〔Abstract〕 ObjectiveTo investigate the causes of false negative results in the detection of Streptococcus agalactiaeusing fluorescent quantitative PCR (qPCR) targeting the CAMP factor gene ( cfb), and to perform a comprehensive analysis of the associated molecular mechanisms. MethodsA total of 76 vaginal secretion samples were evaluated using both qPCR based on cfb gene and bacterial culture methods. Four suspicious strains exhibiting negative qPCR results but positive culture findings were identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), latex agglutination antigen detection, and the CAMP test. Whole genome analysis was conducted utilizing the MGI DNBSEQ-T7 and Nanopore-PromethION 48 sequencing platforms. Phylogenetic and circular evolutionary trees were constructed using the 16S rRNA gene for strain verification. Multilocus sequence typing (MLST) was conducted, and cfb sequences were aligned and analyzed based on spliced sequences and original data. Specific primers targeting the cfbgene were designed for full-length amplification, followed by verification through agarose gel electrophoresis. ResultsThe four strains identified as suspicious were classified as S.agalactiae through MALDI-TOF MS, antigen detection, and 16S rRNA gene analysis, with MLST typing indicating ST862. Phenotypic analysis revealed a negative CAMP test. Whole genome sequence alignment failed to detect the cfb gene or any homologous sequences, and molecular testing confirmed the absence of cfb gene PCR amplification products, thereby confirming its complete deletion. Conclusion This deletion is identified as the molecular mechanism responsible for the false negative qPCR detection of S. agalactiae when targeting this specific gene. It is recommended that the qPCR detection targeting a single cfb gene has limitation, and this may impact clinical diagnosis and treatment decisions. This limitation warrants careful consideration.