| Cell fusion is one of the important methods of industrial microbial fermentation,compared with transgenic technology,cell fusion technology is safer and more efficient,and has been widely used in many fields,such as immunology,biology,genetics etc.Spore is strong resistance,with the features of heat resistant,acid and alkali resistance,drying resistance,uv resistance and resistance to organic solvents,and the "spores-lactic acid bacteria" endow lactic acid bacteria the characteristics of spore.As a kind of living bacteria preparations,it is better for the gastrointestinal colonization,and it can better play to its physiological function than Dead Bacteria.In this study,cell fusion was performed with Bacillus natto as the representative of Bacillus and Lactobacillus acidophilus as the representative of lactic acid bacteria.In order to get the aerobic,easier to cultivate and preparations standardized spore-forming lactic acid bacteria,the research established the rapid filtering method of lactic acid bacteria and bacillus fusant,and provided a rapid and easy high-throughput screening method for cell fusion breeding work of lactic acid bacteria which can generate spores.The research used spores and ?-galactosidase as an index for the phenotype identification of fusants,and filtrated 7 strains positive fusants as primers reference strains.In order to establish Bacillus natto-Lactobacillus acidophilus fusant multiple PCR identification method,primer was designed on the basis of the parent strain of specific gene sequences.With parent strain DNA as a template,PCR system for each pair primers were optimized under appropriate annealing temperature.The specific of each pair of primers were tested with non spore-forming strains.spore-forming strains and lactic acid bacteria as reference strains.Then 50 fusion strains was tested with single PCR identification method.Using positive fusant genomic DNA as template,using parent strain and negative fusant as reference strains,useful primer were filtrated,then multiple PCR reaction system were established.The results showed that,the primers pair P1/P2 designed based on early factor gene spoOA which generated by the spore of Bacillus,could specifically amplified spoOA gene fragment(308 BP),and all the 7 strains of lactic acid bacteria presented negative features.The primers pair P3/P4 designed by conserved sequence of 13 strains Lactic acid bacteria ?-galactosidase gene,could specifically amplified the target gene fragment(576 BP)of all tested Lactic acid bacteria and the positive fusant determined by phenotype identification,but the bacillus has no amplification.It shows that the primers designed had specificity.The single PCR identification of 50 strains were consistent with phenotype identification.The multiple PCR results showed that the co-amplification of 308 BP and 576 BP fragments can be achieved in the optimal system P3/P4.The identification results of 50 strains matched up with the test result of single PCR and of the phenotypic characterization,but only 308 BP fragment can be amplified in the P1/P2 optimal system.So P3/P4 optimal system was selected as multiple PCR reaction system.P3/P4 optimal system contained template DNA 1 ng/?L,each prime r0.4 ?mol/L,dNTPs 0.25 mmol/L,Taq enzyme 0.06 U/?L,and the amplification program were as follows 94? for 5 min for initial denaturation followed by 30 cycles of 94 ? for 30 s,53 ? for 30 s,72?for 60,and a final extension at 72 ? for 5 min followed by rapid cooling to 4 ?.The multiple PCR method is simple,rapid and has better specificity than phenotype identification and single PCR.The designed primer after specific detectioncan is suitable for Bacillus and lactobacillus,so it is suitable for fast identification of all Bacillus and lactic acid bacteria fusants. |
PDF Full Text Request