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Simultaneous Detection and Viral DNA Load Quantification of Different Human Papillomavirus Types in Clinical Specimens by the High Analytical Droplet Digital PCR Method - PubMed

  • ️Wed Jan 01 2020

Simultaneous Detection and Viral DNA Load Quantification of Different Human Papillomavirus Types in Clinical Specimens by the High Analytical Droplet Digital PCR Method

John Charles Rotondo et al. Front Microbiol. 2020.

Abstract

Human papillomaviruses (HPVs) are small DNA tumor viruses that mainly infect mucosal epithelia of anogenital and upper respiratory tracts. There has been progressive demand for more analytical assays for HPV DNA quantification. A novel droplet digital PCR (ddPCR) method was developed to simultaneously detect and quantify HPV DNA from different HPV types. DdPCR was initially tested for assay sensitivity, accuracy, specificity as well as intra- and inter-run assay variation employing four recombinant plasmids containing HPV16, HPV18, HPV11, and HPV45 DNAs. The assay was extended to investigate/quantify HPV DNA in Cervical Intraepithelial Neoplasia (CIN, n = 45) specimens and human cell lines (n = 4). DdPCR and qPCR data from clinical samples were compared. The assay showed high accuracy, sensitivity and specificity, with low intra-/inter- run variations, in detecting/quantifying HPV16/18/11/45 DNAs. HPV DNA was detected in 51.1% (23/45) CIN DNA samples by ddPCR, whereas 40% (18/45) CIN tested HPV-positive by qPCR. Five CIN, tested positive by ddPCR, were found to be negative by qPCR. In CIN specimens, the mean HPV DNA loads determined by ddPCR were 3.81 copy/cell (range 0.002-51.02 copy/cell), whereas 8.04 copy/cell (range 0.003-78.73 copy/cell) by qPCR. DdPCR and qPCR concordantly detected HPV DNA in SiHa, CaSki and Hela cells, whereas HaCaT tested HPV-negative. The correlation between HPV DNA loads simultaneously detected by ddPCR/qPCR in CINs/cell lines was good (R 2 = 0.9706, p < 0.0001). Our data indicate that ddPCR is a valuable technique in quantifying HPV DNA load in CIN specimens and human cell lines, thereby improving clinical applications, such as patient management after primary diagnosis of HPV-related lesions with HPV-type specific assays.

Keywords: CIN 3; cervical intraepithelial neoplasia; ddPCR; droplet digital PCR; human papillomavirus; infection; quantitative PCR; viral DNA load.

Copyright © 2020 Rotondo, Oton-Gonzalez, Mazziotta, Lanzillotti, Iaquinta, Tognon and Martini.

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Figures

FIGURE 1
FIGURE 1

Flow diagram. The ddPCR set up and development for the detection and quantification of HPV16/18/11/45 DNAs from clinical specimens is represented in this diagram.

FIGURE 2
FIGURE 2

Optimization of ddPCR parameters. (A) Optimization of the GP5+/GP6+ primers annealing temperature using 103 copies/μL pUC19_HPV16 plasmid. Vertical lines represent the gradient of the eight following annealing temperatures: 46, 48, 50, 52, 54, 56, 58, and 60°C. The optimal annealing temperature was 48°C, which resulted in a distinct fluorescence amplitude difference between positive (blue) and negative (gray) droplets. (B) Optimization of the GP5+/GP6+ primers concentration. Optimization was performed using 103 copies/μL pUC19_HPV16 plasmid. Vertical lines represent the different primers concentrations: 112, 225, 450, and 900 nM. The optimal primer concentration resulted 225 nM, based on better amplitude fluorescence spread between positive (blue) and negative (gray) droplets.

FIGURE 3
FIGURE 3

Representative quantification of pUC19_HPV16 plasmid with or without restriction enzyme digestion by ddPCR. (A) Copy number of pUC19_HPV16 plasmid without enzyme digestion. The circular pUC19_HPV16 plasmid was serially diluted from 105 to 100 copies/μL and ddPCR amplified. (B) Copy number of pUC19_HPV16 plasmid after restriction enzyme digestion. The enzyme-digested linearized pUC19_HPV16 plasmid was serially diluted from 105 to 100 copies/μL and ddPCR amplified. In A and B panels, vertical lines represent the fluorescent amplitude of the different plasmid concentrations: 100, 101, 102, 103, 104, 105 copies/μL.

FIGURE 4
FIGURE 4

Representative analysis of ddPCR specificity. Vertical lines represent the fluorescent amplitude of pUC19 and pGEM1 plasmids, which contain the complete genomes of HPV16/18/11 and HPV45, respectively, as well as pUC19, which contains the complete genome of HPV52, employed as negative control. Two others negative controls are included: one control contains the salmon sperm DNA (ssDNA), whereas the additional negative control is represented by ddH2O without DNA.

FIGURE 5
FIGURE 5

Representative ddPCR amplifications of pUC19/pGEM1 plasmids with HPV16/18/11/45 genomes, together with the correlation between theoretical and detected plasmid concentrations. Ten-fold serial dilutions of pUC19_HPV16 (A), pUC19_HPV18 (C), pUC19_HPV11 (E), pGEM1_HPV45 (G) plasmids ranging from 100 to 105 copies/μL are showed. All panels: vertical lines represent the different plasmid concentrations: 100, 101, 102, 103, 104, 105 copies/μL. The correlation was assessed by plotting the log copy/reaction against the log starting concentration of 10-fold serial dilutions of pUC19_HPV16 (B), pUC19_HPV18 (D), pUC19_HPV11 (H), pGEM1_HPV45 (F) plasmids. The ddPCR gave high accuracy for pUC19_HPV16, pUC19_HPV18, and pUC19_HPV11, with an R2 of 1 and a P = 0.0001, whereas pGEM1_HPV45 was detected with good accuracy, with an R2 of 0.9999 and a p = 0.0001. Linear regression analysis was performed using GraphPad Prism software.

FIGURE 6
FIGURE 6

Correlation between ddPCR and qPCR results for HPV viral DNA load in CIN specimens and cervical cancer cell lines. Quantitative results obtained by both ddPCR and qPCR in HPV-positive samples (n = 21), including CIN specimens (n = 18) and SiHa, CaSKi, and HeLa cervical cancer cell lines, were compared between the two respective assays. Quantitative results are reported as Log 10 of HPV DNA copy/cell. Results from ddPCR and qPCR significantly correlated with a R2 = 0.9706 and P = 0.0001. Linear regression analysis was performed using GraphPad Prism software.

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