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An effort to optimize molecular diagnosis of Nipah virus (NiV) by PCR for the detection of infection
3rd World Congress and Expo on Applied Microbiology
November 07-09, 2016 Dubai, UAE
Farhina Pasha
University of Tabuk, KSA
Posters & Accepted Abstracts: J Microb Biochem Technol
Abstract:
The present study was aimed at optimization of PCR as molecular diagnosis tool for the detection of Nipah virus infection and expression of recombinant protein for which �??G�?? gene of NiV was targeted. Different primer sets were designed with the help of Bioinformatics software DNAStar (Lasergene) and recombinant protein was expressed in E.coli cells. A total of 98 clinical samples were screened for the desired sequence of NiV �??G�?? gene by PCR. The results indicated that, firstly the primers designed were found to be specific for �??G�?? region of NiV and could be used to amplify various fragments of NiV �??G�?? region. Also when these primers were used in different combinations, a large fragment of Niv �??G�?? gene (1 kb) could be amplified. Secondly of the 98 clinical samples screened none of them were found to be positive for NiV infection in conventional PCR. Amplification and cloning of 1 kb fragment of �??G�?? gene of NiV was done in pGEMT-Easy vector. 1 kb fragment of �??G�?? gene of NiV could be sub cloned in two different expression vector systems i.e., pET-28(+) B and pGex-5x-1. In both the cases the recombinant protein could be obtained in the form of soluble fraction. The yield of expressed protein was more in case of pET 28(+) vector but the stability was less in second and third passages while in case of pGex-5x-1 the yield of the expressed protein was less but the stability was more in second and third passages. The specificity of recombinant protein was confirmed by Western Blot analysis using penta his-HRP and GST-HRP conjugate in case of pET 28(+) B and pGex-5x-1 respectively. The protein would be used for raising antibodies and further used as diagnostic reagent for the detection of NiV antibodies.
Biography :
Email: fbasha@ut.edu.sa