Comparison of Reduced PCR Volume PowerPlex Fusion 6C Kit Validations on Manual and Automated Systems

DNA Pub Date : 2024-02-04 DOI:10.3390/dna4010003
Eszter É. Lőrincz, Norbert Mátrai, Katalin A. Rádóczy, Tamás Cseppentő, Nóra M. Magonyi, Attila Heinrich
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Abstract

The PowerPlex Fusion 6C PCR™ amplification kit provides a strong discriminatory power for human identification. We have validated the kit with a reduced volume (12.5 µL) and as part of the validation we compared the efficiency of the polymerase chain reaction (PCR) prepared manually and on Hamilton Microlab® Autolys STAR Biorobot. Three years of casework data has been also included in the validation. Optimisation was carried out on different types of samples (blood, saliva, semen) and DNA was extracted robotically. Tests were conducted at two different cycle numbers (30;32), followed by analysis on both the Applied BiosystemsTM 3500 and 3500 xL Genetic Analyzer instruments (Applied Biosystems®, Foster City, CA, USA). When the PCR was prepared manually, no allele dropout was observed over 0.15 ng input DNA. Whereas when the PCR was prepared robotically, dropout already appeared at the level of 0.15 ng input DNA. In cases when increased cycle number was utilised, an increasing number of dropouts started to arise from 0.075 ng total input DNA. Despite the fact that robotically prepared PCR produced more missing alleles than the manually prepared PCR, using the optimal 0.5 ng input DNA, both methods proved to be reliable. Based on the results, our half-volume protocol is robust, and after three years of application it has proven to be effective with respect to a large number of casework samples.
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减少 PCR 容量的 PowerPlex Fusion 6C 套件在手动和自动系统上的验证比较
PowerPlex Fusion 6C PCR™ 扩增试剂盒具有很强的人体识别鉴别能力。我们已对该试剂盒进行了验证,试剂盒的容量有所减少(12.5 µL),作为验证的一部分,我们比较了手动和 Hamilton Microlab® Autolys STAR Biorobot 上聚合酶链反应 (PCR) 的效率。验证还包括三年的案例工作数据。对不同类型的样本(血液、唾液、精液)进行了优化,并使用机器人提取 DNA。测试在两个不同的周期数(30;32)下进行,然后在应用生物系统 3500 和 3500 xL 基因分析仪(Applied Biosystems®, Foster City, CA, USA)上进行分析。手工制备 PCR 时,输入的 DNA 超过 0.15 纳克就不会出现等位基因丢失。而用机器人进行 PCR 时,在 0.15 纳克输入 DNA 的水平上就已经出现了等位基因丢失。当循环次数增加时,从 0.075 纳克总输入 DNA 开始出现越来越多的脱落。尽管在使用最佳 0.5 纳克输入 DNA 时,机器人 PCR 比人工 PCR 产生更多的等位基因缺失,但两种方法都证明是可靠的。根据这些结果,我们的半体积方案是可靠的,经过三年的应用,它已被证明对大量的个案样本是有效的。
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DNA
DNA
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