Exo-LAMP: A Rapid Extraction-Free Isothermal Assay for Exosomal mRNA Detection

Abstract

Preeclampsia arises from early placental dysfunction that precedes clinical presentation, creating a need for molecular readouts that enable timely risk assessment. Placenta derived exosomes carry nucleic acid biomarkers with diagnostic potential, yet current analytical workflows rely on multistep isolation and extraction procedures that are slow and resource intensive. Here, we report Exo-LAMP, an extraction-free isothermal assay for detecting placental exosomal KISS1 mRNA, representing the first isothermal approach for exosomal mRNA detection. Placental alkaline phosphatase (PLAP) positive syncytiotrophoblast derived exosomes are selectively captured from biofluids using PLAP antibody conjugated magnetic beads within 20 minutes. Captured exosomes are treated with RNase inhibitor and lysed at 90C for 5 minutes, enabling direct RNA release while preserving transcript integrity and eliminating conventional extraction steps. The released RNA is analyzed in the same vessel by colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP), providing semi-quantitative readout within 25 to 30 minutes. The assay quantifies KISS1 mRNA across 103 to 108 exosomes per reaction, with an estimated limit of detection of about 2 exosomes and a limit of quantification of 103 exosomes. Stoichiometric analysis indicates approximately one KISS1 transcript per 27 exosomes, compared with one per 38 exosomes measured by reverse transcription quantitative polymerase chain reaction (RT-qPCR). In both the exosome-depleted serum spike-in and the plasma spike-in samples containing mixed vesicle populations, the assay detects target derived transcripts at 1% abundance and retains measurable signal at the quantification limit in the presence of up to 108 non-target exosomes. These results establish Exo-LAMP as a rapid and simplified platform for exosomal mRNA analysis that integrates selective capture, lysis, and amplification in a single workflow completed in less than 1 hour. The method provides a general framework for semi-quantitative detection of exosomal transcripts and can be adapted to other coding and non-coding RNA targets for liquid biopsy applications.