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Fluorescence in situ hybridization, or FISH, was developed in the early 1980s and has quickly become one of the most widely used techniques in cytogenetics. Labeled probes are used to bind complementary DNA or RNA sequences on a chromosome or in a region within a cell. Earlier, the probes could only be obtained by cloning or reverse transcription of a DNA template. Currently, the probe oligonucleotides can be synthesized synthetically. Additionally, with the advancement of optical techniques,...

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Published on: May 25, 2015

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

Yanyan Zhou1, Wen-Jing Zhong2, Xin-Li An3

  • 1State Key Laboratory of Regional and Urban Ecology, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen, 361021, China; Guangdong Provincial Key Laboratory of Marine Biotechnology, Shantou University, Shantou, 515063, China.

Environmental Pollution (Barking, Essex : 1987)
|June 25, 2026
PubMed
Summary
This summary is machine-generated.

Insertion sequences (IS) are mobile genetic elements driving antimicrobial resistance spread. A new qPCR array, ISChip, enables their absolute quantification across environmental samples, revealing hotspots and a conserved core assemblage in human feces.

Keywords:
Absolute quantificationBiological hazardGenomic plasticityHigh-throughput qPCRInsertion sequencesOne Health

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Area of Science:

  • Microbiology
  • Genomics
  • Environmental Science

Background:

  • Insertion sequences (IS) are mobile genetic elements that influence bacterial genome evolution and facilitate the spread of antimicrobial resistance (AMR).
  • Accurate quantification of IS elements in diverse environmental samples is challenging due to their repetitive nature and low abundance, limiting traditional metagenomic approaches.
  • Understanding the distribution and dynamics of IS elements is crucial for assessing environmental genetic hazards and AMR dissemination.

Purpose of the Study:

  • To develop a high-throughput method for the absolute quantification of multiple insertion sequence (IS) elements.
  • To assess the distribution and abundance of IS elements across various environmental matrices within a One Health framework.
  • To identify key environmental compartments that act as reservoirs for IS elements and potential drivers of AMR spread.

Main Methods:

  • Development and validation of ISChip, a high-capacity quantitative PCR (qPCR) array designed for multiplexed absolute quantification of 183 prevalent IS elements.
  • Rigorous validation of the platform using 119 primer sets to assess specificity, efficiency, and absolute sensitivity (limit of quantification: 23-28 copies per reaction).
  • Application of ISChip to 69 anthropogenically impacted environmental samples from 13 matrices, including air, wastewater, soil, and feces.

Main Results:

  • ISChip demonstrated high specificity, efficiency, and superior absolute sensitivity compared to conventional qPCR.
  • Analysis of environmental samples revealed distinct compartmentalization of IS communities, with wastewater, sludge, sediments, and human feces identified as primary IS hotspots.
  • A highly conserved "core IS assemblage" was identified in human feces, indicating a specialized niche for IS-driven microbial evolution.

Conclusions:

  • ISChip provides a scalable and robust framework for the absolute quantification of IS elements, overcoming limitations of short-read metagenomics.
  • The study highlights the significant spatial distribution of IS elements as biological hazards across the One Health continuum.
  • This quantitative approach serves as a valuable tool for monitoring genetic pollution and understanding the role of IS elements in AMR dissemination.