Nano-PCR for the detection of tomato leaf curl virus (Record no. 290912)
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| 000 -LEADER | |
|---|---|
| fixed length control field | 05193nam a22002297a 4500 |
| 003 - CONTROL NUMBER IDENTIFIER | |
| control field | OSt |
| 082 ## - DEWEY DECIMAL CLASSIFICATION NUMBER | |
| Classification number | 660.6 |
| Item number | DEV/NA PG |
| 100 ## - MAIN ENTRY--PERSONAL NAME | |
| Personal name | Devika, P P |
| 245 ## - TITLE STATEMENT | |
| Title | Nano-PCR for the detection of tomato leaf curl virus |
| 260 ## - PUBLICATION, DISTRIBUTION, ETC. (IMPRINT) | |
| Place of publication, distribution, etc | Vellayani |
| Name of publisher, distributor, etc | Department of Department of Molecular biology and biotechnology , College of Agricultureand botechnology , College of Agriculture |
| Date of publication, distribution, etc | 2023 |
| 300 ## - PHYSICAL DESCRIPTION | |
| Extent | 71p. |
| 502 ## - DISSERTATION NOTE | |
| Dissertation note | MSc |
| 520 3# - SUMMARY, ETC. | |
| Abstract | The study entitled "Nano-PCR for the detection of Tomato leaf curl virus<br/>(ToLCV)" was conducted at the Department of Molecular Biology and <br/>Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram during <br/>2022-23. The objective of the study was to evaluate the improvement of detection <br/>efficiency of Tomato leaf curl virus (ToLCV) in Solanum lycopersicum L. by NanoPCR by the inclusion of magnesium oxide and silver nanoparticles.<br/>ToLCV – partial CP gene cloned in plasmid DNA (pMD 20-T) (Athira et al., <br/>2022) and maintained in E. coli cells (strain DH5-α) was used for standardization <br/>of concentration of nanoparticles for viral detection in PCR. Plasmid DNA was <br/>isolated from overnight grown cultures of E. coli using alkaline lysis method <br/>(Birnboim and Doly, 1979), and the good quality was confirmed using nanodrop <br/>spectrophotometric analysis. A universal primer for geminiviruses designed using <br/>the CP gene was used for viral detection (Deng et al., 1994). <br/>The minimum concentration of plasmid DNA at which virus amplification is <br/>obtained using PCR was standardized as 1.5ng and was used for further treatments <br/>using nanoparticles in PCR. Silver nanoparticles (AgNPs) of size 20 nm (Sigma <br/>Aldrich, USA) and magnesium oxide nanoparticles (MgONPs) of size ≤ 50 nm <br/>(Sigma Aldrich, USA) was used for the study. In treatments 1-4, different <br/>concentration of AgNPs (1 ng/µL, 2.5 ng/µL, 3 ng/µL, 3.5 ng/µL and 4 ng/µL) alone <br/>was used in PCR. In treatments 5-8, different concentration of MgONPs (100 ng/µL, <br/>150 ng/µL, 200 ng/µL, and 250 ng/µL) was used in PCR. Combinations of AgNPs <br/>and MgONPs (3 ng/µL +200 ng/µL, 3 ng/µL +150 ng/µL, 2.5 ng/µL +200 ng/µL, <br/>2.5 ng/µL +150 ng/µL) were used for treatments 9-12. In treatments 13-16, different <br/>concentrations of MgONPs (100 ng/µL, 200 ng/µL, 250 ng/µL, 275 ng/µL, and 300 <br/>ng/µL) by replacement of magnesium chloride (MgCl2) in PCR buffer were tried. <br/>Combinations of AgNPs and MgONPs (3 ng/µL +275 ng/µL, 3 ng/µL +250 ng/µL, <br/>2.5 ng/µL +275 ng/µL, 2.5 ng/µL +250 ng/µL) by the replacement of MgCl2 in PCR <br/>buffer were used for treatments 17-20. PCR was performed at 95⁰C for 3 min <br/>followed by 34 cycles of 95⁰C for 30s, 53⁰C for 90s, 72⁰C for 90s, and final <br/>extension at 72⁰C for 10 min. Control was kept without any nanoparticles. Three <br/>100<br/>replications were done. The efficiency and specificity of PCR were checked by <br/>analyzing the intensity of the expected amplicon (500bp) in agarose gel <br/>electrophoresis and comparing the band area with that of the control using Image <br/>Lab/ImageJ software. <br/>The inclusion of AgNPs and MgONPs in PCR reaction mix at concentrations <br/>of 3 ng/µL and 200 ng/µL respectively, exhibited a 4-fold and 7.6-fold increase in <br/>the intensity of the band. Simultaneous inclusion of both AgNPs and MgONPs at <br/>concentrations of 3 ng/µL and 200 ng/µL respectively in PCR exhibited a 4.5-fold <br/>increase in the intensity of the band. The inclusion of MgONPs (275 ng/µL) <br/>replacing the MgCl2 in PCR buffer resulted in a 13-fold increase in band intensity <br/>and the simultaneous inclusion of both AgNPs and MgONPs replacing the MgCl2<br/>in PCR buffer exhibited a 12-fold increase in band intensity. The inclusion of <br/>nanoparticles in PCR resulted in the production of the visible band even at 25 cycles, <br/>thereby reducing the duration of PCR by 26%.<br/>The results were further confirmed by using genomic DNA isolated from <br/>ToLCV-infected tomato plants as the template for PCR, by the inclusion of <br/>MgONPs (275 ng/µL) replacing MgCl2 in PCR buffer with optimized PCR cycles <br/>(25 cycles). Leaf samples for the isolation of genomic DNA of ToLCV-infected <br/>tomato plants were collected from Instructional Farm, College of Agriculture, <br/>Vellayani. The modified cetyltrimethylammonium bromide (CTAB) method was <br/>performed for isolating genomic DNA and the good quality was confirmed using <br/>nanodrop spectrophotometric analysis.<br/>To conclude, the inclusion of MgONPs at a concentration of 275 ng/µL<br/>replacing MgCl2 in PCR buffer, exhibited maximum improvement (13-fold <br/>increase) in the sensitivity of PCR. The cycle number in PCR is reduced to 25 cycles, <br/>thereby decreasing the duration of PCR by 26%. Evaluation of ToLCV-infected <br/>samples at different stages can be studied by challenge inoculation to make detection <br/>possible at the earliest stage for diagnostic purposes<br/> |
| 650 ## - SUBJECT ADDED ENTRY--TOPICAL TERM | |
| Topical term or geographic name as entry element | Molecular biology |
| 650 ## - SUBJECT ADDED ENTRY--TOPICAL TERM | |
| Topical term or geographic name as entry element | Biotechnology |
| 650 ## - SUBJECT ADDED ENTRY--TOPICAL TERM | |
| Topical term or geographic name as entry element | Molecular biology and biotechnology |
| 700 ## - ADDED ENTRY--PERSONAL NAME | |
| Personal name | Swapana Alex (Guide) |
| 856 ## - ELECTRONIC LOCATION AND ACCESS | |
| Uniform Resource Identifier | https://krishikosh.egranth.ac.in/handle/1/5810225075 |
| 942 ## - ADDED ENTRY ELEMENTS (KOHA) | |
| Source of classification or shelving scheme | Dewey Decimal Classification |
| Item type | Theses |
| 500 ## - GENERAL NOTE | |
| General note | B.SC- M.SC |
| 546 ## - LANGUAGE NOTE | |
| Language note | English |
| Not for loan | Collection code | Home library | Current library | Shelving location | Date acquired | Full call number | Barcode | Date last seen | Koha item type |
|---|---|---|---|---|---|---|---|---|---|
| Not For Loan | Thesis | KAU Central Library, Thrissur | KAU Central Library, Thrissur | Theses | 01/02/2024 | 660.6 DEV/NA PG | 175893 | 01/02/2024 | Theses |