The ISChip: A high-throughput qPCR array for absolute quantification of insertion sequences across the One Health...

Insertion sequences (IS) are pivotal mobile genetic elements that shape bacterial genome plasticity and act as critical drivers of environmental genetic hazards by accelerating the dissemination of antimicrobial resistance. However, high-throughput, absolute quantification of IS elements across diverse environmental matrices remains a significant technical challenge, as conventional short-read metagenomics often lacks the sensitivity and resolution required for profiling low-abundance and highly repetitive targets. Here, we developed ISChip, a high-capacity qPCR array for the multiplexed absolute quantification of 183 prevalent IS elements, serving as a robust quantitative complement to metagenomics. The platform was rigorously validated using 119 primer sets, demonstrating high specificity, efficiency, and a superior absolute sensitivity (limit of quantification: 23-28 copies per reaction) compared to conventional qPCR. We applied ISChip to 69 anthropogenically impacted samples spanning 13 matrices, including air, wastewater, soil, and human/animal feces, representing a comprehensive One Health continuum. Our results revealed a distinct compartmentalization of IS communities and identified wastewater, sludge, sediments, and human feces as primary IS hotspots. Notably, we discovered a highly conserved "core IS assemblage" in human feces, suggesting a unique niche for IS-driven microbial evolution. By providing a scalable and absolute quantitative framework, this study uncovers the extreme spatial magnitude of these biological hazards, serving as a powerful tool for monitoring genetic pollution across the One Health framework.