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. 2023 Dec 6;14(1):5. doi: 10.1007/s13205-023-03842-2

Nano-PCR for the early detection of tomato leaf curl virus

P P Devika 1, Swapna Alex 1,✉, K B Soni 1, K P Sindura 1, R Ayisha 2, R V Manju 3
PMCID: PMC10700262  PMID: 38074290

Abstract

Nano-PCR is a potential tool for the early detection of plant viruses. In the current study, different concentrations of silver nanoparticles (20 nm) and magnesium oxide nanoparticles (50 nm) were included in the PCR mixture to improve the sensitivity of PCR for the detection of tomato leaf curl virus. The inclusion of nanoparticles in single or combination in PCR mixture has resulted in improvement of PCR sensitivity. Four-fold improvement was exhibited by the inclusion of 3 ng/µL silver nanoparticles, whereas the combination of silver and magnesium oxide nanoparticles (3 ng/µL and 200 ng/µL, respectively), resulted in a 4.5-fold improvement. The inclusion of 200 ng/µL of magnesium oxide nanoparticles in the PCR mixture exhibited a 7.6-fold increase in PCR sensitivity. Replacement of magnesium chloride with a combination of silver and magnesium oxide nanoparticles (3 ng/µL and 275 ng/µL, respectively) resulted in a 12-fold increase. A 13-fold improvement in PCR sensitivity was observed by the replacement of magnesium chloride in PCR buffer with 275 ng/µL of magnesium oxide nanoparticles. This could also produce detectable amplicon in PCR with a minimum of 25 cycles, resulting in a 26.5% reduction in the duration of PCR. This is the first report on the use of magnesium oxide nanoparticles in PCR for the early detection and better management of tomato leaf curl virus.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13205-023-03842-2.

Keywords: Nano-PCR, PCR sensitivity, Magnesium oxide nanoparticles, Silver nanoparticles, Viral detection

Introduction

Tomato (Solanum lycopersicum L.) is one of the most widely cultivated vegetable crops in the world (Caruso et al. 2022). Tomato leaf curl disease (ToLCD) caused by the tomato leaf curl virus (ToLCV) results in serious damage to tomato plants (Kumari et al. 2020). ToLCV is a Begomovirus (family Geminiviridae, genus Begomovirus) transmitted by whiteflies (Bemisia tabaci) and has a genome organisation consisting of ssDNA (Fiallo-Olivé et al. 2021). The symptoms of ToLCV infection in plants can range from symptomless to varying degrees of stunting and curling, distortion, mosaic, mottling, vein yellowing of leaves, flower abortion, and small and unmarketable fruits (Rojas et al. 2018). Early detection plays a major role in the management of viruses.

Plant viruses can be detected using a variety of approaches, which can be divided into two categories: immunotechniques (techniques based on proteins) and molecular methods (procedures based on nucleic acids). PCR is the most widely used molecular biology technique for the detection of viruses. Viral detection by PCR was initially reported by Vunsh and co-workers in 1990. An important factor that determines the early detection of viruses is the sensitivity of the PCR, which refers to the capacity of the PCR to detect lower limits of the target nucleic acid (Nurliyana et al. 2018).

PCR-enhancing additives (PCR enhancers) are substances that are included in a PCR reaction mix to enhance the amplification of the product. Betaine, organic solvents such as DMSO, and formamide, sugars like glycerol and trehalose, proteins such as BSA, non-ionic detergents like Tween-20, and nanomaterials, which include metal and non-metal nanomaterials, are some of the widely used PCR enhancers (Simonovic et al. 2012; Karunanathie et al. 2022). The use of nanomaterials to improve PCR, known as nano-PCR, is a relatively new field of biotechnology. Numerous studies have shown the use of nanomaterials such as carbon nanotubes, quantum dots, and metal-based nanoparticles, which include gold nanoparticles (AuNPs), silver nanoparticles (AgNPs), and titanium dioxide nanoparticles (TiO2NPs) for PCR (Wan et al. 2009; Yuce et al. 2014).

Nanoparticles have excellent heat transfer properties, which is a major factor in the improvement of PCR. They can transport the heat flux into the liquid resulting in thermal equilibrium of the liquid. Thermal efficiency is observed to improve with a decrease in the size of nanoparticles (Li et al. 2005). The use of nanoparticles that enhance thermal conductivity may reduce the thermal mass of the reaction solution, enabling faster heat transfer and thereby improving the PCR efficiency (Haber et al. 2008). The metal ion gets embedded inside a double helix structure and stabilizes the DNA template because of its specific binding (Hemanta and Varsha 2014). Nanoparticles can attain high temperatures and a fast ramp rate, and their thermal decay properties make them perfect candidates as nano-heaters in PCR (Kadu et al. 2020). Complex formation between DNA and AgNPs leads to a change in melting temperature, which suggests the possibility of binding of AgNPs to the grooves of DNA (Talebpour et al. 2019).

Among metal PCR enhancers, AuNPs as PCR enhancers have been extensively studied (Lou and Zhang 2013). However, the rising cost of AuNPs presents an important limitation. Hence, other nanoparticles like TiO2NPs, AgNPs, and magnetite nanoparticles (Fe3O4NPs) were studied for their effect on PCR sensitivity (Amadeh et al. 2021; Karunanathie et al. 2022). In the present work, the effects of AgNPs, MgONPs, and their combination in the presence and absence of magnesium chloride (MgCl2) in the PCR reaction mixture, for plant viral detection were thoroughly investigated.

Materials and methods

Materials

Silver and magnesium oxide nanoparticles (Sigma-Aldrich) were used for the study. Magnesium oxide nanoparticles (50 nm) were dispersed in deionized distilled water at a 1 mg/mL concentration. AgNPs (20 nm) were available as dispersion in aqueous buffer and sodium citrate as a stabilizer at a concentration of 0.02 ng/µL. The partial Coat Protein (CP) gene of tomato leaf curl virus (ToLCV) inserted in pMDT-20 was used as the template for optimization of nano-PCR. DNA from leaves of tomato leaf curl virus infected plants were used for validation of the results of nano-PCR.

Preparation of DNA template for PCR

ToLCV, partial CP gene cloned in plasmid DNA (pMD20-T), was maintained in E. coli cells (strain DH5-α). Plasmid DNA was isolated from cloned E. coli cultures using the alkaline lysis method (Birnboim and Doly 1979).

Viral detection by PCR

PCR was carried out using a universal primer for geminiviruses (Deng et al. 1994) and the primer details are provided in the supplementary table (Table S1). The concentration of PCR components and the conditions for PCR are also provided in the supplementary tables S2 and S3, respectively.

The PCR products were analyzed in agarose gel (1.2%) electrophoresis and viewed using the gel documentation system. The relative intensity of the PCR results was analyzed using the Image Lab Software for the determination of the optimum concentration.

Standardization of the concentration of DNA for nano-PCR

Cloned viral DNA with a concentration range of 0–15 ng was used for detection. PCR products using different concentrations of viral DNA were compared using agarose gel electrophoresis. The minimum concentration of DNA at which amplification was obtained using PCR was standardized.

Optimization of nano-PCR

PCR was performed for the standardization of the concentration of nanoparticles for virus detection based on the least concentration of DNA standardized (Table 1). Different concentrations of AgNPs and MgONPs were included in the PCR as shown in Table 2. PCR reactions without nanoparticles were used as a control for the comparison of the PCR products. Three replications of each treatment were maintained. The PCR products were checked using agarose gel (1.2%) and viewed using the gel documentation system. The relative intensity of the PCR amplicon on the gel was analyzed using Image Lab/ImageJ software for the determination of the optimum concentration.

Table 1.

Components and concentration of PCR reaction mix for treatments using nanoparticles

Sl. no. Components Final concentration
1 10 × PCR buffer 1x
2 dNTPs 0.2 mM
3 Primer (forward) 0.5 mM
4 Primer (reverse) 0.5 mM
5 Taq DNA polymerase 1 U
6 Plasmid DNA 1.5 ng
7 Nanoparticles Different concentrations of AgNPs/MgONPs
8 Sterile distilled water Made up to 20 µL mix
Total 20 µL
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Table 2.

Treatments selected for nano-PCR

Treatment no. Nanoparticles Concentration of nanoparticles MgCl2 in PCR buffer
1 AgNPs

1 ng/µL

2.5 ng/µL

3 ng/µL

3.5 ng/µL

4 ng/µL

5 ng/µL

7 ng/µL

  + 
2 MgONPs

100 ng/µL

150 ng/µL

200 ng/µL

250 ng/µL

  + 
3 AgNPs + MgONPs Combination of the best treatments selected from treatment 1 & 2  + 
4 MgONPs

50 ng/µL

100 ng/µL

150 ng/µL

200 ng/µL

225 ng/µL

250 ng/µL

275 ng/µL

300 ng/µL

 −
5 AgNPs + MgONPs Combination of the best treatments selected from treatment 1 and 4 −
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Rapid and enhanced PCR sensitivity using nano-PCR for viral detection

The rapidity and improved sensitivity of nano-PCR were analyzed by two methods, viz., reducing the number of PCR cycles and template DNA concentration.

  1. Optimization of PCR cycles for nano-PCR

    Nano-PCR with cycle number 15, 20, and 25 was carried out, and the PCR cycles were optimized for nano-PCR.

  2. Validation of nano-PCR at different concentrations of template DNA

    Template DNA concentrations ranging from 3.5 to 0.001 ng were added to the reaction mixture, and nano-PCR was carried out with 35 cycles of PCR. The minimum concentration of template DNA required for the amplification using nano-PCR was identified. A control was kept with 5 ng viral DNA as template using conventional PCR mixture.

Detection of viruses in infected samples by nano-PCR

Leaf samples of six tomato plants showing symptoms of ToLCV infection were collected and genomic DNA was isolated (Vasudeva and Samraj 1948). Viral detection using conventional PCR was performed to confirm the presence of ToLCV. The increase in the sensitivity of nano-PCR was assessed by reducing the template DNA concentration of virus-positive samples using the standardized concentration of nanoparticles obtained in the study. A virus-positive sample was kept as control.

Results

Optimization of nano-PCR

Plasmid DNA cloned with the partial CP gene of ToLCV and isolated using alkaline lysis method was used for the optimization of nano-PCR. Among the different concentrations of plasmid DNA used as a template for PCR, 1.5 ng of plasmid DNA was selected as the standard concentration of template DNA for nano-PCR (Supplementary file Fig. S1).

AgNPs of concentrations ranging between 1 and 7 ng/µL of the PCR reaction mixture and MgONPs of concentrations ranging between 50 and 300 ng/µL were included in the PCR mixture, and the results of the PCR reaction visualized after agarose gel electrophoresis showed that the inclusion of nanoparticles improved the sensitivity of PCR remarkably.

The best concentration of AgNPs was identified as 3 ng/µL of reaction mixture (here, 20 µL reaction mixture was used) from the gel profile as shown by using Image Lab/ImageJ software. In this study, the band area obtained from ImageJ software was used for comparison in addition to visual observation. The band areas obtained in ImageJ software are arbitrary numbers that can be used for comparison across a single gel profile but cannot be converted to nanograms of DNA (Stael et al. 2022). Figure 1A shows that inclusion of 3 ng/µL of AgNPs in PCR mixture resulted in maximum PCR amplification with a band area of 9378 mm2. Further experiments were carried out with AgNPs concentrations such as 2.5 ng/µL, 3 ng/µL, 3.5 ng/µL, and 4 ng/µL and the gel profile (Fig. 1B) showed the maximum band area for treatment containing 3 ng/µL AgNPs. Hence, 3 ng/µL of AgNPs was selected as the concentration of AgNPs to be included in the PCR mixture, exhibiting maximum improvement in PCR, followed by the addition of 2.5 ng/µL of AgNPs.

Fig. 1.

Fig. 1

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Effect of AgNPs on the sensitivity of PCR. A AgNPs of concentration ranging between 1 and 7 ng/µL was used in PCR. M: 100 bp DNA marker; lane 1: 1 ng/µL; lane 2: 3 ng/µL; lane 3: 5 ng/µL; lane 4: 7 ng/µL; lane 5: Control. B AgNPs of concentration ranging between 2.5 and 4 ng/µL was used in PCR. M: 100 bp DNA marker; lane 1: 2.5 ng/µL; lane 2: 3 ng/µL; lane 3: 3.5 ng/µL; lane 4: 4 ng/µL

Different concentrations of MgONPs ranging between 100 and 250 ng/µL were included in PCR, among which PCR amplification by the inclusion of 200 ng/µL MgONPs exhibited maximum amplification as shown in Fig. 2. The band area of the PCR product in the agarose gel of treatment containing 200 ng/µL MgONPs was obtained as 10,443 mm2, followed by 8915 mm2 for treatment containing 150 ng/µL.

Fig. 2.

Fig. 2

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Effect of MgONPs on the sensitivity of PCR. M: 100 bp DNA marker; lane 1: 100 ng/µL; lane 2: 150 ng/µL; lane 3: 200 ng/µL; lane 4: 250 ng/µL; lane 5: control

A combination of AgNPs (3 ng/µL and 2.5 ng/µL) and MgONPs (200 ng/µL and 150 ng/µL) was treated in PCR, and the combination of 3 ng/µL AgNPs and 200 ng/µL MgONPs resulted in the maximum amplification in PCR as shown in Fig. 3 with a band area of 10,151 mm2 for the PCR product.

Fig. 3.

Fig. 3

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Effect of AgNPs and MgONPs on the sensitivity of PCR. M: 100 bp DNA marker; lane 1: AgNPs (3 ng/µL) and MgONPs (200 ng/µL); lane 2: AgNPs (2.5 ng/µL) and MgONPs (200 ng/µL); lane 3: AgNPs (3 ng/µL) and MgONPs (150 ng/µL); lane 4: AgNPs (2.5 ng/µL) and MgONPs (150 ng/µL); lane 5: Control

Magnesium chloride in the PCR buffer was replaced by MgONPs of concentrations ranging from 50 to 300 ng/µL, and the PCR was carried out (Fig. 4). Maximum amplification was exhibited by the inclusion of 275 ng/µL of MgONPs, followed by the inclusion of 250 ng/µL of MgONPs in the PCR mix. Band areas obtained from the gel profiles of PCR products of treatments including 275 ng/µL and 250 ng/µL of MgONPs are 11,560 mm2 and 11,448 mm2, respectively. A combination of the best two concentrations of AgNPs (3 ng/µL and 2.5 ng/µL) with the above-mentioned concentrations of MgONPs was included in the PCR mix and the best combination was found to be the combination of 3 ng/µL AgNPs and 275 ng/µL MgONPs, as shown in Fig. 5 with a band area of 9422 mm2 as mentioned in Fig. 5.

Fig. 4.

Fig. 4

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Effect of MgONPs by replacing MgCl2 on the sensitivity of PCR. A MgONPs of concentration ranging between 50 and 250 ng/µL was used in PCR. M: 100 bp DNA marker; lane 1:50 ng/µL; lane 2: 100 ng/µL; lane 3: 150 ng/µL;lane 4:200 ng/µL; lane 5: 250 ng/µL; lane 6: control. B MgONPs of concentration ranging between 225 and 300 ng/µL was used in PCR. M: 100 bp DNA marker; lane 1: 225 ng/µL; lane 2: 250 ng/µL; lane 3: 275 ng/µL; lane 4: 300 ng/µL

Fig. 5.

Fig. 5

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Effect of AgNPs and MgONPs with replacement of MgCl2 on the sensitivity of PCR. M: 100 bp DNA marker; lane 1: AgNPs (3 ng/µL) and MgONPs (275 ng/µL); lane 2: AgNPs (2.5 ng/µL) and MgONPs (275 ng/µL); lane 3: AgNPs (3 ng/µL) and MgONPs (250 ng/µL); lane 4: AgNPs (2.5 ng/µL) and MgONPs (250 ng/µL); lane 5: Control

Concentrations of nanoparticles that gave the maximum improvement in PCR among treatments 1–12 were compared in a single gel profile (Fig. 6). The maximum improvement in PCR amplification among all the treatments tried was observed with the addition of MgONPs (275 ng/µL) and the replacement of MgCl2 in PCR buffer, with a band area of 15,383 mm2 in the gel profile. The band area of the PCR product of treatment using a combination of AgNPs (3 ng/µL) and MgONPs (275 ng/µL) by replacing MgCl2 in PCR buffer was obtained as 14,258 mm2, which also represents a higher improvement in PCR. The amplification efficiency of the treatments is much higher than that of the control.

Fig. 6.

Fig. 6

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Comparison of effect of inclusion of AgNPs, MgONPs, and their combination on PCR sensitivity. M: 100 bp DNA marker; lane 1: 3 ng/µL AgNPs; lane 2: 200 ng/µL MgONPs; lane 3: AgNPs (3 ng/µL) and MgONPs (200 ng/µL); lane 4: Replacement of MgCl2 in PCR buffer by 275 ng/µL MgONPs; lane 5: replacement of MgCl2 in PCR buffer by 275 ng/µL MgONPs and 3 ng/µL AgNPs; lane 6: control 1—buffer with MgCl2; lane 7: control 2—buffer without MgCl2

Optimization of PCR cycles with respect to the optimized concentrations of nanoparticles for virus detection

Viral detection using PCR as well as standardization of nanoparticles for PCR were carried out with 35 cycles of PCR, including the initial denaturation and final extension. A standardized concentration of nanoparticles (275 ng/µL of MgONPs replacing MgCl2 in buffer) was used for the optimization of PCR cycles. Cycle number was reduced to 15, 20, and 25 PCR cycles, among which PCR amplification was visually detected from 25 cycles onwards using the treatment with MgONPs by replacing MgCl2 in PCR buffer (Fig. 7).

Fig. 7.

Fig. 7

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Optimization of PCR cycles for nano-PCR using template DNA of concentration 170 ng. A 25 cycles of PCR; B 20 cycles of PCR; C 15 cycles of PCR

Improved PCR sensitivity using nano-PCR

Among the different concentrations of template DNA (3.5–0.001 ng) added to the PCR mixture, detection was possible even at 0.01 ng of template DNA (Fig. 8), by using optimized concentration of nanoparticles (275 ng/µL of MgONPs replacing MgCl2 in PCR buffer) in nano-PCR.

Fig. 8.

Fig. 8

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Gel profile of PCR amplification using different concentrations of template DNA in nano-PCR (275 ng/µL MgONPs without MgCl2 in PCR buffer). M: 100 bp DNA marker; lane 1: 3.5 ng template DNA; lane 2: 0.8 ng template DNA; lane 3: 0.2 ng template DNA; lane 4: 0.04 ng template DNA; lane 5: 0.01 ng template DNA; lane 6: 0.008 ng template DNA; lane 7: 0.004 ng template DNA; lane 8: 0.001 ng template DNA; lane 9: control-conventional PCR using 5 ng genomic DNA as template

Detection of viruses in infected samples by nano-PCR

All six collected virus samples were confirmed as virus-positive using the conventional PCR method. Two samples produced faded amplicons compared to the rest of the samples (Fig. 9). The DNA concentration of the virus-positive samples was diluted to thrice its volume for nano-PCR (275 ng/µL of MgONPs replacing MgCl2 in PCR buffer), and amplicons were produced with improved band intensity (Fig. 10).

Fig. 9.

Fig. 9

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Gel profile of PCR amplification to confirm viral detection in field samples. M: 100 bp DNA marker; lane 1: control-PCR using virus free plant genomic DNA; lane 2–7: PCR using genomic DNA of tomato plants showing disease symptoms

Fig. 10.

Fig. 10

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Gel profile of PCR amplification to confirm the improvement of detection efficiency by nano-PCR (275 ng/µL MgONPs without MgCl2 in PCR buffer) using genomic DNA of infected sample M: 100 bp DNA marker; lane 1: control-PCR using virus free plant genomic DNA; lane 2–7: PCR using genomic DNA of tomato plants showing disease symptoms

Discussion

Nanoparticles offer various applications in biotechnology, such as biolistic transformation, bacterial transformation, etc. (Rajkumari et al. 2021). Functionalized nanoparticles are reported to improve the sensitivity of viral detection techniques (Ramesh and Viswanathan 2021). During the past decade, various nanomaterials have been used for improving PCR sensitivity, including gold, silver, and graphene oxide nanoparticles (Karunanathie et al. 2022). The tomato leaf curl virus has a high rate of disease transmission. Hence, early detection is crucial for its management. Improvement of PCR sensitivity can aid in the detection of viruses during the initial stages of infection, even at low titre values. Here, AgNPs and MgONPs were used to improve the PCR sensitivity for the detection of tomato leaf curl virus.

Reports on AgNPs and MgONPs in nano-PCR, as well as their interactions with PCR components, are limited. The properties of nanoparticles vary largely with their particle size and concentration. Hence, optimization of the size and concentration of nanoparticles have to be carried out specifically for each molecular experiment. The current study was able to optimize nanoparticle concentration and PCR cycle optimization for viral detection using PCR.

In this study, among the different concentrations of AgNPs used (1–7 ng/µL), 3 ng/µL of AgNPs gave the maximum improvement in PCR (fourfold increase). In an earlier report by Qun and co-workers in 2007, AgNPs of particle size 70 nm required a concentration of about 100–200 ng/µL to enhance PCR amplification. According to Li et al. (2005), as the size of the nanoparticles decreases, their thermal properties tend to increase. The size of the AgNPs used in the present study was 20 nm, and this might be the reason that the silver nanoparticles have exhibited maximum improvement in sensitivity at a concentration as low as 3 ng/µL.

Inclusion of MgONPs in PCR reactions gave a maximum improvement in PCR sensitivity compared to AgNPs for viral detection. According to Kambili and Kelkar-Mane (2016), nanomaterials of lower conductance in PCR resulted in a higher yield compared to the inclusion of nanoparticles of higher conductance. In this present study, the lower conductance of MgONPs in comparison to AgNPs can also be a possible reason for the remarkable improvement of PCR sensitivity by MgONPs (Arkel et al. 1953; Chen et al. 2007).

Inclusion of 200 ng/µL of MgONPs exhibited a maximum amplification of PCR with a 7.6-fold increase in PCR improvement. Mg2+ as a co-factor is necessary for the activity of Taq DNA polymerase (Schmidt et al. 2014). Magnesium ions in MgONPs might have also played a similar function as a co-factor for Taq DNA polymerase and might have contributed to the enhancement of the PCR reaction.

In the present study, combinations of AgNPs and MgONPs that showed maximum improvement in PCR amplification were included in the PCR mixture simultaneously. Among the combinations of AgNPs and MgONPs tested in PCR, a combination of 3 ng/µL AgNPs and 200 ng/µL of MgONPs showed a 4.5-fold improvement in PCR sensitivity. The combination of AgNPs and MgONPs improved the PCR amplification by 12.5% compared to the treatment of AgNPs alone. There are earlier reports that a combination of AgNPs and titanium dioxide nanoparticles (TiO2NPs) recorded an improvement in efficiency (Wan et al. 2009).

Inclusion of MgONPs of concentrations ranging between 50 and 300 ng/µL by replacing magnesium chloride (MgCl2) in PCR buffer exhibited a significant increase in the improvement in PCR. A 13-fold increase in PCR with the addition of 275 ng/µL of MgONPs was noticed. Magnesium salt in PCR buffer can lead to the aggregation of metallic nanoparticles (Li et al. 2005). Replacement of MgCl2 in the PCR reaction mixture might have resulted in a decrease in the Mg ion salt concentration and reduced the tendency for aggregation of nanoparticles. This led to an improvement in PCR amplification by 41% compared to the treatment of MgONPs with PCR buffer containing MgCl2. Various combinations of AgNPs and MgONPs by the replacement of MgCl2 in PCR buffer were also tested, among which 3 ng/µL of AgNPs and 275 ng/µL of MgONPs gave maximum improvement. The combination resulted in a 12-fold increase in PCR compared to the control. The same combination of AgNPs and MgONPs resulted in a 4.5-fold increase in treatment containing PCR buffer with MgCl2, which indicates a 62.5% improvement in PCR amplification by replacing MgCl2 in PCR buffer with respect to the treatment containing MgCl2 in PCR buffer. MgONPs of concentration 275 ng/µL replacing MgCl2 in PCR buffer showed the maximum improvement among all the treatments and were selected as the standardized concentration of nanoparticles for further treatments.

For PCR cycle optimization, a PCR mix containing a standardized concentration of nanoparticles (275 ng/µL MgONPs) by replacing MgCl2 in PCR buffer was used for PCR reactions at 15, 20, and 25 cycles. Here, the inclusion of a standardized concentration of nanoparticles of MgONPs in PCR resulted in the production of the visible band even after 25 cycles. Bansod et al. (2013) have also reported optimization of PCR by reducing PCR cycles using nanoparticles of gold and silver. Apart from the rapidity of nano-PCR confirmed by reducing PCR cycles, improved PCR sensitivity was confirmed by reducing the template DNA concentration used for nano-PCR. 0.5 ng of template DNA was required for viral detection in conventional PCR whereas, nano-PCR improved the PCR sensitivity such that viral detection was possible at a template DNA concentration of 0.01 ng.

Improved PCR sensitivity using MgONPs (275 ng/µL) by replacing MgCl2 in PCR buffer was confirmed using genomic DNA of infected field samples. Nano-PCR was able to detect viruses in much lower quantities of template DNA without compromising the band intensity.

Conclusion

The inclusion of MgONPs at a concentration of 275 ng/µL, replacing MgCl2 in PCR buffer, exhibited a 13-fold improvement in the sensitivity of PCR for ToLCV detection. Inclusion of MgONPs could reduce the cycle number to 25 cycles, resulting in a 26.5% reduction in time for PCR. Template DNA required for viral detection could be reduced to 0.01 ng. Thus, detection of ToLCV could be improved by nano-PCR and aid in better management of the virus.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 120 KB) (120.5KB, docx)

Acknowledgements

Research facilities provided by Kerala Agricultural University is gratefully acknowledged.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Research involving in human participants

Human subjects are not involved in the experiment.

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