[Real Time PCR]#17 CT(Chlamydia trachomatis) amplification test

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24 Nov 2021
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Contents

1.Introduction

2.Materials and equipment

3. Procedure

4. Results

5. Precautions

1. Introduction


1. Introduction

An experiment to see whether the amplification results are affected by the storage period or not.

2. Materials and equipment


2. Materials and equipment 

  • Reagents

TaqMan Gene expression master mix, Primer set (Forward, Reverse, *Probe), Chlamydia trachomatis DNA, Nuclease free water (Qiagen)

  • Sequence of Primer, Probe

※ The primer and probe sequence are the same as the #3 NAAT for CT

  • Equipment

Real-time PCR (LightCycler 480 – Roche Ltd. Switzerland)

3. Procedure


3. Procedure 

1) Prepare Chlamydia trachomatis DNA(10^5 copy)by diluting 10 to 100 times.

2) A sample prepared according to the following PCR sample reagent  composition is injected into the 8-strip tube.

ReagentsSamplesNegative control
Master mix15 μL15 μL
Primer set7.5 μL7.5 μL
CT DNA4.5 μL (by conc.)-
Nuclease Free water3 μL7.5 μL
Total30 μL30 μL



3) Set the device as below according to as PCR protocol

StepsTemperatureTimeCycles
Initial denaturation50℃02:001
Denaturation95℃10:001
Primers annealing95℃00:1540
Elongation60℃01:00


4) Check the results after the PCR is over.

4. Results


4. Results

- Results Image

<Roche - LightCycler 480 Results>

1) What is marked as new in the sample is a reagent that was subdivided immediately after warehousing and diluted.  

2) what is marked as old in the sample is a reagent that has been subdivided after warehousing and stored in the freezer at least more than one month or diluted conditions for a long time.


- interpretation of results

1. Even at the same concentration, it can be seen that the CT value of the OLD sample rises late compared to the NEW sample. 

2. When the concentration is low, it can be seen that the CT value rises later.

3. It is considered very important to minimize the freezing and thawing processes by subdividing as aliquot from high concentration of the reagent (for minimizing contamination and deterioration).

5. Precautions


5. Precautions 

1)  Be careful, do not expose too much to the light.

2) Whenever diluting DNA, be careful to pipette the exact amount.

3) Be careful not to create bubbles in mixed samples

4) Be attention, do not get contaminated every steps during test.




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