관리자
7 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 polymerase chain reaction used to detect tuberculosis bacteria.

Using a universal two-part Primer set of Mycobacterium tuberculosis (MTB) total genome, polymerase chain reaction(PCR) is generated and specific parts are amplified to determine whether tuberculosis is amplified.

2. Materials and equipment


2. Materials and equipment 

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

  • Sequence of Primer
Forward primer 1CCT GCG AGC GTA GGC GTC GG
Reverse primer 1CTC GTC CAG CGC CGC TTC GG
Forward primer 2TCC GCT GCC AGT CGT CTT CC
Reverse primer 2GTC CTC GCG AGT CTA GGC CA


  • Equipment

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

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

3. Procedure


3. Procedure 

1) The composition of the reaction tube for PCR is as follows. (for one sample)

Reagent addedAmount of each tube
Template DNA3 uL
PCR PreMix10 uL
primer set2 uL
D.W.5 uL
Total20 uL

2) when testing the above premix, primer sets, D.W., mix the number of samples by one plus tube and put 17uL into the each PCR tube

3) The template DNA 3uL extracted from the clinical sample is added into the above tube.

(Positive DNA and negative DNA were added to the positive and negative control tubes, respectively.)

4) after spin down the test tubes containing each mixture for 30 seconds, put them in PCR instrument and react as follows


ProcessingTemperatureTime (min.)Cycles
Pre-Denaturation94℃05:001
Denaturation94℃00:3040
Annealing / Extension65℃01:00
Final-Extension72℃05:001
End4℃

4. Results


4. Results

NegativePositivePositivePositive

5. Precautions


5. Precautions 

1) Pay attention to pipetting so that DNA or PCR reagents do not contaminated.

2) Be careful not to make mistake in temperature and /or reagent amount during the experimental method.


#Tuberculosis #MTB #DNA # Universal primer #PCR




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