Optimization of real time PCR for checking the activity of siRNA in Dengue Serotypes

Order of Publishing in Issue: 
2
Volume :9
Issue :4
November, 2015
Page No: 
317-327
Authors: 
Anil Babu Korrapati[1& 2*], Mervin Noronha[3] and Muvva Vijayalakshmi[2]
Address: 
[1]Recombinant Gene Products Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India.
Address: 
[2]Department of Botany and Microbiology, Acharya Nagarjuna University, Nagarjuna Nagar - 522510, Guntur, Andhra Pradesh, India.
Address: 
[3]Vaarnika Bio Sciences private limited, Hyderabad, Telangana, India.

Dengue, the most prevalent infectious disease among the arbovirus community is emerging as a first order health complication, especially, in the tropics and in subtropical regions. It is untreatable, since, there is no advanced diagnostic method with which tendency of the infection can be traced with great precision in its acute phase. In our previous studies we showed that, siRNA targeting the 5NTR among all the 4 serotypes of dengue successfully inhibited the replication of respective serotypes to the greatest extent as confirmed by the realtime RT-PCR. In the present study, we monitored the relative expression of dengue genomic RNA in treated (rAdsh-5b) and non treated cells and control (rAdsh-N) to reveal the efficiency of the real-time RT-PCR in detecting the viral load in the presence of antiviral agent (rAdsh-5b) to discriminate the disease severity in the presence and absence of antiviral therapeutics. The cDNA for all the samples were prepared and performed real-time RT-PCR with the aid of SYBR green 1 and the post PCR amplicons were analyzed with melting curve analysis to check the specificity and efficiency of the process. The results showed that, the relevant viral RNA expression was found to be less in rAdsh-5b treated sample than the non treated sample as marked by the Ct-values and the melting curve peaks. This indicates that real-time RT-PCR can serve as a gold standard for diagnosing the dengue infection in the acute phase and in distinguishing the relative viral content in the sample treated with antiviral agent.

Keywords: 
Dengue, siRNA, real-time RT-PCR, rAdsh-5b, rAdsh-N, Ct-values, cDNA.
AttachmentSize
Paper-2.pdf1.42 MB

 
To download full text articles and issues in PDF at no cost.All you have to do is fill out a brief registration form. It's easy and it's free. At any time, you may request removal of your contact information from our database.
Click here to Register | Click here if you have already registered

2018 ABAP Awards

Invitation for Nominations to 2019 ABAP Awards
The Association of Biotechnology and Pharmacy invites nominations for the following awards from the eligible talented people with proven track record and achievements on or before August 31, 2019 in the prescribed format.

DOWNLOAD CIRCULAR | APPLICATION FORM

AWARDS

Upcoming 0 events

  • No upcoming events available

User login