Z Q Yang1,2,3, Y F Wei4, S Q Rao4, L Gao4, Y Q Yin4, F Xue5, W M Fang4, R X Gu6, X A Jiao7. 1. College of Food Science and Engineering, Yangzhou University, Yangzhou, Jiangsu, China. yangzq@yzu.edu.cn. 2. Jiangsu Key Laboratory of Dairy Biotechnology and Safety Control, Yangzhou University, Yangzhou, Jiangsu, China. yangzq@yzu.edu.cn. 3. Jiangsu Key Laboratory of Zoonosis, Yangzhou, Jiangsu, China. yangzq@yzu.edu.cn. 4. College of Food Science and Engineering, Yangzhou University, Yangzhou, Jiangsu, China. 5. Plant, Animal and Food Inspection Center, Jiangsu Entry-Exit Inspection and Quarantine Bureau, Nanjing, China. xuef1@jsciq.gov.cn. 6. Jiangsu Key Laboratory of Dairy Biotechnology and Safety Control, Yangzhou University, Yangzhou, Jiangsu, China. 7. Jiangsu Key Laboratory of Zoonosis, Yangzhou, Jiangsu, China.
Abstract
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.
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.