Abundance of Yersinia pestis Nucleic Acids in Soil from Rattus tanezumi Plague Foci in Yunnan Using ddPCR

To determine whether Yersinia pestis (Y. pestis) signals were present in the soil, 78 soil samples were collected from 10 counties identified as Rattus tanezumi plague foci and another six counties in non-foci areas in Yunnan, China, from August 2024 to May 2025. Nucleic acids were extracted using the DNeasy PowerSoil Pro Kit (Qiagen) and detected with a Bio-Rad QX200 droplet digital PCR (ddPCR) system targeting two Y. pestis genes (caf1 and ypo0392). Our sampling design also considered two additional dimensions: nine soil types and three habitat types. The raw data obtained from ddPCR were copy numbers. A gene was considered positive when its copy number exceeded the limit of detection (LoD). A sample was considered positive if the copy number of ypo0392 exceeded its LoD (regardless of caf1), or if both genes exceeded their respective LoDs. No correlation was detected between the positivity rate of Y. pestis and the division of sampling sites into plague focus and non-focus regions (p = 0.758). Similarly, the Mann–Whitney U test revealed non-significant differences in pathogen copy number across the two site categories, with p = 0.603 for the caf1 gene and p = 0.372 for the ypo0392 gene. No statistical difference in positivity rate was found for either soil types or habitat types. However, for both the grouping variables, a statistical difference in copy number was observed. Our results indicated that quantifying nucleic acid abundance by copy number provided richer information than a simple positive/negative determination. The detection of Y. pestis signals was associated with soil type, rather than with whether an area was classified as a focus or non-focus region. Accordingly, future research on the preservation mechanism of Y. pestis should not be restricted to the concept of natural foci but should adopt a broader perspective.