Development of Dengue Multiplex RTPCR

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Development of Dengue Multiplex RT-PCR

• By Yong Yean Kong

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Outline.

• Dengue disease
• Theory of PCR
• How bioinformatics help in development of a PCR
  assay.

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Dengue disease

• Dengue or the African word denga meaning bone
  breaking is an acute infectious disease
  transmitted by the female Aedes aegypti mosquito.
• There are four known serotypes DEN-1, DEN-2,
  DEN3 and DEN-4.
• This disease is endemic in over 100 countries
  throughout the Americas, South-East Asia, the
  Western Pacific Islands, Africa and Eastern
  Mediterranean.
• The typical clinical symptoms are high fever,
  severe headache, pain behind the eyes, muscles
  and joint pain and rash. The scary fact here is
  infection of one serotype of dengue virus does
  not protect a person against secondary attack by
  another serotype.

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2. Theory of PCR

• Invented by Kary Mullis
• PCR polymerase chain reaction
• PCR in-vitro synthesis of DNA
• Building block of PCR
• dNTPs (A, G, C, T)
• MgCl2
• Taq DNA polymerase (PCR buffer)
• Primers (forward and reverse)

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PCR Amplification
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PCR vs. RT-PCR
PCR utilized DNA as initial
template
RT-PCR utilized RNA as initial
template
ssRNA
Reverse Transcription
Taq DNA polymerase
dsDNA
dsDNA
Taq DNA polymerase
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3. Development of a PCR assay
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Primer Design

• Bioinformatics
• NCBI genomics database
• Clustal X multiple alignment
• FastPCR primer designing software

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Multiple Alignment
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FastPCR
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Gel Electrophoresis
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Thank you