Devika, P P creator Swapana Alex (Guide) text Vellayani Department of Department of Molecular biology and biotechnology , College of Agricultureand botechnology , College of Agriculture 2023 monographic 71p. The study entitled "Nano-PCR for the detection of Tomato leaf curl virus (ToLCV)" was conducted at the Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram during 2022-23. The objective of the study was to evaluate the improvement of detection efficiency of Tomato leaf curl virus (ToLCV) in Solanum lycopersicum L. by NanoPCR by the inclusion of magnesium oxide and silver nanoparticles. ToLCV – partial CP gene cloned in plasmid DNA (pMD 20-T) (Athira et al., 2022) and maintained in E. coli cells (strain DH5-α) was used for standardization of concentration of nanoparticles for viral detection in PCR. Plasmid DNA was isolated from overnight grown cultures of E. coli using alkaline lysis method (Birnboim and Doly, 1979), and the good quality was confirmed using nanodrop spectrophotometric analysis. A universal primer for geminiviruses designed using the CP gene was used for viral detection (Deng et al., 1994). The minimum concentration of plasmid DNA at which virus amplification is obtained using PCR was standardized as 1.5ng and was used for further treatments using nanoparticles in PCR. Silver nanoparticles (AgNPs) of size 20 nm (Sigma Aldrich, USA) and magnesium oxide nanoparticles (MgONPs) of size ≤ 50 nm (Sigma Aldrich, USA) was used for the study. In treatments 1-4, different concentration of AgNPs (1 ng/µL, 2.5 ng/µL, 3 ng/µL, 3.5 ng/µL and 4 ng/µL) alone was used in PCR. In treatments 5-8, different concentration of MgONPs (100 ng/µL, 150 ng/µL, 200 ng/µL, and 250 ng/µL) was used in PCR. Combinations of AgNPs and MgONPs (3 ng/µL +200 ng/µL, 3 ng/µL +150 ng/µL, 2.5 ng/µL +200 ng/µL, 2.5 ng/µL +150 ng/µL) were used for treatments 9-12. In treatments 13-16, different concentrations of MgONPs (100 ng/µL, 200 ng/µL, 250 ng/µL, 275 ng/µL, and 300 ng/µL) by replacement of magnesium chloride (MgCl2) in PCR buffer were tried. Combinations of AgNPs and MgONPs (3 ng/µL +275 ng/µL, 3 ng/µL +250 ng/µL, 2.5 ng/µL +275 ng/µL, 2.5 ng/µL +250 ng/µL) by the replacement of MgCl2 in PCR buffer were used for treatments 17-20. PCR was performed at 95⁰C for 3 min followed by 34 cycles of 95⁰C for 30s, 53⁰C for 90s, 72⁰C for 90s, and final extension at 72⁰C for 10 min. Control was kept without any nanoparticles. Three 100 replications were done. The efficiency and specificity of PCR were checked by analyzing the intensity of the expected amplicon (500bp) in agarose gel electrophoresis and comparing the band area with that of the control using Image Lab/ImageJ software. The inclusion of AgNPs and MgONPs in PCR reaction mix at concentrations of 3 ng/µL and 200 ng/µL respectively, exhibited a 4-fold and 7.6-fold increase in the intensity of the band. Simultaneous inclusion of both AgNPs and MgONPs at concentrations of 3 ng/µL and 200 ng/µL respectively in PCR exhibited a 4.5-fold increase in the intensity of the band. The inclusion of MgONPs (275 ng/µL) replacing the MgCl2 in PCR buffer resulted in a 13-fold increase in band intensity and the simultaneous inclusion of both AgNPs and MgONPs replacing the MgCl2 in PCR buffer exhibited a 12-fold increase in band intensity. The inclusion of nanoparticles in PCR resulted in the production of the visible band even at 25 cycles, thereby reducing the duration of PCR by 26%. The results were further confirmed by using genomic DNA isolated from ToLCV-infected tomato plants as the template for PCR, by the inclusion of MgONPs (275 ng/µL) replacing MgCl2 in PCR buffer with optimized PCR cycles (25 cycles). Leaf samples for the isolation of genomic DNA of ToLCV-infected tomato plants were collected from Instructional Farm, College of Agriculture, Vellayani. The modified cetyltrimethylammonium bromide (CTAB) method was performed for isolating genomic DNA and the good quality was confirmed using nanodrop spectrophotometric analysis. To conclude, the inclusion of MgONPs at a concentration of 275 ng/µL replacing MgCl2 in PCR buffer, exhibited maximum improvement (13-fold increase) in the sensitivity of PCR. The cycle number in PCR is reduced to 25 cycles, thereby decreasing the duration of PCR by 26%. Evaluation of ToLCV-infected samples at different stages can be studied by challenge inoculation to make detection possible at the earliest stage for diagnostic purposes B.SC- M.SC MSc English Molecular biology Biotechnology Molecular biology and biotechnology 660.6 DEV/NA PG https://krishikosh.egranth.ac.in/handle/1/5810225075 https://krishikosh.egranth.ac.in/handle/1/5810225075