Rapid Identification Of Bifidobacterium-Design And Validation Of Genus-Specific And Species-Specific Primers

Human intestinal flora and its metabolites have beneficial effects in the prevention and intervention treatment of irritable bowel syndrome,diabetes,obesity and other chronic metabolic and immune system diseases.It can even affect nervous systems diseses.Those researches further stimulate the prosperity of related probiotic product market.Bifidobacterium,as the most popular probiotics,has emerged in a variety of foods,health care products and even drugs.But so far there is no ideal detection and identification methods.Our current technology can not keep up with the actual work demand of the increasingly popular market demand for probiotics and diversified products.The relevant technology and inspection standards need to be improved immediately.Therefore,the focus of this study is to develop rapid and accurate identification and differencial methods for Bifidobacterium in genius and species levels.We take six standard strains(B.bifidum GDMCC 1.1256,B.longum subsp.infantis GDMCC 1.1258,B.longum GDMCC 1.248,B.adolescentis GDMCC 1.278,B.animalis GDMCC 1.169,and B.breve GDMCC 1.206)from Guangdong Provincial Microbe Culture Preservation Center as test materials.More than 10 pairs of specific primers(P1-P14)were designed using software Primer Premier5 to target fragments of house genes(tuf、gro EL、atp D),and the designed primers were screened specifically.The 14 pairs of specific primers were used to extract DNA from 6 standard strains of tested strains and a large number of standard strains and isolates of lactic acid bacteria and enterobacter isolated or preserved in laboratory.DNA obtained by PCR methods to verify the species specificity of these primers.The research results are as follows:(1)primer p7,targeting house-keeping gene-gro EL,can product a significant single amplification band in all six species standard strains of Bifidobacterium,while no band in other probiotic bacteria such as Lactobacillus,and enterbacteria in similar niche.So it’s suitable for quickly identify Bifidobacterium in genius level;(2)primer p1,targeting tuf gene,only produced a 586 bp specific amplification band in B.animalis GDMCC 1.169,while no band in the other 5 Bifidobacterium species standard strains.So it shows specificity for identifying B.animalis in species level.While p2,p4,p5 and P6 produce the specific amplification of B.adolescentis,B.longum,B.bifidum and B.breve,individually.In this study,rapid and accurate PCR methods were established for the identification of Bifidobacteria and 6 species in the Bifidobacteria from the List of Bacteria Available for Food issued in China in 2010.The results showed that this method could improve the identification efficiency of Bifidobacteria,and provide an efficient and low-cost analysis method.