Suppression subtractive hybridisation and real-time PCR for strain-specific quantification of the probiotic Bifidobacterium animalis BAN in broiler feed

摘要

To ensure quality management during the production processes of probiotics and for efficacy testing in vivo, accurate tools are needed for the identification and quantification of probiotic strains. In this study, a strain-specific qPCR assay based on Suppression Subtractive Hybridisation (SSH) for identifying unique sequences, was developed to quantify the strain Bifidobacterium animalis BAN in broiler feed. Seventy potential BAN specific sequences were obtained after SSH of the BAN genome, with a pool of closely related strain genomes and subsequent differential screening by dot blot hybridisation. Primers were designed for 30 sequences which showed no match with any sequence database entry, using BLAST and FASTA. Primer specificity was assessed by qPCR using 45 non target strains and species in a stepwise approach. Primer T39_S2 was the only primer pair without any unspecific binding properties and it showed a PCR efficiency of 80% with a C-q value of 1732 for 20 ng BAN DNA. Optimised feed-matrix dependent calibration curve for the quantification of BAN was generated, ranging from 628 x 10(3) cfu g, to 1.61 x 10(6) cfu g(-1). Limit of detection of the qPCR assay was 2 x 10(1) cfu g(-1) BAN. Applicability of the strain-specific qPCR assay was confirmed in a spiking experiment which added BAN to the feed in two concentrations, 2 x 10(6) cfu g(-1), and 2 x 10(4) cfu g(-1). Results showed BAN mean recovery rates in feed of 1.44 x 10(6) +/- 4.39 x 10(6) cfu g(-1) and 1.59 x 10(4) +/- 1.69 x 10(4) cfu g(-1), respectively. The presented BAN specific qPCR assay can be applied in animal feeding trials, in order to control the correct inclusion rates of the probiotic to the feed, and it could further be adapted, to monitor the uptake of the probiotic into the gastrointestinal tract of broiler chickens. (C) 2016 Elsevier B.V. All rights reserved.