Design of qPCR primers for the early region of Human Papillomavirus oncogenic types 16 and 18

Primer pairs for the HR-HPV16 and 18 were designed to target the open reading frames (ORF) E6, E7, E1 and E2 in real-time PCR assays. This data is provided in the folders: Figures, Tables and HPV Alignments

1. HPV alignments folder contain the following files:
For gene-specific qPCR primer design, poorly conserved nucleotide regions were identified from HPV alignments of full-length genomes of types HPV16, 31, 35 and HPV 18, 45 and 59 in FASTA format. The HPV clusters are available: “HPV16_31_35_alignment.fa”; “HPV18_45_59_alignment.fa”.

2. A list of qPCR primers proposed is presented in Table 1. A graphic representation of amplification strategy is shown in Figure 1. Briefly, primers that produce overlapping amplicons less than 600 bp, that passed the secondary structure tests, designed with annealing temperatures above 58°C, were selected for experimental evaluation. Calibration curves were generated using SYBR Green chemistry and serial dilutions of a DNA standard as template for each genotype evaluated. For ORF HPV18 E7, the amplification plots, including the melt curve analysis are shown in Figure 2. Primer pairs producing multiple products (Figure 3), are not recommended. Complete information for primer pair used in real-time PCR assays are described in Table 2. All this information is presented in Figures and Tables folders.

针对人乳头瘤病毒16型和18型的高灵敏度引物对旨在针对实时聚合酶链反应（PCR）分析中的开放阅读框（ORF）E6、E7、E1和E2进行靶向设计。该数据集包含于以下文件夹中：图表、表格和HPV对齐。HPV对齐文件夹包含以下文件：针对基因特异性定量PCR引物设计，从HPV16、31、35型及HPV 18、45和59型的全基因组对齐中识别出低保守性核苷酸区域，这些对齐文件以FASTA格式提供。HPV簇对齐文件包括：“HPV16_31_35_alignment.fa”；“HPV18_45_59_alignment.fa”。表格1展示了提出的qPCR引物列表。扩增策略的图形表示如图1所示。简而言之，选定的引物能够产生小于600碱基对的重叠扩增片段，通过了二级结构测试，且设计时的退火温度高于58°C，并经过实验评估。使用SYBR Green化学和DNA标准品的连续稀释作为模板，为每个评估的基因型生成了校准曲线。针对HPV18 E7的ORF，扩增曲线及熔解曲线分析如图2所示。不推荐使用产生多个产物的引物对（见图3）。实时PCR分析中使用的引物对的信息详见表2。所有这些信息均展示在图表和表格文件夹中。