile data for assessment.DNA yields were determined by qPCR,followed by multiplex STR amplification,CE analysis,and mixture interpretation.The automated method was capable of effective cell separation,producing DNA yields sufficient for STR amplification.Further,sperm fraction human:male DNA ratios from the QIAcube samples were consistently closer to the desired 1:1 and STR profiles were less likely to result in mixtures,with 6–8fewer female alleles detected(median 1.5 alleles).Ultimately,using the QIAcube for automated differential processing of semen-containing mixtures reduces the need for downstream mixture interpretation and improves STR profile quality with substantially less hands-on time.
