Development of National Medical Diagnostic Kits from Locally Produced Enzymes

The effective control and treatment of chronic diseases requires access to diagnostic tests that are rapid, reliable and very sensitive. Nucleic acid based methods of pathogen detection technologies offer very rapid and accurate results with high sensitivity and specificity, at relatively low cost. This study aims to develop very high-sensitivity nucleic acid based diagnostic kits using locally produced enzymes that will enable rapid and accurate detection of Hepatitis B and Hepatitis C viruses from blood and also biological fluids at very low cost. Transformed E. coli cells with pTOL recombinant plasmids were cultured in 3 litres LB triple medium supplemented with 100 µg/ml ampicillin at 37 oC in bioreactor. When the optical density at 600 nm IPTG (Isopropyl β- D -1-thiogalactopyranoside) was added to a final concentration of 1 mM in order to express Pfu DNA Polymerase, Taq DNA Polymerase and MMLV Reverse Transcriptase. After 5 hours, the cells were harvested by centrifugation. Cell pellets were suspended in lysis buffer and disrupted by sonication. Soluble protein was collected using ultra-centrifugation. Hexa histidine tag on the N-terminus of proteins were used for purification of recombinant enzymes. The expression levels of proteins was assessed using 10% (w/v) SDS-PAGE and UV spectroscopy. Enzymatic activities of the produced DNA polymerase and MMLV reverse transcriptase enzymes were tested by PCR and RT PCR reactions using specific primers for Hepatitis B and C viruses. Rapid, highly sensitive and Nucleic acid based diagnostic kits for hepatitis pathogen detection were developed in this research, which specifically responds to Hepatitis B and Hepatitis C viruses will be useful in detecting and monitoring infections from blood and biofluids. Production of these molecular diagnostic kits that are based entirely on using domestically produced enzymes are fully intended to reduce the dependence of our country abroad on this sector. Reference: Isa Gokce, Gregor Anderluh, Jeremy H Lakey, Patent, "Fusion proteins" 2005 US20050130269 A1