Comparison of digital PCR platforms using the molecular marker

Abstract

Assays of clinical diagnosis and species identification using molecular markers are performed according to a quantitative method in consideration of sensitivity, cost, speed, convenience, and specificity. However, typical polymerase chain reaction (PCR) assay is difficult to quantify and have various limitations. In addition, to perform quantitative analysis with the quantitative real-time PCR (qRT-PCR) equipment, a standard curve or normalization using reference genes is essential. Within the last a decade, previous studies have reported that the digital PCR (dPCR) assay, a third-generation PCR, can be applied in various fields by overcoming the shortcomings of typical PCR and qRT-PCR assays. We selected Stilla Naica System (Stilla Technologies), Droplet Digital PCR Technology (Bio-Rad), and Lab on an Array Digital Real-Time PCR analyzer system (OPTOLANE) for comparative analysis among the various droplet digital PCR platforms currently in use commercially. Our previous study discovered a molecular marker that can distinguish Hanwoo species (Korean native cattle) using Hanwoo-specific genomic structural variation. Here, we report the pros and cons of the operation of each dPCR platform from various perspectives using this species identification marker. In conclusion, we hope that this study will help researchers to select suitable dPCR platforms according to their purpose and resources.

Keywords: digital PCR platforms; molecular marker; point-of-care testing.

Fig. 1.

Polymorphic patterns of the Del_96 region in 9 Hanwoo (A) and Holstein (B) genomes. As a result of gel chromatography, heterozygous alleles at 680 bp and 310 bp were identified in the 9 Hanwoo samples, but no deleted alleles were confirmed in the Holstein sample.

Fig. 2.

Probe designs for each equipment. (A) The FAM dye was designed to detect all bovine genomes through digital PCR analysis, and the VIC dye was designed at the Del_96 boundary to detect only Hanwoo. (B) The VIC dye was designed to detect all cattle genomes, and the FAM dye was designed to detect only Hanwoo at the Del_96 boundary. (C) The Cy5 dye was designed to detect all cattle genomes, and the FAM dye was designed to detect only Hanwoo at the Del_96 boundary of Hanwoo.

Fig. 3.

Comparison of each positive droplet rate through three repeated experiments. (A) X-axis and Y-axis show the name of each sample and the number of positive droplets formed with FAM dye and VIC dye using the Stilla Naica System. (B) X-axis and Y-axis indicate the name of each sample and the number of positive droplets formed with FAM dye and VIC dye using Bio-Rad equipment. (C) X-axis and Y-axis indicate the name of each sample and the number of positive droplets formed with FAM dye and Cy5 dye using OPTOLANE equipment.

Fig. 4.

Absolute copy number comparison of each equipment using Hanwoo-specific probe. The X-axis represents sample information, Y-axis represents concentration (copy/μL). (A–C) The fluorescence of the Hanwoo-specific probe was detected in Hanwoo but not in Holstein.