A multi-tube singleplex qPCR assay for detection of the Corynebacterium kroppenstedtii complex, Corynebacterium spp., and other bacteria in granulomatous mastitis

Objective To establish a multi-tube singleplex qPCR assay for detecting the Corynebacterium kroppenstedtii complex (CKC), the genus Corynebacterium , and other bacteria in pus specimens from patients with granulomatous mastitis (GM). Methods CKC-specific and Corynebacterium genus-specific primers were designed based on 16S rRNA, rpoB , and fusA sequences using MEGA 11 and DNAMAN V6.0. Primer specificity was evaluated using Primer-BLAST and screened by PCR and qPCR. qPCR standard curve was constructed using clinical isolate LCNY-20. Pus specimens from 86 patients with GM were tested by qPCR using CKC-specific, Corynebacterium genus-specific, universal bacterial, and β-actin primers. Pathogens were isolated using optimized culture medium and identified by sequencing. Receiver operating characteristic (ROC) curve analysis was used to derive an exploratory, cohort-specific Cq cut-off value for the universal bacterial assay. Based on qPCR results, patients were classified into CKC-detected, non-CKC Corynebacterium -detected, other bacteria-detected, and no-bacteria-detected groups. Clinical characteristics and associations between antibiotic use and clinical outcomes were then explored. Results The optimal primers were selected from 20 self-designed and 10 literature-derived candidate pairs. The limit of detection (LOD) for both the CKC-specific and Corynebacterium genus-specific primers was 1×10 4 CFU/mL, and the cohort-specific Cq cutoff derived for the universal bacterial assay was 22.43. Among 86 clinical GM specimens, the detection proportions of CKC-specific qPCR and Corynebacterium genus-specific qPCR were 10.4 and 34.9 percentage points higher, respectively, than those of the optimized culture method ( P = 0.0039 and P < 0.001, respectively). The detection proportion of universal qPCR was 8.2 percentage points higher, but the difference did not reach statistical significance ( P = 0.2100). Based on qPCR grouping, overall comparisons of all analyzed clinical variables across the four groups did not reach statistical significance (all P > 0.05). Among patients with Corynebacterium detected, no statistically significant associations were observed between empirical antibiotic use and the analyzed clinical outcomes. Conclusion This multi-tube singleplex qPCR system enables hierarchical detection of CKC, the genus Corynebacterium , and other bacteria in GM pus, with a higher bacterial detection proportion than the optimized culture method.