MSc

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
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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

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