Bluetongue virus diagnosis in Argentina, primarily via RT-qPCR, faces challenges due to the limited number of laboratories, many of which are located in regions where BTV is exotic and far from primary sample collection areas. Consequently, blood samples must be transported over considerable distances for analysis, posing significant logistical challenges due to the specific transport conditions needed. Therefore, the development of methodologies that optimize sample collection, preservation, and safe transport is of vital importance. The objective of this work was to standardize a sampling protocol for BTV using filter paper cards, to achieve adequate preservation and transport of blood samples for subsequent diagnosis. Hydration with TE at 37°C yielded the lowest Cq among tested conditions, with a detection limit of 2.75 10² gc/µL. We evaluated the stability of the sample on the cards by storing them at 25°C, 4°C, -20°C, and -80°C for 1, 7, 30, 60, 120, and 360 days. Stability was verified up to 360 days at all temperatures. Additionally, we confirmed the absence of infectious material in the card eluate. Finally, we conducted a preliminary field evaluation by collecting blood samples from cattle and sheep in tubes and on cards. According to the Cohen´s Kappa index, the agreement between both methods was almost perfect. The standardized methodology represents a significant advance in BTV diagnosis, as it will allow samples to be transported without refrigeration and with adequate biosafety conditions without losing diagnostic capacity.

The diagnosis of Bluetongue Virus (BTV) in Argentina is primarily conducted via RT-qPCR and faces challenges due to the limited number of diagnostic laboratories, many of which are located in regions where BTV is exotic and far from primary sample collection areas. As a result, blood samples must be transported over considerable distances for analysis. This poses significant logistical challenges due to the specific transport conditions that are required. Consequently, the development of methodologies that optimize sample collection, preservation, and safe transport is of vital importance. The objective of this study was to standardize a sampling protocol for the detection of BTV using filter paper cards. This protocol was developed to ensure adequate preservation and transport of blood samples for diagnosis. Hydration with TE at 37 °C resulted in the lowest Cq among the tested treatments, with ~10 TCID₅₀/mL representing the lowest viral concentration that was consistently detected under the experimental conditions. The stability of the sample on the cards was evaluated through a series of storage trials at temperatures of 25 °C, 4 °C, −20 °C, and −80 °C for 1, 7, 30, 60, 120, and 360 days. Stability was verified up to 120 days at all temperatures. Furthermore, the absence of infectious material in the card eluate was confirmed. A preliminary field evaluation was conducted, in which blood samples from cattle and sheep were collected in tubes and on cards. According to the Cohen’s Kappa index, the agreement between both methods was nearly perfect. The standardized methodology signifies a substantial advancement in BTV diagnosis, as it facilitates the transportation of samples without the necessity of refrigeration and with adequate biosafety conditions without losing diagnostic capacity.