Main topic : Animal Health
The development, optimization and validation of the Tetracore VetAlert? Magnetic bead extraction Kit for Oral fluid samples.
RAUH R. 1, SAUNDERS A. 1, ZIMMERMAN J. 2, SILVA A. 2, NELSON W. 1
1 Tetracore, Rockville, United States; 2 Iowa State University, Ames, United States
Oral fluid sampling is an effective tool for swine health monitoring. It can be used to detect the presence of a disease agent or antibody response within a population as well as monitor how these agents and diseases circulate in swine. Oral fluids are a mixture of saliva and fluid entering the mouth from capillaries. The oral fluid specimens are easily collected using a cotton rope which allows for a fast, non-invasive and inexpensive collection. Tetracore began working with Iowa State University (ISU) on Oral fluids in 2012. Studies focused on the detection of PRRS1,2 and Swine Influenza3,4 in Oral fluids. Over the years, Oral fluids became the preferred sample type in the US. Data from the Swine Disease Detection Dashboard shows that the number of oral fluid samples tested since 2019 was 1,617,492 and continues to increase yearly. Extraction is a crucial part of isolating infective agents from oral fluids. Tetracore has validated over the years several commercially available extraction kits with Oral Fluids. All of these methods were not validated by the manufacturer for oral fluids, leaving experimentation and validation in the hands of the end-user. With the Tetracore VetAlert™ Magnetic bead extraction Kit, we present an extraction kit that was developed, optimized and validated on Oral fluids. In the development and optimization of the extraction kit, confirmed positive and negative field samples (PRRSV) from industrial and university partners were used. The final validation was performed with Oral fluid samples with well-characterized Mycoplasma hyopneumoniae (MHP) samples, which were tested and published (“Effect of testing protocol and within-pen prevalence on the detection of Mycoplasma hyopneumoniae DNA in oral fluid samples.5”) The pen-based oral fluid samples (n = 320) were collected daily from -4 to 59 days post-inoculation (DPI) under experimental conditions. Tetracore received 205 of the 320 Oral fluid samples. The samples were extracted using the Qiagen QIAamp Viral RNA Mini Kit, Applied Biosystems MagMAX™ CORE and Tetracore VetAlert™ Magnetic bead extraction Kit. The Applied Biosystems and Tetracore kits both use the KingFisher automated extraction instrument for the extraction of DNA and RNA. All three extraction Kits require a pre-treatment of the Oral fluids samples, which includes centrifugation and disruption of the samples. After analysis by ISU, no false positives were reported with any of the extraction kits. The Tetracore extraction kit showed a numerically higher MHP positivity rate.