[PCR]#12 Salmonellosis PCR

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18 Oct 2021
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Contents

1. Introduction 

2. Materials and equipment 

3. Procedure 

4. Results 

5. Precautions 

1. Introduction


1. Introduction

It is a polymerase chain reaction analysis method used to diagnose salmonellosis using a primer set capable of amplifying two major typhoid genes.

2. Materials and equipment


2. Materials and equipment 

PCR PreMix, Primer set, Salmonella DNA, Agarose gel, TAE or TBE buffer, loading dye, DNA ladder (100bp)

  • Sequence of Primer
Forward primer 1GCT TAA TGT CCA AGA TGC CTA CAC
Reverse primer 1GAG CAA CGC CAG TAC CAT CTG
Forward primer 2CAC GCA CCA TCA TTT CAC CG
Reverse primer 2ACT AAC GGC CTA AAC GGG AT


  • Equipment

Conventional PCR (ex.DuxCycler-Biomedux. Ltd., South Korea)

DNA Electrophoresis (ex.DuxGeldoc- Biomedux. Ltd., South Korea)

3. Procedure


3. Procedure 

1) Prepare a PCR mixture. The PCR method solution is prepared by mixing each reagent with the composition of the following table based on one sample. At this time, the amount of each reagent used is calculated according to the number of reactions of the sample including the control group (positive control group, negative control group)

Reagent addedAmount for each tube
PCR Premix10 uL
Primer mix4 uL
DW3 uL
Total17 uL


2) Mix the PCR mixture 5 times, vortex it, and centrifuge it lightly.

3) Pour the PCR mixture into the PCR tube 17 uL on each tube (close the lid immediately after pouring to prevent contamination)

4) Add template DNA 3 uL extracted from clinical samples to the tube.

(The positive control group DNA provided is added to the positive control group, and D.W. is added to the negative group)

Reagent addedAmount (for each tube)
PCR mixture17 uL
template DNA3 uL
Total20 uL


4) each mixed solution tube is centrifuged for 30 seconds and then put into a PCR machine to react as follows.

ProcessingTemperatureTime(min.)Cycles
UDG treatment37℃02:001
Pre-Denaturation95℃15:001
Denaturation95℃00:3040
Annealing58℃01:00
Extension72℃02:00
Final-Extension72℃05:001
End4℃


5) Check the PCR product 5 uL by electrophoresis in 1~2% agarose gel.

4. Results


4. Results

Lane
SMSize Marker
1Positive
2,3,4Negative

-Both bands are determined to be positive when confirmed at 587bp and 484bp. 

5. Precautions


5. Precautions 

1) Pay attention to pipetting so that DNA or PCR reagents are not contaminated during the testing.

2) Be careful not to make a mistake in temperature and or amount of reagent during the experiment is performed 


#Salmonellosis #DNA # UDG system #PCR







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