[PCR]#9 Dengue virus PCR

관리자
10 Sep 2021
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Contents

1.Introduction 

2.Materials and equipment 

3. Procedure 

4. Results 

5. Precautions 

1. Introduction


1. Introduction

Dengue fever is a infectious disease caused by mosquito with dengue virus that bite a person, and is an acute febrile diseases accompanied fever.

Due to a sudden high fever, this fever lasts for 3 to 5 days and can lead to heart burn sense, muscle pain, joint pain, and loss of appetite. Sometimes red spots can appear thought out the body in the early stages. As the fever drops, skin rashes continue for 1 to 5 days all over the body, and initially millet-shaped rashes appear temporarily on the neck, and chest, and then spread to the arms, legs, and face starting on the 3rd to 4th days. Minor bleeding, such as nose bleeding or gum bleeding, can occur during the course of the disease. Adults may experience bloody stool, menstrual results, symptoms of lymph node edema surround the neck area.


This experiment is reverse transcription polymerization appeal chain reaction analysis method used to detect Dengue viruses. Four types of Dengue viruses can be identified by extracting RNA from dengue–suspecting serum, synthesizing the nuclear acid of Dengue virus through reverse transcription, and amplifying the polymerase chain reaction using one forward primer and four reverse primer pairs.

2. Materials and equipment


2. Materials and equipment 

Materials

2X buffer, Enzyme Mix, Primer Mix, Dengue RNA (Dengue fever virus RNA), Agarose gel, TAE or TBE buffer, loading dye, DNA ladder (100bp)

  • Sequence of Primer
Forward primerTCA ATA TGC TGA AAC GCG CGA GAA ACC G
Reverse primer (Type 1)CGC TCC ATT CTT CTT GAA TGA GC
Reverse primer (Type 2)CGC CAC AAG GGC CAT GAA CAG
Reverse primer (Type 3)TAA CAT CAT CAT GAG ACA GAG C
Reverse primer (Type 4)TGT TGT CTT AAA CAA GAG AGG TC


Equipment

Conventional PCR (ex.DuxCycler-바이오메듀스)

DNA Electrophoresis (ex.DuxGeldoc- 바이오메듀스,한국)

3. Procedure


3. Procedure 

1) The composition of reaction tube for RT-PCR is as follows (for one sample):

Reagent added
tube
Template RNA4uL
2X buffer10uL
enzyme mix1uL
primer mix5uL
Total20uL

2) Except for template RNA, mix the above 2X buffer, enzyme mix, and primer mix by the number +1 to be tested and put 16uL into the PCR tubes

3) Add the extracted template RNA 4uL to the tube.

4) After spin down in each mixed solution tube for 30 seconds, put it in a PCR device and react as follows.

StepsTemperatureTime (min.)Cycles
cDNA Synthesis50℃30:001
Pre-Denaturation95℃15:001
Denaturation95℃00:3040
Annealing60℃00:30
Extension72℃01:00
Final-Extension72℃05:001
End4℃

4. Results


4. Results

GenotypesPCR amplification
product size
Type 1190bp
Type 2
119bp
Type 3
290bp
Type 4
389bp

5. Precautions


5. Precautions 

1) Pay attention to pipetting so that DNA and/or PCR reagents do not become contaminated during the experiment.

2) The experiment should be conducted carefully so that the temperature or reagent amount is not different from the testing method.


#Dengue virus #Gangue fever #RNA # reverse-transcription PCR





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