Development of Rapid Diagnostic Tests for the On-Site Detection of Capripoxvirus

Abstract

Capripoxviruses are responsible for significant transboundary diseases affecting cattle, sheep, and goats, and are notifiable diseases in Kenya due to their economic impact. Early and accurate detection is crucial for effective disease control and surveillance. Current serological assays, while affordable and sensitive, are time-consuming, and molecular techniques such as PCR and qPCR require specialized equipment and expertise. In contrast, loop-mediated isothermal amplification (LAMP) offers a rapid, cost-effective alternative that can be performed with minimal infrastructure.This study aimed to develop and validate a LAMP assay that is efficient, rapid (<30 min), specific, and sensitive for the detection of Capripoxviruses. The study work was a cross-sectional laboratory activity design over two years, 2018 and 2020. Sampling was based on a non-probability sampling technique where convenience sampling was employed. Samples that were suspected to be Capripoxvirus positive were used and analyzed for this study. A total of 62 samples, comprising blood, skin scrapings, and cell culture isolates from cattle, sheep, and goats, were analyzed. The study compared the performance of a newly designed LAMP primer set (10_LSD), developed using the GLAPD software, with a generic Capripoxvirus (CaPV) primer and conventional molecular assays (PCR and qPCR, the gold standard). Appropriate positive and negative controls, which were a pure culture sample of lumpy skin disease virus and nuclease-free water, respectively, were included throughout all assays to ensure diagnostic accuracy and reliability. The 10_LSD LAMP primer demonstrated superior diagnostic performance with a sensitivity of 60% and specificity of 86%, compared to 56% and 50%, respectively, for the CaPV primer. The 10_LSD assay showed substantial agreement with qPCR, with a kappa value of 0.32, indicating fair agreement, while CaPV and PCR had kappa values of 0.04 (agreement equivalent to chance) and 0.17 (slight agreement), respectively. The 10_LSD assay also demonstrated a lower limit of detection (1 ng/µl) than the CaPV primer (10 ng/µl), though qPCR remained more sensitive (10⁻³ ng/µl). Time to positivity for the LAMP assay ranged between 14 and 28 minutes. The 10_LSD LAMP assay stands out as the best diagnostic option for Lumpy Skin Disease Virus (LSDV) due to its high specificity (86%), which minimizes false positives and ensures meaningful results. Its practicality for field use is a major advantage, requiring minimal equipment and providing results within 15–23 minutes. With a high positive predictive value (93.5%) and a strong negative predictive value (85.7%), it reliably guides treatment and control decisions. Additionally, a field-based DNA extraction method using polyethylene glycol (PEG) was developed for skin biopsies and paired with the LAMP assay. This study demonstrates that the 10_LSD LAMP assay is a promising diagnostic tool for rapid, on-site Capripoxvirus detection. The assay demonstrates proof of concept, validating its potential for further optimization and eventual field deployment.