Nóra M. Magonyi PhD, Bálint Megadja MD, Katalin A. Rádóczy MSc, Tamás Cseppentő MSc, Eszter É. Lőrincz MSc, Norbert G. Valis MSc, Norbert Mátrai PhD, Attila Heinrich PhD
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Regardless of the PCR preparation method, the optimal 0.5 ng DNA input produced optimal allelic peak heights with no allelic dropout. The first alleles that failed to amplify started to appear at the level of 0.0375 ng input DNA, although the manually prepared PCRs produced fewer missing alleles. In this case, the raised cycle number produced 1.9% and 4.4% of dropout for manually and for robotically set up PCRs, respectively. In the case of 84 degraded casework samples, PCRs were prepared only by hand. The kit was able to provide informative profiles for 78.57%, 70.37%, and 69.77% for lowly, moderately, and highly degraded samples, respectively. Allelic dropouts were 26.05%, 44.88%, and 51.23% for the same groups. 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引用次数: 0
摘要
NGM Detect™ PCR 扩增试剂盒专为对降解的病例样本进行基因分型而设计。本研究旨在验证该试剂盒的半容量扩增功能,并介绍其成功的长期应用。验证是根据 DNA 分析方法科学工作组和欧洲法医科学研究所网络的相应指南进行的。在灵敏度、再现性和重复性等验证参数方面,聚合酶链式反应(PCR)的设置既有人工操作,也有机器人操作,共进行了 29 个循环。对于次优 DNA 输入(≤0.5 纳克)的 PCR,循环次数增加到 31 次。无论采用哪种 PCR 制备方法,最佳的 0.5 纳克 DNA 输入都能产生最佳的等位基因峰高,且无等位基因丢失。第一批未能扩增的等位基因开始出现在 0.0375 ng 输入 DNA 的水平上,尽管手工制备的 PCR 产生的缺失等位基因较少。在这种情况下,人工和机器人设置的 PCR 在循环次数增加时分别产生了 1.9% 和 4.4% 的丢失。在 84 份降解的个案样本中,PCR 只采用了手工操作。在低度、中度和高度降解的样本中,该试剂盒分别能为 78.57%、70.37% 和 69.77%的样本提供信息概况。同组的等位基因丢失率分别为 26.05%、44.88% 和 51.23%。根据我们的研究结果,我们强烈建议在半容量 PCR 系统中使用 NGM Detect™ 试剂盒,并鼓励在其他试剂盒无法生成完整 DNA 图谱的特殊情况下使用该试剂盒。
Half-volume validation of the NGM Detect™ PCR Amplification Kit and its application on degraded casework samples
The NGM Detect™ PCR Amplification Kit was designed particularly for genotyping degraded casework samples. This study aimed to validate the half-volume amplification of the kit and to present its successful long-term application. The validation was performed in accordance to the corresponding guidelines of the Scientific Working Group on DNA analysis methods and the European Network of Forensic Science Institutes. For validation parameters, such as sensitivity, reproducibility, and repeatability, polymerase chain reactions (PCR) were set up both manually and robotically, applying 29 cycles. For PCRs with sub-optimal DNA input (≤0.5 ng) the cycle numbers were increased to 31. Regardless of the PCR preparation method, the optimal 0.5 ng DNA input produced optimal allelic peak heights with no allelic dropout. The first alleles that failed to amplify started to appear at the level of 0.0375 ng input DNA, although the manually prepared PCRs produced fewer missing alleles. In this case, the raised cycle number produced 1.9% and 4.4% of dropout for manually and for robotically set up PCRs, respectively. In the case of 84 degraded casework samples, PCRs were prepared only by hand. The kit was able to provide informative profiles for 78.57%, 70.37%, and 69.77% for lowly, moderately, and highly degraded samples, respectively. Allelic dropouts were 26.05%, 44.88%, and 51.23% for the same groups. According to our results, we strongly recommend using the NGM Detect™ Kit in half-volume PCR system and encourage the usage of the kit in the particular cases when other kits fail to produce a complete DNA profile.
期刊介绍:
The Journal of Forensic Sciences (JFS) is the official publication of the American Academy of Forensic Sciences (AAFS). It is devoted to the publication of original investigations, observations, scholarly inquiries and reviews in various branches of the forensic sciences. These include anthropology, criminalistics, digital and multimedia sciences, engineering and applied sciences, pathology/biology, psychiatry and behavioral science, jurisprudence, odontology, questioned documents, and toxicology. Similar submissions dealing with forensic aspects of other sciences and the social sciences are also accepted, as are submissions dealing with scientifically sound emerging science disciplines. The content and/or views expressed in the JFS are not necessarily those of the AAFS, the JFS Editorial Board, the organizations with which authors are affiliated, or the publisher of JFS. All manuscript submissions are double-blind peer-reviewed.