17 J. Muhammad M. Rabbani K. Muhammad M. Wasim K. Dewan A. A. Anjum A. A. Sheikh A. Ahmad F. Akhtar M. Nisar B. M. Jayarao G. S Kirimanjeswara6. Journal of Animal and Plant Sciences JAPS 20172017/08/01 27 4 1161-1166 1018-7081 NA <p><em>Francisella tularensis</em>&nbsp;(FT) is a Tier 1 select agent due to its low infectious dose and potential threat of being a bio-weapon while causing zoonotic disease in more than 150 mammalian species. Since there are concerns in its culturing at bio-containment deficient labs in developing countries and its subsequent diagnostics and surveillance accordingly, here we proposed a conventional PCR based test of choice that have been developed and optimized for detection of FT using&nbsp;<em>tul</em>4 gene specific primers. Annealing temperature and genomic DNA concentration was optimized using a live vaccine strain. Specificity of primer showed FT detection in soil DNA in the presence of other cross-reactive organism. Sensitivity was determined in two fold dilutions with detection limit of up to 320 pg/&micro;L. Utilizing pET28a vector, a construct was prepared containing transformed&nbsp;<em>tul</em>4 gene (450bp) showing 100% sequence homology to query gene sequence. These results suggest&nbsp;<em>tul</em>4 gene based constructs in vector could be shared to developing countries while developing diagnostic assays as positive control and could potentially be used for lipoprotein purification for ELISA kit development.</p> tul4 gene, primer, conventional PCR, specificity, sensitivity, transformation Pakistan Agricultural Scientists Forum https://thejaps.org.pk/AbstractView.aspx?mid=2017-JAPS-146