Flores Shahida, Sun Jie, King Jonathan, Budowle Bruce
Institute of Applied Genetics, Department of Forensics and Investigative Genetics, University of North Texas Health Science Center, Fort Worth, TX 76107, USA.
Institute of Applied Genetics, Department of Forensics and Investigative Genetics, University of North Texas Health Science Center, Fort Worth, TX 76107, USA.
Forensic Sci Int Genet. 2014 May;10:33-39. doi: 10.1016/j.fsigen.2014.01.005. Epub 2014 Jan 28.
The GlobalFiler™ Express PCR Amplification Kit uses 6-dye fluorescent chemistry to enable multiplexing of 21 autosomal STRs, 1 Y-STR, 1 Y-indel and the sex-determining marker amelogenin. The kit is specifically designed for processing reference DNA samples in a high throughput manner. Validation studies were conducted to assess the performance and define the limitations of this direct amplification kit for typing blood and buccal reference DNA samples on various punchable collection media. Studies included thermal cycling sensitivity, reproducibility, precision, sensitivity of detection, minimum detection threshold, system contamination, stochastic threshold and concordance. Results showed that optimal amplification and injection parameters for a 1.2mm punch from blood and buccal samples were 27 and 28 cycles, respectively, combined with a 12s injection on an ABI 3500xL Genetic Analyzer. Minimum detection thresholds were set at 100 and 120RFUs for 27 and 28 cycles, respectively, and it was suggested that data from positive amplification controls provided a better threshold representation. Stochastic thresholds were set at 250 and 400RFUs for 27 and 28 cycles, respectively, as stochastic effects increased with cycle number. The minimum amount of input DNA resulting in a full profile was 0.5ng, however, the optimum range determined was 2.5-10ng. Profile quality from the GlobalFiler™ Express Kit and the previously validated AmpFlSTR(®) Identifiler(®) Direct Kit was comparable. The validation data support that reliable DNA typing results from reference DNA samples can be obtained using the GlobalFiler™ Express PCR Amplification Kit.
GlobalFiler™ Express PCR扩增试剂盒采用6染料荧光化学技术，可实现21个常染色体STR、1个Y染色体STR、1个Y染色体插入缺失标记以及性别决定标记牙釉蛋白的多重检测。该试剂盒专为高通量处理参考DNA样本而设计。开展了验证研究，以评估此直接扩增试剂盒在各种可打孔采集介质上对血液和口腔参考DNA样本进行分型的性能，并确定其局限性。研究内容包括热循环敏感性、重现性、精密度、检测灵敏度、最低检测阈值、系统污染、随机阈值和一致性。结果表明，对于血液和口腔样本1.2毫米打孔样本，在ABI 3500xL基因分析仪上，最佳扩增和进样参数分别为27个循环和28个循环，进样时间为12秒。27个循环和28个循环的最低检测阈值分别设定为100 RFU和120 RFU，建议来自阳性扩增对照的数据能更好地代表阈值。27个循环和28个循环的随机阈值分别设定为250 RFU和400 RFU，因为随机效应会随着循环数增加。产生完整图谱的最低输入DNA量为0.5 ng，不过确定的最佳范围是2.5 - 10 ng。GlobalFiler™ Express试剂盒与之前经验证的AmpFlSTR(®) Identifiler(®) Direct试剂盒的图谱质量相当。验证数据支持使用GlobalFiler™ Express PCR扩增试剂盒可从参考DNA样本获得可靠的DNA分型结果。
