Impact of RNA Degradation on Viral Diagnosis: An Understated but Essential Step for the Successful Establishment of a Diagnosis Network
<p>Comparison of RNA load values detected from M gene diagnostic target region under different storage conditions for 57 days post incubation (dpi). Dynamics of degradation at (<b>A</b>) low RNA load, (<b>B</b>) medium RNA load and (<b>C</b>) high RNA load. Each matrix of storage is denoted (S: swab matrix, RSB: RNA safe buffer described by Hoffmann et al. (2006) modified in this study, DP: dry pellet matrix), each temperature of storage is also denoted. Data is mean of qRT-PCR from triplicate samples. Based on the limit of detection of the assay, samples with ct > 35 were considered negative (see <a href="#app1-vetsci-05-00019" class="html-app">Supplementary Material Figure S1A</a>).</p> "> Figure 2
<p>Comparison of RNA load values detected from the HA gene diagnostic target region under different storage conditions for 57 days post incubation (dpi). Dynamic of degradation at (<b>A</b>) low RNA load, (<b>B</b>) medium RNA load and (<b>C</b>) high RNA load. Each matrix of storage is denoted (S: swab matrix, RSB: RNA safe buffer described by Hoffmann et al. (2006) modified in this study, DP: dry pellet matrix), each temperature of storage is also denoted. Data is mean of qRT-PCR from triplicate samples. Based on the limit of detection of the assay, samples with ct > 35 were considered negative (see <a href="#app1-vetsci-05-00019" class="html-app">Supplementary Materials Figure S1B</a>).</p> "> Figure 3
<p>Comparison of the probability of folding for secondary structures. (<b>A</b>) Secondary structure folding and conservation pattern of pairing for M gene. The target region framed by M+25 and M-124 primers is denoted by a rectangle, and the hybridization region for each primer is highlighted. (<b>B</b>) Secondary structure folding and conservation pattern of pairing for HA gene. The target region framed by H155f and H699r is denoted by a rectangle and the hybridization region for each primer is highlighted. In the upper panels, the color scale indicates level of conservation and in the lower panels the bar indicates the scale of probability for folding.</p> "> Figure 4
<p>Comparison of the RNA load values under RNase A treatment for HA and M genes. The dynamics of degradation of RNA by the action of RNase A for each gene (HA gene and M gene) is shown. Data is mean of triplicate samples.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Ethics Statement
2.2. Virus Strain and Matrix Samples
2.3. RNA Isolation
2.4. In Vitro Transcription Reaction
2.5. cDNA Synthesis and Quantitative Real-Time RT-PCR (qRT-PCR)
2.6. RNA Stability Conditions
2.7. RNA Secondary Structure for M and HA Genes
2.8. Analysis Degradation Sensitibity
2.9. Statistical Analysis
3. Results
3.1. Stability of the RNA Transcripts for M Gene Stored under Different Conditions
3.2. Stability of the RNA Transcript for HA Gene Stored under Different Conditions
3.3. Secondary RNA Structures Analysis for M and H5 Genes
3.4. Evaluation of the Stability of the Diagnostic Target Regions for M and H5 Genes
4. Discussion
5. Conclusions
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Relova, D.; Rios, L.; Acevedo, A.M.; Coronado, L.; Perera, C.L.; Pérez, L.J. Impact of RNA Degradation on Viral Diagnosis: An Understated but Essential Step for the Successful Establishment of a Diagnosis Network. Vet. Sci. 2018, 5, 19. https://doi.org/10.3390/vetsci5010019
Relova D, Rios L, Acevedo AM, Coronado L, Perera CL, Pérez LJ. Impact of RNA Degradation on Viral Diagnosis: An Understated but Essential Step for the Successful Establishment of a Diagnosis Network. Veterinary Sciences. 2018; 5(1):19. https://doi.org/10.3390/vetsci5010019
Chicago/Turabian StyleRelova, Damarys, Liliam Rios, Ana M. Acevedo, Liani Coronado, Carmen L. Perera, and Lester J. Pérez. 2018. "Impact of RNA Degradation on Viral Diagnosis: An Understated but Essential Step for the Successful Establishment of a Diagnosis Network" Veterinary Sciences 5, no. 1: 19. https://doi.org/10.3390/vetsci5010019