联苯胺预试验处理血痕后样本DNA定量的研究

目的探讨联苯胺血痕预试验处理后样本DNA含量的变化及对STR分型检测的影响。方法选取10名无关个体EDTA抗凝血液制成滤纸血痕，保存干燥时间分0．5h、1h、3h、6h、12h、24h六个实验组，并采用磁珠提取法、QIAcubeDNA提取纯化法、chelex-100提取法提取样本DNA，应用RT-PCR定量技术检测样本DNA含量，同时应用PCR-STR技术和Idfiler-plus试剂盒检测相应样本STR分型。结果联苯胺血痕预试验后处理样本，随保存时间的延长，其样本DNA含量显示逐渐降低的趋势。回归线性对数分析显示，磁珠提取法：Y=-0．40871n（x）＋0．7044R0—0．7633；QIAcubeDNA提取纯化法：Y=-0．23931n（x）＋0．4764R0—0．8715；chelex-100提取法：Y=-0．11781n（x）＋0．2302R2=0．9571。不同DNA提取方法对同一保存时间的联苯胺血痕预试验试剂处理样本DNA含量间差异有极显著性，P〈O．01。结论联苯胺血痕预试验后对后续STR分型影响较大，联苯胺血痕预实验后的血痕不能继续进行STR分型检测。

Study on DNA analysis and DNA quantification of bloodstain treated with benzidine

Objective To investigate DNA analysis and DNA quantification of samples treated with the agents of benzidine tests. Methods Bloods of ten peoples were collected, and bloodstains were made on the filter paper. The same size and amount bloodstains were made by BSD600-DUET Paper Hole Puncher. Six experimental groups are divided by samples for 30min, lh, 3h, 6h, 12h and 24h after samples treated with benzidine. Template DNA of samples were extracted by three DNA extraction methods （magnetic bead-based DNA extraction protocol, QIAamp DNA Investigator Kit protocol and Chelex-100 extraction）. STR profiling was detected by PCR. DNA quantification of samples treatment was detected by real time PCR. Results The average values of DNA quantification of samples were decreased continuously with times after treatment with benzidine. The linear curves is Y=- 0. 40871n（x）＋0. 7044（R2 =0. 7633）for the group of magnetic bead-based DNA extraction protocol）, Y=-0. 2393In（x）＋0. 4764 （R2 =0. 8715）for the group of QIAamp DNA Investigator Kit protocol, Y=-0. 11781n（x） ＋0. 2302（R2 =0. 9571）for the group of Chelex-100 extractior Statistical comparisons between each of the groups were made by multiple-ways Analysis of variance with SPSS 19. 0 indicated that benzidine can alter DNA amount over time up to 24h（ P 〈0. 01）, and that DNA amount with different DNA extraction Methods have significant differences（ P 〈0. 01）. DNA typing of 16 locis of 8. 33M samples was detected by PCR-STR with Identifiler plus kits and the loss of 232 heterozygosity were detected in 142 samples. Conclusion Benzidine test has significant influence on DNA typing and DNA quantification, so the samples treated by benzidine could not be detected by PCRSTR.