Hudlow William R, Chong Mavis Date, Swango Katie L, Timken Mark D, Buoncristiani Martin R
California Department of Justice Jan Bashinski DNA Laboratory, Richmond, CA 94804, USA.
Forensic Sci Int Genet. 2008 Mar;2(2):108-25. doi: 10.1016/j.fsigen.2007.09.001. Epub 2007 Nov 26.
A quadruplex real-time qPCR assay was developed to simultaneously assess total human DNA, human male DNA, DNA degradation and PCR inhibitors in forensic samples. Specifically, the assay utilizes a approximately 170-190bp target sequence that spans the TH01 STR locus to quantify total human DNA (nuTH01), a 137 bp target sequence directly adjacent to the SRY gene to quantify human male DNA (nuSRY), a 67 bp target sequence flanking the CSF1PO STR locus (nuCSF) to assess degradation (nuCSF:nuTH01 ratio) and a 77 bp synthetic DNA template used as an internal PCR control target sequence (IPC) for the assessment of PCR inhibition. Validation studies, performed on an ABI 7500 SDS instrument using TaqMan and TaqManMGB detection, indicate each of the targets in the quadruplex assay performs effectively and is informative even when challenged with DNase-degraded and hematin-inhibited samples. The nuTH01-nuSRY-nuCSF-IPC quadruplex qPCR assay is envisioned to assist in the choice of the most informative DNA typing system available, which may include standard autosomal STR typing when the results indicate the presence of non-degraded, single gender DNA or non-degraded, male:female mixtures at ratios expected to yield probative alleles; Y STR typing in samples containing a male component that is overwhelmed by the presence of an excess of female DNA; reduced amplicon size STR typing ("MiniSTRs") where the nuCSF:nuTH01 ratio indicates the sample is highly degraded; enhanced STR amplification with additional AmpliTaq Gold/BSA and/or sample clean-up when the presence of PCR inhibitors is suggested by a delayed IPC C(T) value or mitochondrial DNA typing in samples where little to no nuclear DNA is detected. The present study includes evaluations of species specificity, sensitivity, precision, reproducibility, male-female mixtures, population samples and applications to various casework-type samples as indicated by the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines.
开发了一种四重实时定量PCR检测方法，用于同时评估法医样本中的人类总DNA、人类男性DNA、DNA降解情况和PCR抑制剂。具体而言，该检测方法利用一个跨越TH01 STR基因座的约170 - 190bp靶序列来定量人类总DNA（nuTH01），一个紧邻SRY基因的137bp靶序列来定量人类男性DNA（nuSRY），一个位于CSF1PO STR基因座侧翼的67bp靶序列（nuCSF）来评估降解情况（nuCSF:nuTH01比值），以及一个77bp的合成DNA模板用作内部PCR对照靶序列（IPC）以评估PCR抑制。使用TaqMan和TaqManMGB检测在ABI 7500 SDS仪器上进行的验证研究表明，四重检测中的每个靶标即使在受到DNA酶降解和血红素抑制的样本挑战时也能有效发挥作用且提供有用信息。nuTH01 - nuSRY - nuCSF - IPC四重定量PCR检测方法预期可协助选择最具信息量的可用DNA分型系统，当结果表明存在未降解的单性别DNA或未降解的、男性:女性比例预期能产生 probative等位基因的混合物时，可能包括标准常染色体STR分型；在含有被过量女性DNA掩盖的男性成分的样本中进行Y STR分型；当nuCSF:nuTH01比值表明样本高度降解时，进行扩增子大小减小的STR分型（“MiniSTRs”）；当延迟的IPC C(T)值提示存在PCR抑制剂时，使用额外的AmpliTaq Gold/BSA和/或样本净化增强STR扩增，或在几乎未检测到核DNA的样本中进行线粒体DNA分型。本研究包括根据DNA分析方法科学工作组（SWGDAM）指南对物种特异性、灵敏度、精密度、重现性、男女混合物、群体样本以及对各种案件样本类型的应用进行评估。
