Detection Of Mutation In The Acid-resisitant Gene CcpA Of The Fluoride-resistant Strain Of Streptococcus Mutans

Caries is chronic progressive destructive lesions of dental hard tissues, which iscaused by a variety of factors,bacteria in the mouth mainly.It shows as the inorganicdeminearlization and the decomposition of organic matter, With the development ofthe course of the disease, there is a evolution of the lesions from color change tosubstantial. General average caries rate is at about50%,it is the most common diseasein oral cavity and one of the most common human diseases,.A variety of factors caused caries, including bacteria and plaque, food as well asthe environment of the teeth in etc. Cariogenic bacteria in plaque is the primary cariespathogen. Streptococcus mutans is the most important cariogenic bacteria,it is alsothe originating factor for dental caries.Streptococcus mutans is a gram-positive cocci,is an oral natural flora in an account for the largest proportion of Streptococcus spp.After repeated study, we confirmed that the Streptococcus mutans can cause rodentsand primates experimental caries animal model, and also there is evidence that thebacteria is closely related to human caries.Streptococcus mutans’strong cariogenicity depends primarily on the Acidproduction and acid resistance. Streptococcus mutans in plaque can rapidly ferment avariety of carbohydrates to produce large amount of acid, allows local pH to drop to5.5. Acid resistance of Streptococcus mutans is strong,it can still continue to live atpH4.5and produce acid. Streptococcus mutans is not only the crown caries pathogen,but also the root caries pathogen. With the application of molecular biologytechniques, a number of genes related to acid resistance of Streptococcus mutans haveisolated and cloned,including ccpA.Fluoride can be effectively prevent tooth decay,which has been recognizedworldwide.A survey shows that long-term use of fluoride toothpaste can make20%to40%decline in the prevalence of dental caries. However,long-term application offluoride will lead to that cariogenic bacteria has gene mutations,and then the fluorideresistant strains is produced,the study found that fluoride-resistant Streptococcus mutans strain significantly enhanced acid production and acid resistance more thanthe wild-type.However,the exact location of the gene mutation occurred requiresfurther study.In this experiment,according to the the gene sequence of the acid-resistantrelated genes ccpA of the streptococcus mutans in GenBank, a pair of primersdesigned from conserved regions,firstly induced fluoride-resistant strains ofStreptococcus mutans in vitro,and then extracted its genome with the V-gene genomicDNA miniprep reagents box,then PCR with the genome of fluoride-resistant strains asa template,we get the acid-resisitant gene fragments of the fluoride-resistant strain ofstreptococcus mutans,recovery the PCR production,T/A connection by using PMD18-T vector,then we get connection products,then transfer connection products intoE.coli competent state cells(E.coli JM109) to construct the recombinantplasmid,extracted plasmid and identified by PCR.Connect the finishing sequencingresults we can get the the full sequence of the acid-resisitant gene ccpA of thefluoride-resistant strain of streptococcus mutans UA159.Using BLAST tool homology comparisons of the measured sequence in theGenBank, the results is found, the sequences of the acid-resisitant gene ccpA of thefluoride strains of Streptococcus mutans are different from GenBank publishedsequences,there are five base mutations,the mutation points locate at1665(Tâ†'A),1704(Aâ†'G),1934(Gâ†'A),2259(Gâ†'A),2285(Tâ†'G)respectively.The study of the acid-resisitant gene ccpA of the fluoride-resistant strain ofstreptococcus mutans has great significance,not only to lay the foundation for theapplication of fluoride anticaries, but also to provide a theoretical and researchfoundation for anticaries vaccine in the future.