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Related Experiment Video

Updated: Mar 15, 2026

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures
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A streamlined, nanopore-compatible 5PSeq protocol for rapid phenotypic antimicrobial sensitivity testing.

Honglian Liu1, Susanne Huch1, Ryan Hull1

  • 1SciLifeLab, Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, 171 65 Solna, Sweden.

Cell Reports Methods
|March 13, 2026
PubMed
Summary
This summary is machine-generated.

Simplified 5PSeq (s5PSeq) offers rapid antimicrobial resistance diagnosis by profiling mRNA degradation, providing a molecular phenotype without culturing. This method enables fast, accurate identification of resistant bacteria, improving treatment guidance.

Keywords:
5PSeq5′P co-translational mRNA decayC. difficileCP: microbiologyCP: molecular biologynanopore sequencingrapid phenotypic ASTribosome stalls

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Sequencing of mRNA from Whole Blood using Nanopore Sequencing
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Area of Science:

  • Molecular Biology
  • Microbiology
  • Genomics

Background:

  • Antimicrobial resistance (AMR) is a major public health concern requiring rapid diagnostics.
  • Current antimicrobial sensitivity testing often relies on culturing, which is time-consuming.
  • Accurate diagnostics are crucial for effective antimicrobial treatment strategies.

Purpose of the Study:

  • To introduce simplified 5PSeq (s5PSeq), a streamlined protocol for profiling 5' monophosphorylated (5'P) mRNA degradation intermediates.
  • To establish s5PSeq as a molecular phenotypic readout for bacterial growth inhibition, bypassing the need for culturing.
  • To demonstrate the clinical utility of s5PSeq for rapid AMR diagnosis.

Main Methods:

  • Profiling 5' monophosphorylated (5'P) mRNA degradation intermediates to reflect ribosome dynamics.
  • Capturing antibiotic-induced, context-specific ribosome stalling events.
  • Utilizing a novel rRNA blocking strategy and real-time nanopore sequencing.

Main Results:

  • s5PSeq reduces library preparation time to under 4 hours.
  • The method successfully identified erythromycin-resistant and sensitive Clostridioides difficile clinical isolates.
  • Fast AMR diagnosis was achieved with as few as 3,000 nanopore sequencing reads.

Conclusions:

  • s5PSeq provides a rapid, culture-independent molecular phenotypic readout for AMR.
  • Combining s5PSeq with nanopore sequencing significantly enhances diagnostic speed and accuracy.
  • This approach offers a promising tool for improving clinical AMR diagnostics and guiding treatment decisions.