# ColorDetect RT-LAMP Assay for the Rapid, Sensitive, and Specific Detection of Porcine Abortion-Associated Pestivirus (PAAPeV)

## TL;DR

This study developed a fast and accurate test for detecting a virus causing reproductive issues in pigs, which could help control outbreaks in swine populations.

## Contribution

A novel RT-LAMP assay called colordetect RT-LAMP was developed for the specific and sensitive detection of PAAPeV.

## Key findings

- The assay detected as few as 2 copies/μL of PAAPeV RNA in 25 minutes at 65°C.
- It showed 100% agreement with the gold-standard RT-qPCR method in clinical validation.
- The assay had no cross-reactivity with other swine pathogens or pestiviruses.

## Abstract

Emerging porcine pestivirus-associated diseases continue to pose significant challenges to preventive and control measures within the global swine industry. In this study, we developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for a newly identified pestivirus—porcine abortion-associated pestivirus (PAAPeV)—designated as colordetect RT-LAMP. Based on the highly conserved 5′ un-translated region (5′UTR) of PAAPeV, this assay achieved a detection limit of 2 copies/μL under 65 °C incubation for 25 min, and had no cross-reactivity against other known pestiviruses or prevalent swine pathogens. Clinical validation revealed 100% concordance between the colordetect RT-LAMP assay and the gold-standard RT-qPCR method. This assay serves as a rapid, sensitive, and specific diagnostic tool for resource-limited settings and thus holds substantial promise for widespread deployment in on-site and regional veterinary laboratories.

The primary objective of this study was to develop and optimize a colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for porcine abortion-associated pestivirus (PAAPeV)—an emerging pathogen that causes severe reproductive disorders in swine, for which no effective treatments or vaccines are currently available. In this study, four sets of LAMP primers were designed and screened for the colorimetric RT-LAMP assay, targeting the highly conserved 5′ untranslated region (5′UTR) of PAAPeV. Three reaction parameters, including reaction temperature, reaction duration, and inner-to-outer primer ratio, were then optimized based on cycle threshold (Ct) values, fluorescence intensity, and color changes of the endpoint products. Subsequently, the specificity and sensitivity of the optimized colordetect RT-LAMP assay were systematically validated, and its diagnostic performance was compared with that of the gold-standard reverse transcription quantitative polymerase chain reaction (RT-qPCR). The results demonstrated that the optimized assay achieved a detection limit of 2 copies/μL under the conditions of 65 °C incubation for 25 min and an inner-to-outer primer ratio of 8:1, with results amenable to naked-eye interpretation. Furthermore, this assay exhibited high specificity, showing no cross-reactivity with other known pestiviruses or prevalent swine pathogens. Clinical sample testing results showed 100% concordance between colordetect RT-LAMP and RT-qPCR. Collectively, this colordetect RT-LAMP assay represents a rapid, sensitive, and specific tool for PAAPeV RNA detection in both clinical laboratories and field settings.