The overall goal of the following experiment is to identify specific salmonella antigen alleles. This is achieved by targeting PCR to identify specific sequence differences associated with these alleles. Sample template is added to A PCR reaction mix containing allele specific primers and the OH one and H two allele typing.
PCRs are run in the thermocycler. Next PCR samples are separated by size using gel electrophoresis to determine the size of the amplicon if present for each sample or isolate it. Amplicon size is then correlated with the expected size for each of the primer sets used in the PCR results are obtained that show the identification of specific salmonella antigen.
Alleles by PCR SEROVAR designation is then given based on results for OH one and H two multiplex PCR assays. The main advantage of the protocol described in this video over SYP is that it's quick, easier to interpret and less expensive. This method can help answer some key questions in the field of epidemiology, such as determining the distribution and persistence of salmonella VARs within or between poultry or dairy farms.
The implication of this technique extends towards the rapid diagnosis of salmon osis attributed to salmonella, serovars, arius, or urian. Visual demonstration of this method is critical to understanding how to use this PCR based method to type salmonella to the serovar level. Demonstrating this procedure will be Adriana Pedroso and Yin Chang research scientists in the Lee Mauer Laboratories at the University of Georgia To prepare whole cell DNA template for the subsequent PCR reaction.
First inoculate one milliliter of LB broth with salmonella. Isolate from a single colony. Incubate the culture with shaking at 200 to 250 rotations per minute and an incubation temperature of 37 degrees Celsius.
The next day, transfer the one milliliter culture to a 1.5 milliliter micro centrifuge tube and centrifuge the suspension for around two minutes at 4, 500 times gravity to pellet the cells next aspirate the sup natant, and then resuspend the bacterial pellet in one milliliter of 100%Ethanol incubate the ethanol suspended cells at room temperature for 10 minutes and then centrifuge to the cells as before. Resuspend the pellet in one milliliter of PBS After centrifugation aspirate the supinate and resus suspend the pellet in one milliliter of sterile distilled water. Finally dilute the cell suspension.
One in 20 with distilled water and store at minus 20 degrees Celsius for up to one month until ready to perform the PCR preparation and dispensing of the PCR reaction mix needs to be performed in a physically separate room and preferably done in a dedicated PCR workstation equipped with a UV light. First, decontaminate the work surface with UV light and by wiping with 10%bleach. Next, using a pipette designated solely for PCR work and fitted with a barrier pipette tip.
Make a master primer stock by first diluting the primers to five times 10 to the minus four molar with distilled water. Then dilute the stock one in 20 with distilled water to attain the working concentration of 25 micromolar. Retrieve the PCR reagents and whole cell template from the freezer and thaw on ice.
Leave the tac DNA polymerase in the freezer until required. Combine the PCR reagents according to table one of the written protocol. Next, pipette nine microliters of the PCR reaction.
Mix into the wells of a sterile 96 round bottom well polystyrene microtiter plate. Start at the top of the first column and move down the column and then to the top of the next column, add one microliter of distilled water to the first well to serve as a negative control. Then dispense 0.5 microliters of the positive control templates to the PCR mix in the second well and one microliter of e coli K 12 DH five alpha to the third.
Well add one microliter of the third sample template to the remaining wells for the H one I and gyp PCR Salmonella entera, CVaR, TYM and Enteritis will serve as positive controls for the eye and GM alleles respectively. Salmonella CVaR, heidelberg and haar are the positive controls that will be used in the second H one allele typing PCR for detecting the r and z 10 alleles respectively. Next place 10 microliter capacity glass capillaries into designated holders for the Idaho technology.
Rapid cycler once secured in the holder, load each glass capillary with the PCR reaction mix by touching the capillaries to the bottom of the wells. Tilt the holder to allow liquid to move down the tube and provide space between the ends and the PCR mix. Before heat sealing the glass tubes with a butane torch to seal both ends of the capillaries, place the capillary tubes and holder into the Idaho technology rapid cycler and use the thermocycler programs described in table one of the written protocol for the different all ELO type in PCRs.
Once the thermocycler program is complete, proceed to the gel electropheresis step for a 15 by 10 centimeter. 1.5%Molecular biology grade arose gel containing a th dium bromide according to standard procedures. Once the gel has solidified, transfer the gel to a submersible horizontal electrophoresis chamber containing one times TAE electrophoresis buffer premixed 200 microliters of 0.25 milligrams per milliliter.
DNA molecular weight markers with 40 microliters of six times DNA loading dye load, 4.8 microliters of the premixed DNA molecular weight marker to the first middle and last wells. Next, remove a glass capillary containing a PCR sample reaction from the holder and etch both ends of the capillary with a glass cutter. Then place the thumb and forefinger of both hands on either side of the capillary marked by the glass cutter and snap the capillary place the capillary dispenser on the end opposite of the PCR reaction mix and slowly turn the plunger clockwise until the liquid is at the very bottom of the tube.
Place the capillary into the well to be loaded and slowly turn the plunger clockwise to dispense the fol weighted sample into the arose. Well be careful not to puncture the well with the capillary or introduce an air bubble into the well by continuing to turn the plunger after the last of the liquid has left the capillary. Once all the samples and standards are loaded, set the constant voltage of the power supply to 90 volts.
Upon completion of electrophoresis, transfer the gel to a UV trans illuminator to visualize DNA fragments and molecular weight standards capture images on a gel dock XR system. Finally, place the capillary holders in 10%bleach for 15 to 30 minutes. Rinse three times with distilled water and place back in the PCR setup room to air dry.
This first atherium bromide stain gel shows the results of a multiplex PCR for the O allele associated with salmonella entera var, enteritis, hadda Heidelberg.Antifa. Lanes one and 13 contain a 100 base pair. DNA ladder.
Lane two contains the multiplex PCR controls for O alleles B, C one, C two, D one, and E.The remaining lanes three to 12 contain salmonella isolates. An allele designation is given to salmonella isolate based on the amplicon matching the size of the PCR control and as predicted for the allele prima sets used from left to right. These amplicon sizes are as follows, D 1 620 base pairs C two 400 base pairs E 1 280 base pairs, C 1 340 base pairs and B 560 base pairs.
This iridium bromide stain gel shows the results of an H one allele multiplex PCR for identifying the FLA gel in alleles eye and gm. The gel was loaded in the same order as previously shown. Again, an H one allele is assigned to each isolate dependent on the presence of an amplicon matching in size to the PCR control and as predicted for the four H one allele primer sets used from left to right.
The amplicon sizes are GM 310 base pairs and I 510 base pairs. This spinal atherium bromide stain gel shows the results of an H one allele multiplex PCR for identifying the gellin alleles r and z 10. Again, the gel was loaded in the same way as previously shown, and an H one allele is assigned to each isolate dependent on the presence of an amplicon matching in size to the PCR control and as predicted for the four H one allele primer sets used, the amplicon sizes are R 170 base pairs and Z ten three hundred and sixty base pairs Once mastered.
This technique can be done in four hours if performed properly. While attempting this procedure, remember to physically separate template preparation PCR setup and gel electrophoresis from one another so as to minimize the potential for PCR carryover contamination.
