Polymerase Chain Reaction on In-cage Filter Paper at Different Time Points to Detectspp.

Abstract

spp. infections in mice can have broad-ranging effects on gastrointestinal, reproductive, and immune systems. This can introduce significant confounding variables for research and may reduce scientific rigor. Screening mouse colonies forspecies can be accomplished via noninvasive PCR testing on filter paper placed in animal-free dirty bedding sentinel cages. In our facility, one tablespoon of dirty bedding from each cage on a rack is added to a designated sentinel cage every 3 wk at cage change, and PCR testing is performed on in-cage filter paper quarterly. We hypothesized that cages that receivedspp.-positive bedding at later time points would have a lower detection rate ofspp. with PCR testing compared with cages that received positive bedding at earlier time points due to the filter paper becoming saturated. To determine if screening would be able to detect one positive row of cages on a rack, 9 tablespoons ofpositive bedding and 71 tablespoons of negative bedding were added at the 3-, 6-, or 9-wk time points to 14 empty sentinel cages per time point. Negative bedding was added every 3 wk to cages not scheduled to receive positive bedding. Negative controls received 80 tablespoons of negative bedding and positive controls received 80 tablespoons of positive bedding at each time point. Filter paper was tested via PCR forspp. at 12 wk. All positive controls tested positive, and all negative controls tested negative. Two 3-wk cages, two 6-wk cages, and three 9-wk cages were positive, indicating no difference between time points. This resulted in a 16.7%spp. detection rate. These results indicate that PCR on in-cage filter paper may not be reliable in detecting low levels ofspp. nucleic acid in dirty bedding.