High-Throughput Multiplexed T-Cell-Receptor Excision Circle Quantitative PCR Assay with Internal Controls for Detection of Severe Combined Immunodeficiency in Population-Based Newborn Screening
High-Throughput Multiplexed T-Cell-Receptor Excision Circle Quantitative PCR Assay with Internal Controls for Detection of Severe Combined Immunodeficiency in Population-Based Newborn Screening
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DOI：
10.1373/clinchem.2010.144915
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发表时间：
2010-09-01
影响因子：
9.3
通讯作者：
Comeau, Anne Marie
中科院分区：
文献类型：
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作者：
Gerstel-Thompson, Jacalyn L.;Wilkey, Jonathan F.;Comeau, Anne Marie
背景技术背景：实时定量PCR（qPCR）靶向功能性T细胞的特异性标志物，T细胞受体切除环（TREC），检测功能性T细胞的缺乏，并已证明临床有效性，用于检测婴儿的严重联合免疫缺陷（SCID）。需要具有内部对照的qPCR TREC测定来监测在每个反应中采样的特定干血斑穿孔物的DNA质量和相对细胞含量。如果可以对相同的新生儿筛查samples.METHODS进行更多的测试，qPCR TREC检测的实用性也将大大提高：我们通过减弱参考基因的反应来接近TREC的qPCR多重化，重点是保持严格的质量保证，以重现斜率和防止样品间交叉污染。全州新生儿筛查SCID使用的多重检测实施，和质量保证数据recorded.RESULTS：多重qPCR TREC检测显示近100%的扩增效率为每个TREC和参考序列，临床有效性的多种形式的SCID，和分析检测限与预防污染一致。从一个3.2毫米的干血斑的血和残留的鬼可以用作源材料的多重免疫测定和多重DNA测试（Multiplex Plus），没有中断的多重TREC qPCR.CONCLUSIONS：人口为基础的SCID新生儿筛查计划应考虑多重质量保证的目的。使用Multiplex Plus的潜在益处包括能够进行多分析物分析。(C)2010年美国临床化学协会
BACKGROUND: Real-time quantitative PCR (qPCR) targeting a specific marker of functional T cells, the Tcell-receptor excision circle (TREC), detects the absence of functional T cells and has a demonstrated clinical validity for detecting severe combined immunodeficiency (SCID) in infants. There is need for a qPCR TREC assay with an internal control to monitor DNA quality and the relative cellular content of the particular dried blood spot punch sampled in each reaction. The utility of the qPCR TREC assay would also be far improved if more tests could be performed on the same newborn screening sample.METHODS: We approached the multiplexing of qPCR for TREC by attenuating the reaction for the reference gene, with focus on maintaining tight quality assurance for reproducible slopes and for prevention of sample-to-sample cross contamination. Statewide newborn screening for SCID using the multiplexed assay was implemented, and quality-assurance data were recorded.RESULTS: The multiplex qPCR TREC assay showed nearly 100% amplification efficiency for each of the TREC and reference sequences, clinical validity for multiple forms of SCID, and an analytic limit of detection consistent with prevention of contamination. The eluate and residual ghost from a 3.2-mm dried blood spot could be used as source material for multiplexed immunoassays and multiplexed DNA tests (Multiplex Plus), with no disruption to the multiplex TREC qPCR.CONCLUSIONS: Population-based SCID newborn screening programs should consider multiplexing for quality assurance purposes. Potential benefits of using Multiplex Plus include the ability to perform multianalyte profiling. (C) 2010 American Association for Clinical Chemistry