Identification of the novel potential pathogen Trueperella pecoris with interspecies significance by LAMP diagnostics

Abstract

Trueperella pecoris was described as a new species of the genus Trueperella in 2021 and might be pathogenic to various animal species. However, the lack of a suitable diagnostic test system stands in the way of epidemiological surveys to clarify possible causalities. In this study, a Loop-mediated Isothermal Amplification (LAMP) assay was developed and validated that was highly specific for T. pecoris. The assay provided an analytical sensitivity of 0.5 pg/25 µL and showed 100% inclusivity and exclusivity for 11 target and 33 non-target strains, respectively. Three different DNA extraction methods were evaluated to select the most LAMP-compatible method for cell disruption in pure and complex samples. Using an on-site applicable single-buffer DNA extraction with additional heating, the cell-based detection limit was 2.3 CFU/reaction. Finally, the LAMP assay was validated by means of artificially contaminated porcine lung tissue samples in which minimal microbial loads between 6.54 and 8.37 × 10³ CFU per swab sample were detectable. The LAMP assay established in this study represents a suitable diagnostic procedure for identifying T. pecoris in clinical specimens and will help to collect epidemiological data on the pathogenicity of this species.

Figure 1

TP LAMP assay primers. LAMP primers were based on strain T. pecoris 19OD0592 (Acc. No. NZ_CP063212.1). Alignment with other T. pecoris genomes showed the high conservation of the target region. Query = T. pecoris strain 19OD0592, CP071974.1 = T. pecoris strain 15IMDO307, CP063213.1 = T. pecoris strain 15A0121, CP053291.1 = T. pecoris strain 19M2397.

Figure 2

Temperature optimization of the TP LAMP assay. Detection times are shown for 0.5 pg positive control DNA/25 µL from strain T. pecoris DSM 111392^T as a function of the reaction temperature used. Marks and error indicator bars represent the mean and standard deviation, respectively, of three independent measurements.

Figure 3

Analytical sensitivity of TP LAMP assay. (A) Amplification curves for ten-fold serially diluted DNA of T. pecoris DSM 111392^T. Characteristic for LAMP, the amplification curves flattened with decreasing DNA concentration. NTC = non-template control. (B) Detection times for ten-fold serially diluted DNA of T. pecoris DSM 111392^T as a function of DNA concentration per reaction. Marks and error indicator bars represent the mean and standard deviation, respectively, of three independent measurements. With decreasing DNA concentration, detection times did not increase linearly. For this reason, LAMP was not suitable for quantifying the target species.