A LAMP–CRISPR/Cas12a Assay for Specific Detection of Escherichia coli in Whole Blood: Analytical Performance and Clinical Evaluation in Suspected Bloodstream Infection

Background/Objectives: Blood culture is slow and real-time PCR (qPCR) needs a thermal cycler, while loop-mediated isothermal amplification (LAMP) is rapid but prone to non-specific amplification. We developed a LAMP assay with CRISPR/Cas12a read-out targeting the Escherichia coli dxs gene and compared it with qPCR and blood culture. Methods: Reaction conditions were optimised and specificity tested on 15 E. coli and 40 non-target isolates. The limit of detection (LOD) was determined in spiked whole blood (20 replicates per level, 1–10 CFU/mL) by hit rate and probit regression, with conversion to dxs copies by growth phase. Fifty-two patients with suspected bloodstream infection were tested by all three methods. Results: The assay detected all 15 Escherichia coli isolates tested (inclusivity 15/15; 95% CI 79.6–100) and none of 40 non-target isolates (exclusivity 40/40; 95% CI 91.2–100); the exclusivity panel did not include Shigella spp. or Escherichia fergusonii, which the in silico analysis predicts the assay would detect. The probit LOD95 was 14.2 CFU/mL (95% fiducial limits 6.7–156; ≈43 dxs copies/mL for the mid-exponential spike culture used here) for LAMP-CRISPR/Cas12a and 5.8 CFU/mL (2.6–958) for qPCR; a positive was read within 5 min of starting the Cas12a detection step. LAMP-CRISPR/Cas12a was positive in 19/33 qPCR-positive and 0/19 qPCR-negative patients (positive and negative percent agreement 57.6% and 100%; κ = 0.50); all 13 samples at or above the qPCR LOD95 were detected. Conclusions: LAMP-CRISPR/Cas12a detects E. coli in whole blood specifically and rapidly, but its clinical sensitivity is limited by the very low bacterial loads of bacteraemia, so that it is suited to use as a rapid confirmatory test alongside blood culture rather than as a rule-out test.