Droplet digital PCR for rapid enumeration of viral genomes and particles from cells and animals infected with orthopoxviruses

Abstract

Droplet digital polymerase chain reaction (ddPCR) was adapted for quantifying the number of orthopoxviral genomes in purified virus samples, infected cell lysates and tissues of infected animals. In contrast to the more commonly used qPCR, the newer ddPCR provides absolute numbers of DNA copies in samples without need for standard curves and has the ability to detect rare mutants in a population. The genome/infectious unit ratio for several sucrose gradient-purified orthopoxviruses varied from 5 to 10, which correlated well with values obtained using the Virocyt, a dedicated fluorescence flow cytometer. By employing a nuclease step to digest unencapsulated DNA, the genome/infectious unit ratios of virus in crude cell lysates approached that of purified virus particles. The speed, accuracy, sensitivity, and dynamic range of less than one to millions of infectious units in a sample make this semi-automated method well suited to a variety of laboratory, animal and clinical studies.

Keywords: Digital droplet polymerase chain reaction; Poxvirus particle infectivity ratio; Poxvirus quantification; Vaccinia virus quantification.

Fig. 1

Dynamic range of ddPCR. A series of dilutions of sucrose gradient purified VACV strain WR was made to provide a range of 5.6×10⁻¹ to 5×10⁶ PFU. Each dilution was treated with Benzonase nuclease, which was then inactivated with EDTA. Viral DNA was extracted from each sample using the Qiagen Minelute Virus Spin Kit and then amplified with 40 cycles of PCR using primers derived from the VACV E11L gene. The ddPCR analysis was performed on reaction droplets. The plot represents the total number of extracted genomes detected from the corresponding input PFUs. Coefficient of determination (R²) from trend line analysis (dashed line) is provided in the upper left corner of plot.

Fig. 2

Genome/PFU ratios of orthopoxviruses determined from purified particles and cell lysates. Approximately 6×10⁵ to 1.2×10⁶ PFU of VACV-WR, WR-GFP, MVA, and CPXV-BR were treated (+) or mock treated (−) with Benzonase nuclease for 1 h, after which viral DNA was isolated and amplified with 40 cycles of PCR using primers derived from the VACV E11L open reading frame. Genome/PFU ratios were determined for sucrose gradient purified virus particles (A) and crude lysate preparations (B). PFUs were determined in BS-C-1 cells for VACV and CPXV and in chick embryo cells for MVA. All data points were determined in triplicate and error bars indicate standard deviations. Asterisks represent p≦0.01.

Fig. 3

Correlation of viral genomes with total PFU in orthopoxvirus-infected organs. Mice were infected with varying doses of VACV-WR, CPXV-BR or CPXV_GER2002_MKY by intranasal or intraperitoneal routes of inoculation. Infected tissues were harvested on the day of death or euthanasia. Viral DNA from homogenized organs was isolated, amplified with 40 cycles of PCR using primers for the VACV E11L open reading frame, and ddPCR analysis performed in reaction droplets. Organ titers were determined by plaque assay on BS-C-1 cells. The relationship of total genomes and total PFU was determined and plotted for infected organs. Coefficient of determination (R²) from trend line analysis (dashed line) is provided in upper left corner of plot.