# Effect of Pooling and Freeze-Thaw Processes on the PRRSV RNA Detection in TOSc Samples

## TL;DR

This study shows that pooling and freeze-thawing TOSc samples can reduce the detection of PRRSV RNA, especially in samples with high Ct values.

## Contribution

The study provides practical guidance on optimal TOSc sample handling for PRRSV surveillance based on Ct values.

## Key findings

- Pooling TOSc samples with Ct values > 35 significantly reduces PRRSV RNA detection probability.
- Freeze-thaw cycles increase Ct values and lower detection probability in TOSc samples.
- Samples with Ct values < 35 can be pooled up to 1:10 without significant loss in detection.

## Abstract

Tonsil oral scrubbing (TOSc) is a recently developed, easy, and practical sampling method for detecting PRRSV RNA in breeding sows. This study aimed to evaluate whether pooling samples or subjecting them to a freeze-thaw cycle would affect PRRSV RNA detection. We tested pooled samples at various dilution ratios and compared PRRSV RNA detection in fresh versus freeze-thawed TOSc samples. Results showed that pooling reduced probability of PRRSV RNA detection, especially in samples with Ct values > 35, while those with Ct values < 35 remained detectable even after pooling to a ratio of 1:10. Similarly, one freeze-thaw cycle increased Ct values and reduced probability of detection. These findings suggest that pooling may be used in TOSc samples with low Ct values < 35 to reduce testing costs, but should be avoided for samples with Ct values > 35. Freeze- thawed samples should also be minimized during handling. This research provides practical TOSc sample handling guidance for laboratories and veterinarians to optimize PRRSV surveillance using TOSc.

Tonsil oral scrubbing (TOSc) is a simple and practical method recently developed for detecting PRRSV RNA in sows. This study evaluated how pooling and freeze-thaw procedures affect PRRSV RT-rtPCR results in TOSc samples. To assess pooling effects, 22 PRRSV-positive samples were divided into three Ct categories and diluted with negative TOSc samples at various ratios before testing. To assess freeze-thaw effects, 90 TOSc samples were split into two equal aliquots: one tested immediately, and the other tested after one freeze-thaw cycle. Results showed that PRRSV RNA detection probability decreased and Ct values increased as dilution (pooling) ratios rose. Samples with Ct values > 35 had significant drops in probability of detection beginning at a 1:3 dilution (≤71%). In contrast, samples with Ct values <35 maintained a high probability of detection until 1:10 dilution (≥85%). The freeze-thawed samples showed significantly higher Ct values and numerically lower probability of detection compared to non-freeze-thawed ones. In conclusion, pooling and freeze-thaw negatively impact PRRSV RNA detection in TOSc samples. Pooling may be acceptable for samples with a Ct value < 35 for up to 1:10 dilution, but should be avoided when the Ct value > 35. Similarly, the freeze-thaw process should be minimized during TOSc sample handling.