Objective To establish a rapid screening assay for the detection of Escherichia marmotae based on mass spectrometry, and provide technical support for the detection, infection diagnosis, and epidemiological surveillance of emerging pathogens.

Methods A standard reference library and discrimination analysis mathematical model were constructed based on protein level using the global used commercial software Biotyper^® and ClinProTools^® for the identification of E. marmotae. A total of 474 strains of pathogenic bacteria from 60 genera and 193 species were used for the assay validation. The assay was used for the rapid screening of E. marmotae in pure culture colonies. By using multi PCR-mass spectrometry microsequencing technology, we conducted a comparative analysis on screen specific target genes and designed multiple PCR primers as well as quality probe extension primers. Furthermore, we optimized the detection system to establish a direct nucleic acid-based screening assay for the detection of E. marmotae in non-cultured samples. Subsequently, we validated this assay using 66 strains of pathogenic bacteria from 8 genera and 13 species, along with simulated urine samples andfaecal samples.

Results A standardized reference database for E. marmotae was constructed, which was validated with 474 strains of pathogenic bacteria from 60 genera and 193 species, with an accuracy of 100.00%, a specificity of 99.80% for the validation strains, and a specificity of 100.00% for E. marmotae, indicating that this method can accurately identify E. marmotae. An E. marmotae identification model was constructed through parameter optimization using genetic algorithm. The model was validated using 474 strains of pathogenic bacteria, with an accuracy of 100% and specificities of 96.40% for the validation strains and 100.00% for E. marmotae, allowing accurate identification of E. marmotae. Using multiple PCR-mass spectrometry microsequencing technology, five mutation loci were identified, i.e. gyrA-1716, gyrA-2172, secA-1362, era-126, and era-474. Three pairs of specific primers and quality probe extension primers were designed for the mutation loci, and an E. marmotae detection system was established. The system was validated using 66 strains of pathogenic bacteria, with the accuracy, specificity, and specificity of 100%, meeting the requirement for the iddentification of E. marmotae. The detection limit for simulated urine samples contaminated with with E. marmotae was 450 bacteria colonies and the detection limit for simulated stool samples was 4.5×10³ bacteria colonies.

Conclusion Three screening assays were established in this study for the detection of E. marmotae based on matrix-assisted laser desorption/ionization time of flight mass spectrometry, thereby providing technical support for the identification and clinical diagnosis of E. marmotae, and reference for the development of reverse pathogen detection methods.