| Bifidobacterum species are important probiotics in gastrointestinal tract of human,and are closely related to human metabolism and health.As an important probiotic supplement,the market potential of Bifidobacterium species is very huge.However,the distribution of bifidobacteria in the intestine showed species diversity,and the genetic relationship among Bifidobacterium species is very close,the high-throughput detection of bifidobacteria in complicated samples can not be achieved by immunology,nucleic acid hybridization or PCR,even amplicon sequencing based on 16S rRNA is not effective in identifying bifidobacteria at species level.Therefore,the high-throughput detection of bifidobacteria in complicated samples at species and subspecies levels is of great significance to help us understand the relationship between Bifidobacterium species and human health,and to realize the targeted and precise intervention of Bifidobacterium species.Based on composition analysis of Bifidobacterium species,there is still lack of effective methods for the isolation of Bifidobacterium species with low abundance in complicated samples.In this study,a high-throughput and accurate method was established for the detection of bifidobacteria at species and subspecies levels,and an efficient and specific method for the isolation of bifidobacteria at species level is achieved.The main results of this study are as follows:(1)A new method was established for the detection of bifidobacteria at species and subspecies level using groEL gene on the basis of Miseq high-throughput sequencing.Based on the complete genome of Bifidobacterium species,the core genes shared with all Bifidobacterium species were obtained by CD-HIT.Further analysis of the evolutionary rate revealed that the core gene groEL had the potential to identify bifidobacteria at species and subspecies level.The phylogenetic tree of the groEL gene sequences of 120 Bifidobacterium strains were constructed by MEGA software,the results showed that the groEL gene could be used to analyze the evolutionary relationship of bifidobacteria at species and subspecies levels.Therefore,we designed a pair of Bifidobacterium-specific primers which target a hypervariable sequence region within the groEL gene,primers Bif-groEL-F(5-TCC GAT TAC GAY CGY GAG AAG CT-3)/Bif-groEL-R(5-CSG CYT CGG TSG TCA GGA ACA G-3).When the groEL gene sequences of all Bifidobacterium species in the samples was determined by the MiSeq sequencing,the groEL gene database and bioinformatics analysis were used to determine the structural composition of bifidobacteria at the species and subspecies level.The new method could simultaneously analyze the structural composition of Bifidobacterium species from more than 300 samples and achieve high-throughput detection of Bifidobacterium species.The method had a high accuracy and specificity in the detection of Bifidobacterium species.When the concentration of Bifidobacterium species was from 10~4 CFU to 10~7 CFU,there was a good linear relationship,y=0.9943x-2.048(R~2=0.978),and the limit of detection was 10~4 CFU.(2)Bifidobacterium species were analyzed using the new method established on the basis of the MiSeq high-throughput sequencing.To evaluate the efficacy of the method,commercial probiotics containing Bifidobacterium species were detected and structural composition of Bifidobacterium species in fecal samples were analyzed.The detection of 14 commercial probiotics showed that only half of them are composed of consistent Bifidobacterium species with the ones on their labels,and Bifidobacterium species are inconsistent in other 7 products,which were suspected of fraud using B.lactis instead of Bifidobacterum species on the lables.The above results indicate that the high-throughput detection method of Bifidobacterium species based on MiSeq sequencing could be applied to detect probiotics containing Bifidobacterium species,which is an effective supplement to GB4789.34-2012.Particularly,the predominant bifidobacteria of these products was B.animalis subsp.lactis,and the amounts of other Bifidobacterium species were relatively small and undetectable.The effectiveness of the new method was further proved in the detection of Bifidobacterium species from different fecal samples.The results showed that the method could be used to analyze the structural composition of Bifidobacterium species in human and rat fecal samples at species and subspecies level.Based on the specific detection of qPCR,the high-throughput detection method could accurately determin the percentage of Bifidobacterium species.These results suggested that the new detection method established in this study provided a high-throughput,low-cost analytical method for the structural composition of Bifidobacterium species in the intestine and solved the technical neck bottle for analyzing the relationship between human health and Bifidobacterium species using a large amount of samples.(3)The single-stranded DNA(ssDNA)aptamers binding to Bifidobacterium species with high avidity and selectivity were selected using an improved whole-bacterium-based systemic evolution of ligands by exponential enrichment(SELEX).In this study,a random oligonucleotide library was constructed,and B.breve and B.bifidum were used as the target during the whole-bacterium SELEX(Systematic Evolution of Ligands by Exponential Enrichment).Based on the results from flow cytometry,one aptamer,BB16p with high avidity and specificity,was selected for B.breve,and one aptamer CCFM641-5 with high avidity and selectivity was selected for B.bifidum.On the basis of the nucleic acid sequence analysis and secondary structure prediction,the aptamers BB16P and CCFM641-5 were optimized.The BB16p was optimized to BB16-11f with 29 bp.The results proved that after the optimization,BB16-11f has a higher affinity and more specify to B.breve and the K_d values between them was 18.90±2.27 nM.For B.bifidum,the primer binding sites of CCFM641-5 are very important for its binding affinity for B.bifidum,the aptamer can not be optimized,and the dissociation constant K_d between CCFM641-5 and B.bifidum was calculated to be 10.69±0.89nM.To search for the material basis of the binding between the Bifidobacterium species and its aptamer,trypsin and protease K were used to treat B.breve and B.bifidum.The results showed lower affinity between the Bifidobacterium species and its corresponding aptamer.Therefore,we assume the membrane protein might be essential for the binding.To verify the potential of the aptamer as a commercial product,we used ELAA(Enzyme Linked Aptamer Assay)to establish visualization method,a sandwich-type structure of aptamer/target/aptamer,against B.breve and B.bifidum.For B.breve,the linear relationship is y=0.0588x+0.3423(R~2=0.984),the detection range is from 10~3 cfu/mL to 10~7 cfu/m L,and the limit of detection is 10~3 cfu/mL.For B.bifidum,the linear relationship is y=0.07663x+0.1471(R~2=0.983),the detection range is 10~4 cfu/mL to 10~7 cfu/mL,and the limit of detection is 10~4 cfu/mL.(4)A novel method of efficient and specific isolation of Bifidobacterium species was established using targeting recognition of aptamers and directional separation of magnetic beads.A novel and effecient isolation method was established preliminarily based on the aptamers with high avidity and selectivity and the help of streptavidin paramagnetic particles.The results proved that the effecient method established can improve separation efficiency for B.breve or B.bifidum with quite an advance. |
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