Title:   Two Methods of Detection for Streptococcus mutans
Students:   Judy Ball and Leesa Gabrielsen 
Team:   Team 1
Faculty Mentor:   Scott Wright
Date:  October 4, 2004

Abstract:
Streptococcus mutans has been shown to be a major causative agent for dental caries.  Two rapid methods of detection have been developed to identify S. mutans: one involving polymerase chain reaction (PCR) and another using Dentocult SM Strip Mutans.  The first phase of the project will be to optimize and validate the PCR procedure to work in the Clinical Laboratory Science microbiology laboratory.  For the PCR procedure, four primers specific to S. mutans will be used to identify the presence of the bacterium.  The second phase will be to validate the Dentocult SM Strip Mutans.  This requires a 48 hour incubation for detection of S. mutans.   Phase three will be to compare the two methods for specificity and sensitivity. 

Introduction:
Streptococcus mutans is a Gram positive organism that is a member of the Streptococcus viridans group.  This bacterium is a predominant organism of oral flora in most individuals.  Because of its ability to convert sugars and carbohydrates into lactic acid, it is a major cause of tooth decay and dental caries.  The presence, or even absence, can be a strong predictor of high or low susceptibility to dental caries (Orion).   Since the human mouth contains a large variety of oral flora, standard nutrient cultures tend to be very time consuming and non-specific for the detection of S. mutans.  Other tests have been developed for rapid and specific identification of the bacterium.  In this study, we will compare two methods, PCR and Dentocult SM Strip Mutans.
       
Polymerase chain reaction has greatly expanded the abilities of molecular biology by providing a quick, easy, and specific method for generating copies of any DNA fragment.  It involves a repeating three-step process using primers to amplify a specific DNA sequence (Mahon 199).  The presence of S. mutans in saliva and plaque samples can be detected using two sets of primers specific for the organism (Oho 259).
       
The Dentocult SM Strip mutans have been designed so that the dentist can perform the sample collection in the dental office.  Both saliva and plaque are inoculated onto two plastic strips that are treated to simulate a tooth surface.  The strips are then incubated for 48 hours and examined for S. mutans.

Objective:
The first phase of the project will be to validate the PCR procedure for S. mutans.  Using published procedures, the sample collection, DNA extraction, and PCR conditions will be optimized and validated.  The second phase of the project will be to validate the Dentocult SM Strip Mutans.  Phase three will be to statistically compare the two methods by calculating the specificity and sensitivity.

Methods:
Phase 1 : PCR Optimization and Validation
Optimization and validation of the PCR project requires the use of four S. mutans specific primers (GTFB-F, GTFB-R, GTFB-FIN, GTFB-RIN) which will be purchased from Idaho Technology in Salt Lake City, Utah (Oho 259).  The S. mutans strain used for the control will be, ____.  This control strain will be used as the DNA template for the PCR procedure and will be taken from a pure culture on an MSB plate (mitis-salivarius agar with bacitracin and sucrose).
PCR Amplification
The protocol for the first part of the PCR procedure uses two primers, GTFB-F and GTFB-R.  These two primers will amplify the 517-bp DNA fragment of the gtfB sequences of S. mutans.  Each sample will consist of 10mM Tris-HCl buffer (pH 8.3), 1.5 mM MgCl2, 50 mM KCl, 200uM each of dATP, dTTP, dGTP, and dCTP, 1uM oligonucleotide primers (GTFB-F and GTFB-R), 1 U Taq DNA polymerase, and one colony of S. mutans from an MSB plate.  The PCR conditions are: denaturation at 95°C for 30 seconds, followed by annealing at 59ºC for 30 seconds, and extension at 72ºC for one minute.  This amplification will be repeated for 30 cycles (Oho 259).

The second part of the PCR protocol includes two different primers, GTFB-FIN and GTFB-RIN.  These two primers will target the internal sequence of the GTFB primer products from the first PCR.  0.1ul of the GTFB primer product will be used as the template for the second PCR using the same conditions from the first PCR.  The amplified products will be electrophoresed on 1.8% agarose gel and stained with ethidium bromide, and then photographed (Oho 259).

Phase 2: Dentocult SM Strips Mutans Validation
The same strain of S. mutans used in the PCR validation will be used for the Dentocult SM Strip Mutans validation.  A colony of a pure culture from an MSB plate will be used to inoculate the strips.
Procedure
A bacitracin disc must be placed in the selective culture broth vial 15 minutes prior to the sample collection.  After shaking the vial to evenly distribute the bacitracin, the strips can be placed in the vial with the smooth surfaces clipped and attached to the cap.  The vial will then be placed in an air incubator at 35º-37° with the cap a quarter of a turn open.  After 48 hours, the strips can be examined for the presence of S. mutans.  Any dark-blue to light-blue colonies on the rough surface of the strip indicate the presence of the bacterium (Orion). 

Phase 3: Comparison
Collection of Saliva/Plaque
50 college aged students (19-25 yrs) will be asked to chew paraffin for one minute, which will stimulate the secretion of saliva and transfer S. mutans from tooth surfaces into the saliva (Orion).  They will then be asked to spit at least one milliliter of saliva into a 7 ml test tube.  A saliva sample will also be collected by placing the rough surface of the Dentocult SM Strip Mutans on the tongue.  Participants will then need to scrape 4-5 teeth with a tooth pick two separate times.  One plaque sample will go into the tubes with saliva for the PCR procedure, and the other plaque sample will be added to the rough side of the Dentocult SM Strip Mutans.

PCR
DNA Extraction
In order to extract the DNA, one milliliter of saliva will be centrifuged at 12,000 x g for 15 minutes, then 200ul of lysis solution (10mM Tris-HCl buffer, 1mM EDTA, and 1% Triton X-100, pH 8.0) will be added to the precipitate, vortexed, and placed in a 95° water bath for 10 minutes.  The sample will then be centrifuged at 12,000 x g for 5 minutes.  The supernatant will be decanted and saved for DNA amplification (Oho 259).

The exact protocol used for the validation of the PCR amplification will be followed to detect S. mutans in the saliva/plaque sample.  However; instead of using one colony of the pure S. mutans culture, 2ul of the supernatant from the saliva DNA extraction will be used (Oho 259).

Dentocult SM Strips Mutans
The same procedure used in the Dentocult SM Strip Mutans validation will be used for this part of the project.  However; instead of using the control strain of S. mutans on the strips, the participant samples will be used.

Comparison
Data collected will be statistically calculated to compare the specificity and sensitivity of the two methods using the Predictive Value Theory.

Materials:
PCR
Primers: GTFB-F, GTFB-R, GTFB-FIN, GTFB-RIN
1 bottle of agarose
Bacterial stock of S. mutans
Taq polymerase
Pipette tips
Glass tubes:  $41.95 / 1,000 tubes
 
Dentocult SM
7 boxes with 10 kits each: 5 boxes for participants, 2 boxes for validation 
 $45.95 / box (includes shipping and handling)  Total = $321.65

References:
Mahon, Connie R. and George Manuselis.  Textbook of Diagnostic Microbiology.  Philadelphia: W.B. Saunders Company, 2000.
Orion Diagnostica.  “Dentocult SM Strip mutans.”  Package Insert.
T. Oho, et al.  “Simple and rapid detection of Streptococcus mutans and Streptococcus sobrinus in human saliva by polymerase chain reaction.”  Oral Microbiology and Immunology 15 (2000): 258-262. <http://library.weber.edu>  20 Sept. 2004. \

Reviewers:
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Name: _____________________________  Date: ____________