A human papillomavirus whole genome plasmid repository: A resource for HPV DNA quality control reagents

Abstract

Well characterized reference reagents are useful for assay validation, proficiency/competency assessment, daily run controls, and to improve inter-laboratory comparisons. Synthetic human papillomavirus (HPV) DNA fragments and plasmid clones are available, but synthetic fragments include limited segments of the HPV genome and many HPV plasmids have interrupted coding regions or contain partial genomes. As a result, they are not compatible with all HPV DNA detection and typing assays. To address this need, we are establishing an HPV plasmid repository of HPV clones containing the whole genome of each type with no interruptions in coding regions. To date, HPV plasmid clones for 16 HPV types, (including all vaccine types and 14 types in clinical assays: HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68) have been constructed using a Gibson assembly method and validated by sequencing and the Novaplex HPV typing assay. The newly constructed HPV whole genome plasmids can serve as a quality control reagent resource for HPV DNA assays and are available for public health and research laboratories.

Keywords: HPV typing; Human papillomavirus; Quality control reagents; Whole genome plasmid.

Figure 1.

HPV58 genome and HPV58 plasmids. (A) Map of HPV58 circular genome based on sequence from PaVE database. PGMY primers bind to contiguous HPV58 L1 sequence to give 449 bp PCR amplicon. (B) Map of non-CDC HPV58 (HPV58/pLink) whole genome plasmid. The L1 gene is split into two regions of the vector backbone (black arrows) and the primer binding sites for PGMY primers in L1 sequence is highlighted. (C) PCR with PGMY primers using non-CDC HPV58 plasmid as DNA template. Lane1: DNA standard, lane 2: control PCR reaction without DNA template and lane 3: PCR reaction with non-CDC HPV58 plasmid resulting in 4259 bp PCR amplicon. (D) PCR with PGMY primers using CDC HPV58 (HPV58/pGEMT Easy-01) plasmid as DNA template. Lane1: DNA standard, lane 2: control PCR reaction without DNA template and lane 3: PCR reaction with CDC HPV58 plasmid resulting in 449 bp PCR amplicon.

Figure 2.

Laboratory workflow to construct CDC HPV whole genome plasmids in a standard pGEMT Easy-01 vector using a Gibson assembly method. HPV58 is shown here as an example.

Figure 3.

Construction of CDC HPV31 whole genome plasmid. (A) Map of non-CDC HPV31 (HPV31/pUC19) whole genome plasmid. The E2 gene is split into two regions of the vector backbone (black arrows). (B) Map of CDC HPV31 (HPV31/pGEMT Easy-01) whole genome plasmid without interruption at coding genes. (C) Propagation of Gibson assembly reaction product to construct CDC HPV31 plasmid in E.coli DH5α strain resulted in clones containing partial genome sequence of HPV31 genome. Representative example of a clone containing 1568 bp of HPV31 sequence is shown. (D) Propagation of Gibson assembly reaction product in E.coli Stbl2 strain resulted in clones containing whole genome sequence of HPV31. Representative example of a clone containing full length HPV31 genome is shown.