目前法医DNA检验应用的短串联重复序列(short tandem repeat,STR)复合扩增系统均依赖于荧光标记的多重PCR方法。常规DNA检验流程包括提取、定量、扩增、分离和检测,而多重PCR扩增常常是整个过程中的限速步骤,完成28~30个循环大约需要3 ...目前法医DNA检验应用的短串联重复序列(short tandem repeat,STR)复合扩增系统均依赖于荧光标记的多重PCR方法。常规DNA检验流程包括提取、定量、扩增、分离和检测,而多重PCR扩增常常是整个过程中的限速步骤,完成28~30个循环大约需要3 h。以往常利用商业化的热循环仪和热稳定DNA聚合酶,但需要很长的PCR循环时间才能够确保100~400 bp的目标片段得到有效且均衡的复合扩增。随着各种缩短扩增时间方法的出现,STR复合扩增不再需要3 h,而是可以减少到几十分钟甚至十几分钟。现主要总结了快速PCR、直接PCR、芯片PCR以及其他快速扩增等方法的研究现状与进展,尤其是在法医DNA检验领域,同时对比了几种常见快速PCR扩增方法,可见缩短扩增时间对DNA检验的意义重大。未来,以芯片为主导、快速检验为目的的全自动、便携式、集成化的DNA分析系统,将使得法医DNA检验从实验室走进案件现场,甚至日常生活,实现真正的快速即时检验。展开更多
目的为了便于食管癌大规模流行病学筛查工作的开展,探讨一种能快速敏感地检测口腔牙龈卟啉单胞菌(Porphyromonas gingivalis,P.gingivalis)DNA的免核酸抽提直接PCR方法。方法收集我院门诊100例体检人员的200份口腔拭子(每人双份),以TE b...目的为了便于食管癌大规模流行病学筛查工作的开展,探讨一种能快速敏感地检测口腔牙龈卟啉单胞菌(Porphyromonas gingivalis,P.gingivalis)DNA的免核酸抽提直接PCR方法。方法收集我院门诊100例体检人员的200份口腔拭子(每人双份),以TE buffer(10 mM Tris,0.1 mM EDTA,pH 8.0)、商品化裂解液以及核酸抽提试剂盒等作为基因组提取试剂,使用P.gingivalis特异引物探针进行直接定量PCR检测。结果与裂解液-直接qPCR法相比,TE-直接qPCR法的灵敏度为94.12%,特异性为100%;与试剂盒-qPCR法相比,TE-直接qPCR法的灵敏度与特异性均为100%;TE-直接qPCR法与裂解液-直接qPCR法以及与试剂盒-qPCR法检测P.gingivalis均具有高度一致性(分别为Kappa=0.954,Kappa=1);TE-直接qPCR法与试剂盒-qPCR法检测阳性P.gingivalis的Ct均值无显著差异(P=0.907)。结论TE-直接qPCR法检测口腔P.gingivalis灵敏度高、特异性强,是快速高效检测口腔P.gingivalis的理想方法,可用于食管癌大规模分子流行病学研究。展开更多
Agrobacterium-mediated transformation through floral dip and rapid selection process after transgenic event had become a preference as it will overcome the difficulties faced in tissue culturing procedures and lengthy...Agrobacterium-mediated transformation through floral dip and rapid selection process after transgenic event had become a preference as it will overcome the difficulties faced in tissue culturing procedures and lengthy time for screening transformed progenies. Therefore, in this study, three constructs, p5b5 (14,289 bp), p5d9 (15,330 bp) and p5f7 (15,380 bp) in pDRB6b vector which has hygromycin as a selectable marker gene were introduced individually into Agrobacterium tumefaciens strain (AGL1). The cell suspension was applied to Amaranthus inflorescence by drop-by-drop technique and was left to produce seeds (T1). The T1 seeds were germinated and grown to produce seedlings under non-sterile condition. Hygromycin selection on seedling cotyledon leaves results in identification of 12 putative transformants, three from p5b5, four from p5d9 and five from p5f7. All positive putative transformants that were selected at the first stage through hygromycin spraying showed positive result in leaf disk hygromycin assay and in a construct specific polymerase chain reaction-based assay. A ~750 bp amplified hygromycin gene was further verified through sequencing. Our results suggest that Amaranthus inflorescences were able to be transformed and the transformed progenies could be verified through a combination of simple and rapid methods .展开更多
文摘目的为了便于食管癌大规模流行病学筛查工作的开展,探讨一种能快速敏感地检测口腔牙龈卟啉单胞菌(Porphyromonas gingivalis,P.gingivalis)DNA的免核酸抽提直接PCR方法。方法收集我院门诊100例体检人员的200份口腔拭子(每人双份),以TE buffer(10 mM Tris,0.1 mM EDTA,pH 8.0)、商品化裂解液以及核酸抽提试剂盒等作为基因组提取试剂,使用P.gingivalis特异引物探针进行直接定量PCR检测。结果与裂解液-直接qPCR法相比,TE-直接qPCR法的灵敏度为94.12%,特异性为100%;与试剂盒-qPCR法相比,TE-直接qPCR法的灵敏度与特异性均为100%;TE-直接qPCR法与裂解液-直接qPCR法以及与试剂盒-qPCR法检测P.gingivalis均具有高度一致性(分别为Kappa=0.954,Kappa=1);TE-直接qPCR法与试剂盒-qPCR法检测阳性P.gingivalis的Ct均值无显著差异(P=0.907)。结论TE-直接qPCR法检测口腔P.gingivalis灵敏度高、特异性强,是快速高效检测口腔P.gingivalis的理想方法,可用于食管癌大规模分子流行病学研究。
文摘Agrobacterium-mediated transformation through floral dip and rapid selection process after transgenic event had become a preference as it will overcome the difficulties faced in tissue culturing procedures and lengthy time for screening transformed progenies. Therefore, in this study, three constructs, p5b5 (14,289 bp), p5d9 (15,330 bp) and p5f7 (15,380 bp) in pDRB6b vector which has hygromycin as a selectable marker gene were introduced individually into Agrobacterium tumefaciens strain (AGL1). The cell suspension was applied to Amaranthus inflorescence by drop-by-drop technique and was left to produce seeds (T1). The T1 seeds were germinated and grown to produce seedlings under non-sterile condition. Hygromycin selection on seedling cotyledon leaves results in identification of 12 putative transformants, three from p5b5, four from p5d9 and five from p5f7. All positive putative transformants that were selected at the first stage through hygromycin spraying showed positive result in leaf disk hygromycin assay and in a construct specific polymerase chain reaction-based assay. A ~750 bp amplified hygromycin gene was further verified through sequencing. Our results suggest that Amaranthus inflorescences were able to be transformed and the transformed progenies could be verified through a combination of simple and rapid methods .
