Semi-quantitative digital analysis of polymerase chain reaction-electrophoresis gel: Potential applications in low-income veterinary laboratories

Abstract

Aim: The interpretation of conventional polymerase chain reaction (PCR) assay results is often limited to either positive or negative (non-detectable). The more robust quantitative PCR (qPCR) method is mostly reserved for quantitation studies and not a readily accessible technology in laboratories across developing nations. The aim of this study was to evaluate a semi-quantitative method for conventional PCR amplicons using digital image analysis of electrophoretic gel. The potential applications are also discussed.

Materials and methods: This study describes standard conditions for the digital image analysis of PCR amplicons using the freely available ImageJ software and confirmed using the qPCR assay.

Results and conclusion: Comparison of ImageJ analysis of PCR-electrophoresis gel and qPCR methods showed similar trends in the Fusobacterium necrophorum DNA concentration associated with healthy and periodontal disease infected wallabies (p≤0.03). Based on these empirical data, this study adds descriptive attributes ("more" or "less") to the interpretation of conventional PCR results. The potential applications in low-income veterinary laboratories are suggested, and guidelines for the adoption of the method are also highlighted.

Keywords: ImageJ software; applications; digital image analysis; polymerase chain reaction amplicon; polymerase chain reaction-electrophoresis; quantitative polymerase chain reaction.

Figure 1

Screenshots of the ImageJ window showing the selected polymerase chain reaction (PCR) bands and molecular ladder (in rectangles). (a) Lanes 1-4=PCR positive bands (250 bp fragment of the Fusobacterium necrophorum encoded hemagglutinin-related-gene); Lane 5=Positive control; Lane 6=Molecular ladder (Hyperladder I) (Bioline; Australia). No amplification was observed in the negative control test sample. (b) ImageJ generated peaks of the corresponding PCR bands in Figure-1a. The peaks were based on the PCR band densities. The arrow indicates the line that specifies the “area” of measurement within the peak. The PCR band density is automatically generated by the ImageJ software using the specified “area.”

Figure-S1

A three-dimensional image showing the trend of Fusobacterium necrophorum DNA concentration in healthy and periodontal disease (PD) samples obtained from captive wallabies. ImageJ analysis of endpoint polymerase chain reaction (PCR)-electrophoresis gel was used to estimate the density of the PCR bands (amplicons) which was then compared to a band of known DNA concentration. F. necrophorum DNA concentration was higher in PD (mean=27 ng/μl) than in healthy participants (mean=8.9 ng/μl). The samples were run in duplicates and equal volume from each replicate was pooled, and 5 μl was loaded onto the agarose gel.

Figure-S2

A three-dimensional image showing the trend of Fusobacterium necrophorum DNA concentration in healthy and periodontal disease (PD) samples obtained from captive wallabies. F. necrophorum DNA was higher in PD (mean=6.24%) than in healthy participants (mean=0.0029%). The quantitative polymerase chain reaction estimated concentration was expressed as a percentage of the template DNA concentration (10 ng) of DNA extract from oral swab sample). The samples were run in duplicates.

Figure 2

Polymerase chain reaction (PCR) phases in log view showing the region of ethidium bromide detection of PCR amplicons. The quantitative PCR method is detected at the exponential phase.