Exploration of Novel Target Via Genome Mining and Establishment of Proofman-LMTIA Assay for the Rapid Detection of Chlamydia abortus

Abstract

Chlamydia abortus is an obligate intracellular Gram-negative pathogenic bacterium widely distributed globally. This pathogen primarily infects humans and various domestic animals, causing abortions in pregnant animals. It poses a significant threat to public health and the animal husbandry industry, leading to substantial economic losses. In this study, specific primers and a Proofman fluorescent probe for LMTIA (Ladder Melting Temperature Isothermal Amplification) were designed based on the genome of the C. abortus S26/3 strain through sequence alignment and screening of specific target sequences. These primers and probes were used to develop a rapid Proofman-LMTIA assay for detecting C. abortus . The reaction system and temperature were optimized, and the specificity, sensitivity, and reproducibility of the assay were evaluated. The optimal amplification temperature for Proofman-LMTIA was 59.5 °C, and the reaction time was less than 30 minutes. The established assay had a minimum detection limit of 101 fg/μL per test, indicating a high analytical sensitivity. The assay also showed excellent stability, with coefficients of variation below 3% in inter-group and intra-group reproducibility tests. In summary, the rapid and effective Proofman-LMTIA method for detecting C. abortus developed in this study provides a novel technical tool for the clinical diagnosis and control of C. abortus infections.