Study On Identification Of Bifidobacteria By Random Amplied Polymorphic DNA Method

Random Amplified Polymorphic DNA (RAPD) technology was used to identify bifidobacteria. Two simple,rapid methods were developed to extract template DNA from bifidobacteria.. A random primer S256 was screened out and used to identify a long shelf-life bifidobacterium strain. In addition, many factors which may affect RAPD patterns were studied. The result are as follows: 1. Two DNA extraction methods --- lysozyme-SDS-KAC method and lysozyme-boiling method were used. Compared with traditional lysozyme-SDS- phenol/chloroform method,these two methods were rapid,simple and needed no poisonous reagent. There were good results and high reproducibility when DNA extracted by these two methods was used as template in RAPD. 2. Using two steps of optimization---orthogonal array test and crossing- combining test , the optimum condition of RAPD is as follows: 5Ong DNA, 3mM Mg2~ , 1 5pmol random primer,200 ~i M dNTP, 1 U Taq ploymerase,predenaturing at 940C for lOmin,44 amplification cycles of 940C for 30 sec,360C for 60 sec and 720C for l2Osec,at last ,a final extension at 720C for 7mm is neede4. 3. A random primer S256 was screened out which sequence is CTGCGCTGGA. When it was used in RAPD, there is good discrimination of 47 RAPD patterns at the species and strains level. 4. A long shelf-life bifidobacterium strain was identified as JCM1 192 B.breve strain by traditional PCR at genus level,reaction of physio-biochemistry at species level and RAPD at strains level. 5. Many factors which would affect the RAPD patterns were studied. The result shows: the use of different kind of thermocycler may affect the result remarkably. There may be some difference in RAPD patterns which were produced by different kind of Taq polymerase. Although DNA template extracted by various methods will lead to the difference in RAPD patterns, the reproducibility of amplification is good when only a kind of DNA was used. There seem to be little effect on the result when annealling temperature varies from 300C to 4O~C .Good reproducibility will be obtained in repeat analysis, different operator test and PCR tube of different volume test when other conditions are the same.