Immunomagnetic separation combined with colony immunoblotting for selective enrichment and detection of piliated Lactobacillus rhamnosus strains
2016
Yang, Z.Q. | Wei, Y.F. | Rao, S.Q. | Gao, L. | Yin, Y.Q. | Xue, F. | Fang, W.M. | Gu, R.X. | Jiao, X.A.
AIMS: Piliated Lactobacillus rhamnosus (pLR) strains have attracted much attention owing to their excellent mucus adhering capacity and immunomodulatory effects. Here, we aimed to develop a rapid, sensitive method for isolating pLR strains in complex ecosystems using immunomagnetic separation (IMS) with colony immunoblotting (CIB). METHODS AND RESULTS: Magnetic nanobeads (diameter: 180 nm) conjugated with anti‐pLR SpaA pilin antibodies (anti‐SpaA) were prepared and used to preconcentrate pLR strains in samples, followed by confirmation with anti‐SpaA‐based CIB analysis. Under optimized experimental conditions, IMS‐CIB selectively recovered pLR strains from 10⁷ CFU ml⁻¹ of faecal microbiota samples spiked with 2·9 × 10¹ to 2·4 × 10⁶ CFU ml⁻¹ of pLR strains. No positive colonies were detected in samples without addition of pLR strains. The detection limit of IMS‐CIB was 29 CFU pLR ml⁻¹ of faecal microbiota, which is much lower than that of CIB without IMS preconcentration (2·0 × 10⁴ CFU ml⁻¹). CONCLUSIONS: IMS‐CIB allowed selective preconcentration of pLR strains in highly heterogeneous bacterial suspensions and direct detection of pLR colonies, which remained readily available for subsequent isolation. SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings established an effective method for selective enrichment and detection of pLR strains.
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