Development of an RT-RAA-CRISPR/Cas12a fluorescent detection method for the detection of bovine viral diarrhea virus...

Bovine viral diarrhea virus (BVDV) is a viral pathogen that may cause reproductive disorders, health, and performance. BVDV serotypes have undergone dynamic changes due to sustained genetic diversity, particularly in BVDV type 1 (BVDV1), which has compromised the specificity and sensitivity of diagnostic assays. Furthermore, BVDV1 is prevalent worldwide, causing significant losses to the livestock industry. Therefore, in order to further enhance the control and elimination of BVDV, it is necessary to detect and prevent the spread of BVDV. Therefore, establishing a faster and more accurate on-site detection method is particularly important.

Traditional PCR and ELISA methods are limited by cost, sensitivity, and equipment dependence. In this study, we designed primers based on the conserved 5'Untranslated Region (5'UTR) of BVDV1 and developed a method that combines one-step reverse transcription recombinase assisted amplification (RT-RAA) and CRISPR/Cas12a based fluorescent substrates in a mixture to quickly screen and accurately distinguish BVDV1 for the diagnosis and differentiation of common bovine viruses such as Bovine Coronavirus (BCoV) and Bovine Herpesvirus type 1 (BoHV) et al. The established RT-RAA-CRISPR/Cas12a scheme can achieve the entire inspection process in a portable metal bath at 40℃. We further optimized the reaction conditions of RT-RAA-CRISPR/Cas12a to detect its sensitivity, specificity, and repeatability. The established RT-RAA-CRISPR/Cas12a and conventional PCR were used to detect 300 clinical samples of cattle, verifying the actual detection ability.

This method did not show any cross-reactivity with the other nine common bovine viruses, demonstrating excellent specificity. The minimum template concentration required to trigger significant trans cleavage activity without RT-RAA amplification is 10 copies/µL of BVDV template. However, combined with RT‑RAA amplification, the detection limit was significantly decreased to one copy of the BVDV template per reaction. In simulated sample testing, this method achieved 100% accuracy, and the entire detection process can be completed within 30 min. In clinical sample testing, RT-RAA-CRISPR/Cas12a showed a higher sensitivity rate for BVDV1, as the positive detection rate was 1.85 times higher than that of conventional PCR.

In summary, the BVDV-1 RT-RAA-CRISPR/Cas12a detection method established in this study offers several advantages, including fast operation, high sensitivity, strong targeting, and good repeatability. Moreover, the same test tube reaction system makes it widely applicable in resource-limited environments, making it of great value in large-scale screening during BVDV outbreaks. This method not only expands the diagnostic toolbox of BVDV but also provides an up-and-coming solution for controlling the spread of BVDV in cattle herds.