Application of Random Amplified Polymorphic DNA Molecular Marker Technique in Typing of Enterococcus
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Abstract:
The effect of random amplified polymorphic DNA (RAPD) molecular marker technique on rapid typing of Enterococcus species was investigated. An interspecific typing system of Enterococcus was constructed through screening the template, single primer, double primer and RAPD reaction system. The standard of strain similarity typing was established by typing 32 known strains of Enterococcus. Interspecific typing of 46 unknown strains of Enterococcus and testing of the house-keeping gene rpoA were performed to verify the effect of typing. The results showed that microwave-assisted extraction of the genomic DNA satisfied the experimental requirements, and seven single primers were selected for the paired combination of two primers. The optimal two-primer reaction system was: 2× PCR Taq Mix 12.5 μL; Template DNA and primer 1 μL; ddH2O 9.5 μL.The optimal two-primer pair was Primer 1 and Primer 5, which allowed the distinguishment of Enterococcus species with 80% similarity. The primer pair of Primer1 and Primer3 can amplify the characteristic DNA segment of Enterococcus hirae at 500 bp, which can be used as the identification index for the rapid identification of Enterococcus hirae.
