Detection of Parvovirus B19 genome in human heart tissue samples

Abstract

Objective Identifying viral genomes in human heart tissues is critical for disease diagnosis and assessment of cardiovascular damage. Human heart tissue samples obtained during a biopsy procedure are routinely used to test for the presence of viruses, as guided by clinical manifestations and prognosis. Furthermore, heart tissue samples obtained post-mortem or during a cardiac transplant procedure serve as a valuable research tool, as they allow for an in-depth assessment of cardiac pathology that can aid in our understanding of molecular pathways associated with disease. Because viral nucleic acid constitutes only a small portion of each sample’s genetic material, appropriate methods are necessary for positive viral genome identification. Results Snap-frozen heart tissue samples obtained either post-mortem or during a cardiac transplant procedure were used to develop conditions for detection of Parvovirus B19. Briefly, total DNA was isolated from the heart tissue under varying conditions. A PCR-based assay with Parvovirus B19 specific primers was implemented to detect the presence of the viral genome, followed by Sanger Sequencing. The mechanical disruption of the heart tissue, as well as the cardiac tissue processing methods, had a significant effect on the DNA quality and the ability to detect the Parvovirus B19 genome.

Analysis of Parvovirus B19 persistence and reactivation in human heart layers

“…PRO Scientific Bio-Gen homogenizer was used to dissociate the heart tissue samples, and DNA and RNA were extracted using a Quick-DNA/RNA Miniprep Plus Kit (Zymo Research, D7003). RNA samples were treated with DNAse I at the time of the procedure to eliminate DNA contamination according to manufacturer's instructions (31). The absence of residual DNA was independently verified in a PCR reaction to ensure no contamination in the RNA samples.…”