Comparison of a quantitative Real-Time PCR assay and droplet digital PCR for copy number analysis of the CCL4L genes
Comparison of a quantitative Real-Time PCR assay and droplet digital PCR for copy number analysis of the CCL4L genes
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DOI：
10.1016/j.meegid.2014.03.028
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发表时间：
2014-07-01
影响因子：
3.2
通讯作者：
Tiemessen, Caroline T.
中科院分区：
文献类型：
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作者：
Bharuthram, Avani;Paximadis, Maria;Tiemessen, Caroline T.
关于CCL3编码基因的拷贝数变异影响HIV-1感染和疾病进展这一发现的争议，部分归因于从拷贝数评估方法获得的不同结果。与CCL3一样，编码CC趋化因子CCL4的基因也是CCR5受体的天然配体，被发现以群体特有的多拷贝数量出现，并已被证明对HIV-1具有保护作用。本研究对定量实时聚合酶链式反应(QPCR)和滴状数字聚合酶链式反应(DdPCR)检测CCL4L基因拷贝数的标准方法进行了评价。CCL4编码基因是CCL4，每个二倍体基因组(PDG)存在两个拷贝，而非等位基因CCL4L包括CCL4L1和CCL4L2，它们都存在于多个拷贝的PDG中。用定量聚合酶链式反应和直接聚合酶链式反应检测了南非黑人(n=23)和高加索人(n=32)人群中未感染HIV-1的人群中CCL4L、CCL4L1和CCL4L2的拷贝数。与定量聚合酶链式反应(r=0.87，p<；0.0001)相比，动态聚合酶链式反应产生的CCL4L拷贝数与CCL4L1和CCL4L2拷贝之和之间的相关性更强(r=0.99，p<；0.0001)。实时定量聚合酶链式反应在拷贝数较高时表现出更大的不准确性，这与我们的黑人队列特别相关，这些黑人个体的CCL4L拷贝范围(3-6)比高加索人(0-4)更大，人口中位数(分别为4和2)更高。在黑人群体中，CCL4L1(黑人：2，0-4，高加索人：0，0-2)和CCL4L2(黑人：2，1-5，高加索人：2，0-3)的中位数和范围也较高。Droplet数字聚合酶链式反应是一种远优于定量聚合酶链式反应的方法，其准确性对于研究可变基因拷贝数对宿主感染和病程的表型结果的贡献至关重要。(C)2014爱思唯尔B.V.保留所有权利。
The controversy surrounding the findings that copy number variation, of the CCL3 encoding genes, influences HIV-1 infection and disease progression has been in part attributed to the variable results obtained from methods used for copy number evaluation. Like CCL3, the genes encoding the CC chemokine CCL4, also a natural ligand of the CCR5 receptor, are found to occur in population-specific multiple copy number and have been shown to play a protective role against HIV-1. This study evaluated the standard method of quantitative Real-Time PCR (qPCR) and droplet digital PCR (ddPCR) for CCL4L gene copy number determination. The CCL4 encoding genes are CCL4, occurring in two copies per diploid genome (pdg), and the non-allelic CCL4L genes, comprised of CCL4L1 and CCL4L2, which are both found in multiple copies pdg. Copy number of CCL4L, CCL4L1 and CCL4L2 was determined in a cohort of HIV-1-uninfected individuals from the South African Black (n = 23) and Caucasian (n = 32) population groups using qPCR and ddPCR. A stronger correlation between the number of CCL4L copies and the sum of CCL4L1 and CCL4L2 copies generated by ddPCR (r = 0.99, p < 0.0001) compared to qPCR (r = 0.87, p < 0.0001) was observed. Real-Time qPCR exhibited greater inaccuracy at higher copy numbers which is particularly relevant to our cohort of Black individuals who have a higher range of CCL4L copies (3-6) compared to Caucasians (0-4) and a higher population median (4 and 2, respectively). Medians and ranges of CCL4L1 (Black: 2, 0-4, Caucasian: 0, 0-2) and CCL4L2 (Black: 2, 1-5, Caucasian: 2, 0-3) were also higher in the Black population. Droplet digital PCR was shown to be a far superior method to qPCR for assessment of CCL4 gene copy number variation, the accuracy of which is essential for studies of the contribution of variable gene copy number to phenotypic outcomes of host infection and disease course. (C) 2014 Elsevier B.V. All rights reserved.