Utilization of Corn Silk for GMO Detection Through Real-Time PCR

Corn silk is a fiber-rich byproduct obtained during corn processing. The present investigation deals with the purpose of GMO detection and quantification and comparison of DNA extraction in corn silk which is considered generally a waste product. A comprehensive analysis of extracted DNA quality, extraction cost, time, and PCR amplification was performed. Samples were homogenized using three different methods i.e. tissue lyser (CSK-TL) liquid nitrogen (CSK-LN), and grinder (CSK-RG). The homogenized samples from different methods were subjected to scanning electron microscopy (SEM) to access the effectiveness of homogenization methods. Afterward, DNA extraction was carried out using three different Kits QIAGEN DNeasyMericon Food Kit, eurofins|GeneScan GENESpin DNA isolation Kit, and PENICON EZ-10 Spin Column Genomic DNA Miniprep Kit. Furthermore, the fluorometric assay was done using Qubit fluorometer 3.0 for quantification of the extracted DNA. The quality of the DNA was accessed on 1.5% agarose gel. The scanning electron microscopy (SEM) technique displays observable differences among the three homogenization methods. As compared to the other two methods tissue lyzer (CSK-TL) shows the finest particle image. Whereas, the images of liquid nitrogen (CSK-LN), as well as a grinder (CSK-RG) treated sample, show granulated particles. DNA extraction of homogenized samples was carried out using three different kits named QIAGEN DNeasyMericon Food Kit (Kit 1), eurofins|GeneScan GENESpin DNA isolation Kit (Kit 2), and PENICON EZ-10 Spin Column Genomic DNA Miniprep Kit (Kit 3). Kit 1 showed a yield of 122.4 ng/µl, whereas Kit 2 and Kit 3 gave 99.6 and 40.1 ng/µl respectively. By using gel electrophoresis technique the band size of corn silk genomic DNA was above 1000 bp. DNA extracted using waste material (corn silk) can be used for GMO analysis through the Real-Time PCR technique. Among different grinding techniques compared, the Tissue lyzed sample (CSK-TL) displayed the best homogenization and the highest quantity of DNA using the QIAGEN DNeasyMericon Food Kit. This method demonstrates the utility of the waste products for tracing the GMO status.


Introduction
Corn (Zea mays), also called Indian corn or maize, is the third most significant crop produced and consumed globally after rice and wheat.It has versatile applications in industries including pharmaceutical, food, and non-food industries, etc. [1].In Pakistan, maize is harvested by mid-December.
According to FAO stats, the total harvested area, yield, and production for maize in Pakistan was 1413246 ha, 51203 hg/ ha, and 7,236,313 tons, respectively in 2019 [2].
Waste utilization has always been an attractive subject for all researchers.Processing of yellow or green maize (Zea mays L.), also yields some byproducts i.e. husk, cobs, silk, and stalks.Among all, corn silk (Stigma maydis) is the most common waste material produced during corn processing that is not utilized and is typically discarded.Nutritionally, it consists of protein, fat, CHO, steroids, saponin, tannin, etc. [3].However, corn silk is medicinally important as it is effective to treat nephritis, hypertension, prostatitis, and urinary tract infections [4].Many countries around the world including China, Turkey, the United States, and France have used it as a common medicine.It's also used to treat cystitis, edema, kidney stones, bladder diseases, and obesity [5].Its potential therapeutic benefits are due to the chemical composition and mode of action of bioactive elements including phenolics and terpenoids, as well as polysaccharides and glycoproteins.Flavonoids in corn silk have shown varied pharmacological effects.These are known for their antioxidant and free radical scavenging capability [6].
Maize has the most approved events (single and stacked traits) among Genetically Modified (GM) crops and is the second largest crop in terms of global adoption, after soybean.Efforts have been made in Europe during the past few years to develop PCR techniques for the detection of a variety of GMOs such as the FLAVR SAVR tomato, glyphosatetolerant soybeans, Bt-maize, a transgenic potato with altered starch content, and marker genes.According to the Novel Food Regulation in Europe, the distinction between traditional food and food containing GMOs should be detected using suitable scientific methods.
In Pakistan, Bacillus thuringiensis (bt) cotton is the only designated GM crop that is largely farmed in the southern Punjab and used for commercial purposes.In 2005, biosafety rules were notified according to that some of the regulations were developed which underline the procedures to undertake all related activities.For the monitoring and implementation of system of national biosafety guidelines, 3 layers are being served i.e.National biosafety committee (NBC), Technical advisory committee (TAC) and Institutional biosafety committee (IBC).These guidelines were formulated in accordance to the guidelines of FAO, WHO, UNIDO and UNEP.Besides that, Pakistan also maintains a plant protection department to monitor GM crops [7].
The Polymerase Chain Reaction (PCR) is suitable for food analysis and seems to be the preferred method for detecting GMOs in food [8].Many genetically modified (GM) crops and products have been introduced into the market for food and feed use, due to the rapid advancement of recombinant DNA techniques [9].Globally, 53.6 million hectares of GMO maize were planted in 2015, representing about a third of the 185 million ha of maize planted worldwide [10].GMOs can also be detected in soybean and cotton processing by-products and waste using the qPCR technique [9].
The present investigation aims to use corn wastage i.e. corn silk, for the detection of GMOs using the Real-Time (qPCR) technique.To the best of our knowledge, little study has been done on GMO detection from corn silk.We studied the effects of grinding methods for DNA isolation and their morphological difference through scanning electron microscopy.Three different Kit based methods were also compared in terms of extraction of DNA and the time and cost were also estimated.

Sample Collection and Preparation
For the extraction of DNA and morphological study of corn silk, samples (A1, A2, and A3) were collected from three different areas of Karachi, Pakistan.All samples were ground and oven dried at 55 °C overnight.All samples were homogenized/ground using three methods (a) simply by grinding in a laboratory grinder (CSK-RG) (Ciatronic, KSW 3306), (b) grinding with TissueLyser II (CSK-TL) (QIAGEN, Germantown, MD, US) at frequency 30 (s −1 ) for 10-15 s and (c) thirdly with liquid nitrogen (CSK-LN) using mortar and pestle.The samples were stored in corning tubes (Nest, Biotechnology Co., Ltd, China, 15ml) at (− 20 °C) (SIEMENS, Munich Germany, GS32NA23) until the completion of the analysis.

Morphological Characteristics
Morphological analysis of all corn silk samples (grounded by different methods) was performed through analytical scanning electron microscopy (JSM, 6380 A, Jeol, Japan).All samples were precipitated on an aluminum specimen holder and then thinly coated with gold by ion splutter (JFC-1500) in a vacuum evaporator.After gold coating, the stubs were placed and fixed onto the sample holder and then placed in the sample chamber of SEM.Subsequently, corn silk samples were examined under magnifications of 200×, 1000×, 3500×, 5000×, and 10,000×, respectively.

DNA Extraction
The genomic DNA (gDNA) of all samples was extracted using three different Kits.A brief description of the methods is as follows.
The first kit (Kit 1) used for DNA extraction was QIA-GEN DNeasy Mericon Food Kit (QIAGEN, Germantown, MD, US).Approximately 2 g of each sample was weighed on analytical balance (RADWAG, XA 110/X).After grinding in blender, TissueLyser II and by liquid nitrogen the gDNA was extracted by manufacturers protocol [11].
Secondly, we used Eurofins|GeneScan GENESpin DNA isolation Kit (Kit 2) (Eurofins GeneScan Technologies GmbH, Germany).200 mg of sample was taken from each grinding technique i.e. by blender, TissueLyser II, and liquid nitrogen then 1ml of lysis buffer and 10 µl of proteinase K was added.The protocol of the kit was followed and the DNA was eluted in Tris EDTA (TE) buffer.
The third Kit (Kit 3) PENICON EZ-10 Spin Column Genomic DNA Miniprep Kit (BIO BASIC INC, Canada) was used for the isolation of DNA from a sample that was previously homogenized by the three grinding techniques as described earlier.Approx 100 mg of ground sample was transferred to a 15ml tube.For lysis 600 µl of plant cell lysis buffer with 12 µl of β-mercaptoethanol was followed by 0.6ml of chloroform and centrifugation at 12000×g for 2 min.Following the manufacturer's protocol DNA was eluted in the Tris-EDTA buffer.

Quantification
Purified DNA was quantified by Qubit Fluorometer 3.0 (Invitrogen Life Technologies, US). Qubit™ ds DNA HS standard 1 and Qubit™ ds DNA HS standard S2 were prepared by adding 189 µl of buffer with 1ul of fluorescent dye and 10 µl of each standard provided with the Kit [12].For sample preparation, 198 µl of buffer was dispensed into the tube (Qubit Assay Tubes, Thermo Fisher SCIENTIFIC) containing 1 µl of dye. 1 µl of the extracted DNA sample was added and the tubes were then incubated at room temperature (25 °C) for 1 min and inserted in a Qubit fluorometer for observation.

Agarose Gel Electrophoresis
The quality of the extracted gDNA was accessed on agarose gel electrophoresis for the evaluation of band size.1.5% of Agarose was prepared in 1× TAE (Tris Acetate EDTA) buffer with 3 µl ethidium bromide EtBr (10 mg/ml, Scharlau) [13] using 120 V for 60 min (V/m).Interpretation of results was done by visualizing agarose gel on Gel Documentation (azure Biosystems c150) using Second Generation C series capture software.

Real-Time PCR
For the detection of GMO events, DNAeurofins|GeneScan GMOScreen RT PCR Kit was used.The real-time PCR assays were performed on real-time PCR (QIAGEN Rotor-Gene Q, Germany) under the following conditions: initial denaturation step (10 min at 95 °C), 45 cycles at 95 °C for 15 s, and annealing and extension at 60 °C for 90 s [14].The results were interpreted with the help of QIAGEN rotor gene Q series software (version 2.3.1).

Estimation of Cost and Time
The period (hr.) and expected cost (USD) for extraction of one sample using three Kit methods were estimated.Sample preparation and pretreatment steps were eliminated.Based on the cost of DNA extraction Kits, chemical reagents, enzymes, and disposable items, the cost of each method was calculated.Comprehensive time & cost was also calculated by using the following formula [15].

Scanning Electron Microscopy (SEM)
To understand the granular image of corn silk samples, scanning electron microscopy (SEM) was performed that represents the morphological images of corn silk samples obtained from different grinding techniques.SEM images as shown in Fig. 1a-c revealed that varying the grinding technique leads to different physical appearances with respect to size and shape.Among all, corn silk ground by tissue laser (CSK-TL) displayed the finest appearance, a more compact structure (Fig. 1c).The surface of tissue-lyzed samples was more shiny and smooth.However, CSK-RG showed enlarged fiber particles with clusters and threadlike appearance in Fig. 1a.However, at higher magnification, globular appearance is more obvious on the surface of corn silk in CSK-RG (Fig. 1a).In Fig. 1b (CSK-LN) sample displayed compact like structure at low magnification.However, at higher magnifications rough surface is visible which defines the erosion of particles with liquid nitrogen treatment.Hence it can be suggested that CSK-TL decreased the particle size of the sample.Even at 10,000× the particles shape is less damaged as compared to CSK-LN or CSK-RG.It hence indicates that tissue lyser is the most efficient technique for grinding and homogenization as compared to remaining ones.

Quantification
Quantification of DNA in all samples was done by Qubit Fluorometer 3.0.It is a sensitive technique to measure the concentration of DNA even at the nanogram level.The

=
Expected cost of one method maximum expected cost × Expected time of one method maximum expected time extracted samples were quantified in the range of wavelength from 430-495nm (blue) and 510-580nm (green) to determine the yield and purity of DNA in all samples.A comparison between three homogenization techniques concerning different extraction Kits is presented in table 1.
Based on the results observed, QIAGEN DNeasy mericon Food Kit gave the highest efficacy in all three homogenization methods due to efficient purification with no carryover inhibitors, as compared to other Kit that has lesser potency to extract DNA and minimize inhibitors in samples.
Unlike homogenization by blender and Liquid nitrogen in mortar and pestle, which just disrupts the material, the TissueLyser II performs both disruption and homogenization of corn silk, ensuring that high molecular weight cellular proteins and carbohydrates are not sheared [16].Moreover, tissue-lyzed samples showed more DNA yield as compared to samples that were ground by a normal blender or crushed by Liquid nitrogen because the process of homogenization with tissue lyzer is based on stainless steel jars that converted sample into fine powder that result in high-quality DNA.

Agarose Gel Electrophoresis
The technique of agarose gel electrophoresis helps to separate DNA based on molecular weight.All three samples were loaded on the gel and allowed to run for approximately 1 h at 60 Volts.Afterward, bands were observed as shown in Fig. 2.
All three Kits showed the same band size above the largest band of the ladder which confirms the high molecular weight genomic DNA.Among all Kits, DNA extracted by Kit 1 gave the maximum DNA yield due to which it showed darker bands.

Real-Time PCR
The qPCR technique is one of the most commonly used methods for the detection of genetic material and gene quantification.Reference Material of GMO (CRM) was also run by Real-Time PCR and results are shown in Figs. 3, 4, 5 and 6.This technique gives more precise and accurate results then conventional PCR.35 S promoter, ABII, and Nos terminator were selected as GM events in corn silk [17] [Figs.7, 8, 9 and 10].
After amplification, the GM events showed no calculated threshold (Ct) values in all three samples of corn silk indicating negative results for GMO events 35 S promoter, ABII, and Nos terminator.Whereas, internal positive controls have ct values in each target as mentioned in Figs.

Estimation of Cost and Time
The time and cost of all three Kits was estimated on the basis of extraction time and cost of Kits.As shown   2.
Cost of Kit 1 was higher as compared than other two Kits, as it has more favorable solvents such as lysis buffer that rupture the cell wall to extract DNA or elusion buffer 1 3 that gives higher efficacy.But after time estimation, Kit 3 gave the DNA yield in 1 h due to its less time duration.When both time and cost were analyzed together, Kit 3 showed the most economical outcome followed by Kit 1 and then Kit 2 (Table 2).Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Fig. 4
Fig. 4 ABII= (Agroborder II) GMO target DNA extraction is the most critical step in the field of molecular research.The results quality should be good enough to conduct downstream experiments.In the present study, different Kit based DNA extraction protocols were compared by utilizing the byproduct of corn i.e. corn silk for the detection of GMOs using Real-Time PCR technique.Results

Fig. 7 Fig. 8
Fig. 7 IPC channel of corn silk and corn kernel

Fig. 9
Fig. 9 NOSt channel of corn silk and corn kernel