Studying genetic diseases using a DNA collection kit
DNA and RNA analysis is commonly used for studying genetic diseases, performing diagnostic testing, and improving the health of animals and humans. Achieving sensitivity and specificity essential for this analysis requires a high-quality genomic sample. While traditionally blood was considered the preferred sample type, it can be costly, invasive, and time-consuming to collect. Using saliva samples as an alternative, non-invasive source of genomic DNA or RNA reduces risk and cost, while offering flexibility and convenience for collection.
The PERFORMAgene™(PG-100) DNA collection kit, offered by DNA Genotek, is a reliable, non-invasive device that is used to collect, transport, and store high-quality DNA from animal saliva. The PERFORMAgene collection device is well documented as an alternative to blood for acquiring high-quality DNA samples from livestock,1,2 dogs,3,4 and cats,5 and is consistently referenced in leading animal genomics research.6,7
Using the PERFORMAgene™ DNA collection kit for an RNA-based application
The Coronavirus disease 2019 (COVID-19) has affected hundreds of millions of people worldwide since the SARS-CoV-2 outbreak first began. As a result, SARS-CoV-2 diagnostic tests have been developed at an unprecedented rate to detect and consequently limit the spread of the virus. SARS-CoV-2 is frequently detected using a widely-known laboratory technique, reverse transcriptase polymerase chain reaction (RT-PCR), where viral RNA is transformed into a more stable form and then amplified for detection of specific segments.
Basepaws is a pet genetics company known for its leading feline genetics research and products that help pet parents better understand their cats’ breed profiles, traits, and health predispositions. For years, Basepaws has relied on the quality of molecular DNA samples obtained using PERFORMAgene collection kits for animals.
Like humans, cats are also susceptible to COVID-19, but at the time, testing was limited as reagents had to be understandably prioritized for humans. Due to their long standing relationship with DNA Genotek, the Basepaws science team was curious to see if RNA could also be stabilized and extracted using these kits. They decided to find out if they could accurately detect SARS-CoV-2 using the PERFORMAgene collection kit—originally developed for the collection of DNA from saliva—to attempt to sample RNA from felines.
Sampling and extracting RNA from healthy cats
Healthy cats were sampled using two DNA Genotek products, the PERFORMAgene and the ORAcollect™•RNA sample kit, to compare the quality of RNA obtained from each. To avoid using commercial extraction kits and reagents, Basepaws performed the RNA extraction using the solid-phase reversible immobilization (SPRI) method.8
Gel electrophoresis, and NanoDrop and Quantus spectrophotometers were used to measure the quality and quantity of the extracted RNA. The results obtained from quantitative measurements indicated that more RNA was extracted from the samples collected with PERFORMAgene kits compared to the ORAcollect•RNA kits. However, the quality of the RNA was slightly worse, as judged by the NanoDrop 260/280 and 260/230 readings.9
Next, Basepaws researchers investigated whether the extracted RNA contained both feline and viral RNA. The team amplified the two ubiquitously expressed cat housekeeping genes, GAPDH and RSP19, to determine if there were measurable amounts of feline mRNA in the samples. The RT-PCR analysis detected the GAPDH gene in similar abundance in both RNA samples collected with the PERFORMAgene and ORAcollect•RNA kits, while RSP19 gene abundance was higher in the samples collected with the ORAcollect•RNA kits.9
To detect viral genes, RNA sequencing analysis was performed, identifying 33 viral genomes, of which at least nine were genomes of RNA viruses.9 These results suggest that high-quality viral and feline RNA can be extracted from a PERFORMAgene collection kit, without the need for in-demand reagents.
Amplifying RNA from samples using SYBR RT-PCR
After confirming that high-quality RNA samples could be obtained, the next step was to find an alternative to the traditional TaqMan RT-PCR method, ubiquitously used for COVID-19 testing. To circumvent a possible reagent shortage, Basepaws devised a novel hybrid method combining SYBR RT-PCR and Sanger sequencing for SARS-CoV-2 detection (this methodology is available as a preprint on medRxiv). Optimizing the Sanger sequencing component of the hybrid method allows for robust detection of specific coronaviruses, and can distinguish between different strains of coronaviruses. The accurate identification of SARS-CoV-2 is essential, as cats can be infected by several coronaviruses, including SARS-CoV-2, SARS-CoV (the coronavirus that caused the 2003 Asia epidemic) and Feline Enteric Coronavirus (FECV).
To measure the sensitivity of the hybrid method, SYBR RT-PCR was performed on samples with different amounts of SARS-CoV-2 viral copies, with the number of PCR cycles analyzed for different viral loads. The results indicated that the novel hybrid SARS-CoV-2 detection method can reliably detect viral loads of at least 10 copies per reaction. The drawback of this method is that it cannot detect low viral loads, and thus may not be able to detect the virus at the early stages of the infection.9
The hybrid method developed by Basepaws is versatile, as it can be adapted to a range of diverse laboratory setups and available reagents.9 The Sanger sequencing component of the technique also enables tracking and modeling of the SARS-CoV-2 viral genome evolution as the virus spreads.
As exemplified by the research conducted by Basepaws, a Zoetis company, PERFORMAgene collection kits are not only a reliable non-invasive device to collect, transport, and store high-quality DNA from animals, they are also useful for RNA collection. These types of pre-clinical animal cohort studies play an important role in understanding the transmission of human-to-animal (“zoonotic”) viral infectious diseases.
PERFORMAgene collection kits are a simple, non-invasive, and robust method for obtaining high-quality DNA for diverse genetic analyses in animals. In addition to inherited disease testing, genotyping, breed analysis, and other DNA analyses, samples collected using the PERFORMAgene kits appear to be a valuable tool for RNA studies and research applications as well.
1. Neary MT, Neary JM, Lund GK, Garry FB, Holt TN, Mohun TJ, Breckenridge RA. (2014) Technical note: A comparison of DNA collection methods in cattle and yaks. J Anim Sci. doi: 10.2527/jas.2013-7445
2. Foley C, O'Farrelly C, Meade KG. (2011) Technical note: Comparative analyses of the quality and yield of genomic DNA from invasive and noninvasive, automated and manual extraction methods. J Dairy Sci. doi: 10.3168/jds.2010-3987
3. Marchant TW, Johnson EJ, McTeir L, Johnson CI, Gow A, Liuti T, Kuehn D, Svenson K, Bermingham ML, Drögemüller M, Nussbaumer M, Davey MG, Argyle DJ, Powell RM, Guilherme S, Lang J, Ter Haar G, Leeb T, Schwarz T, Mellanby RJ, Clements DN, Schoenebeck JJ. (2017) Canine brachycephaly is associated with a retrotransposon-mediated missplicing of SMOC2. Curr Biol. doi: 10.1016/j.cub.2017.04.057
4. Friedrich J, Talenti A, Arvelius P, Strandberg E, Haskell MJ, Wiener P. (2020) Unravelling selection signatures in a single dog breed suggests recent selection for morphological and behavioral traits. Adv Genet. doi: 10.1002/ggn2.10024
5. Whitney J, Haase B, Beatty J, Barrs VR. (2019) Breed-specific variations in the coding region of toll-like receptor 4 in the domestic cat. Vet Immunol Immunopathol. doi: 10.1016/j.vetimm.2019.02.009
6. Zapata I, Lilly ML, Herron ME, Serpell JA, Alvarez CE. (2022) Genetic testing of dogs predicts problem behaviors in clinical and nonclinical samples. BMC Genomics. doi: 10.1186/s12864-022-08351-9
7. Raffan E, Dennis RJ, O'Donovan CJ, Becker JM, Scott RA, Smith SP, Withers DJ, Wood CJ, Conci E, Clements DN, Summers KM, German AJ, Mellersh CS, Arendt ML, Iyemere VP, Withers E, Söder J, Wernersson S, Andersson G, Lindblad-Toh K, Yeo GS, O'Rahilly S. (2016) A deletion in the canine POMC gene is associated with weight and appetite in obesity-prone Labrador retriever dogs. Cell Metab. doi: 10.1016/j.cmet.2016.04.012
8. DeAngelis MM, Wang DG, Hawkins TL. (1995) Solid-phase reversible immobilization for the isolation of PCR products. Nucleic Acids Res. doi: 10.1093/nar/23.22.4742
9. Yang J, Salfati E, Kao D, Mihaylova Y. (2020) Use of alternative RNA storage and extraction reagents and development of a hybrid PCR-based method for SARS-CoV-2 detection. medRxiv. doi: 10.1101/2020.11.21.20236216