Root surfaces were aseptically cleaned

Preza’s (2) research assessed the bacteria that cause root caries in elderly patients by using molecular techniques independent of culture to determine the association of specific bacteria within healthy carious roots. Methodology. Bacterial 16s r RNA genes extracted from DNA were amplified using PCR machine. Cloning and sequencing ensued to know the species identity. Of the 3,544 clones, 245 were prominent. This represented 8 microbial phyla. A large number of species detected are yet to be cultivated. Two men and nineteen female patients were involved in the research. Their mean age was 89 years. Participants were first clinically examined a couple of days before sampling. They were then divided into control group consisting of ten people and RC group having 11 participants. The control groups did not have RC while the RC group had RC lesion during the process of examination. Lesions on exposed root surfaces were treated as carious when they felt leathery. The candidates had to clean their teeth the day before or in the morning before sampling is done. From the non-RC group, plaques from a healthy root, carious root and dentin from the same carious root were used. There was a random sampling of healthy root sites. Plaque samples were picked using relevant apparatus. Root surfaces were aseptically cleaned. With a spoon excavator the layer of infected dentin was taken and the inner side of RC lesion sampled. The samples were put in 300 microliter TE buffer and dipped into frozen ice. This is stored at -80 degrees Celsius. Bacterial DNA was then extracted using QIAamp DNA mini kit. Extracts were then stored at -20 degrees Celsius. The genes were then amplified using universal forward primer. Polymerase chain reaction was then conducted in thin-walled tubes in PCR machine. Two microliters of DNA template were added to the reaction mixture containing each of the primer, deoxynucleoside triphosphates, magnesium ions and platinum taq polymerase. The samples were preheated at 95 degrees Celsius for 4 minutes, followed by 30 cycles of amplification. The solution was denatured at 95 degrees Celsius for 45 seconds, annealed at 60 degrees Celsius for 45 seconds and then elongated at 72 degrees Celsius for 60 seconds with additional 15 seconds for each cycle. In chain elongation step is done at 72 degrees Celsius for 15 minutes. The results were then observed using gel electrophoresis. Cloning was then done using TOPO TA cloning kit. E. coli TOPO10 cells were used to do the transformation. Luria- Bertani agar plates were used to plate the transformed cells. The plates were incubated at 37 degrees Celsius in the incubator. Amplification was done with m-13 primers. Bacterial profiles of the 21 candidates were investigated. A total of 8 bacterial phyla were observed 46 percent of which were cultivable species in a known genus. Fifty-four pr cent sequences have not yet been identified. The Control group had lower number of bacterial incidence on root surfaces. At relatively high clone levels in few healthy subjects were Veillonella, Selenomonas, and S. Gondi. RC group had no bacteria associated with poor health conditions like Kingella oralis, Fusobacterium nucleatum subsp. Polymorphum, Leptotricha spp., Selenomonas noxia. The microflora overlying the root surface in RC subjects had lower diversity than the control sample. On such surfaces, bacteria species like Campylobacter gracilis, Selenomonas spp. The study offers description of microbes associated with rood surfaces in the elderly based on culture-independent method. Of the 21 people who participated in the study, different bacteria profiles were observed. Diversity was less evident when bacteria moved from healthy to diseased individuals. RC group exhibited lowered bacterial diversity on healthy root surfaces. Some species of bacteria were considered important to the health of the candidates because they were never detected. The prevalent bacterial species were Fusobacterium nucleatum subsp. Polymorphum, S. sputigena, Selenomonas and Propionibacterium sp. Strain FMA5. The bacteria associated with RC were more complex than initially imagined. These bacterial species that were detected included S. mutans, lactobacilli and Actinomycetes.

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