The experiments were conducted during 2021–22 in the Entomology Laboratory of the Crop Protection Division at ICAR–Indian Institute of Wheat and Barley Research (IIWBR), Karnal, India. Disease- and insect-free wheat germplasm of the cultivar HS490, essential for the study, was obtained from the Germplasm Resources Unit (GRU) at ICAR–IIWBR. Prior to experimentation, the grains were thoroughly cleaned and inspected to eliminate any damaged kernels, thereby ensuring the absence of contamination.

The initial stock population of S. oryzae adults used in the study was sourced from the experimental laboratory of ICAR–IIWBR, Karnal, and subsequently reared for several generations on healthy wheat grains in the Insect Rearing Room of the Entomology Laboratory. For experimental purposes, the S. oryzae culture was maintained on undamaged wheat grains stored in 1-liter glass jars (20 cm height x 5 cm diameter) under controlled environmental conditions within a Biochemical Oxygen Demand (BOD) incubator, set at a temperature of 28 ± 2°C and relative humidity of 70 ± 5%.

Genomic DNA was extracted from adult S. oryzae individuals (used as positive controls) obtained from the maintained pure culture. Insect tissues were lysed using TNES buffer (comprising Tris-HCl, NaCl, EDTA, and SDS) in a microtube homogenizer. The resulting DNA pellet was resuspended in 1% TE (Tris-EDTA) buffer. To eliminate RNA contamination, 3 µl of RNase solution (10 mg/ml) was added to each sample, followed by incubation in a water bath at 37°C for 30 minutes. Additionally, genomic DNA was isolated by crushing the whole grains (5 g) of the wheat genotype HS490 and then following modified CTAB (cetyltrimethylammonium bromide) extraction protocol, based on the method described by Saghai-Maroof et al. (1984), with slight modifications.

Furthermore, genomic DNA was also isolated from contaminated wheat grains. For contaminating the wheat grains, two hundred gram of wheat grains, in triplicate, after conditioning, were maintained at 25 ± 2°C and 65 ± 5% relative humidity in round plastic containers of capacity 500 grams. One hundred pair of S. oryzae adults, were transferred in each container holding the grains and was covered with muslin cloth and tightened using rubber bands. Contaminated and infested grains were sub-sampled (5 g) for DNA isolation at 30 days after infestation through the same modified CTAB extraction method, resulting in contamination level of 1000 insects in one kg of grains.

The quantity and purity of DNA was tested using BioDrop Touch PC + Spectrophotometer (BioDrop, Cambridge shire, UK) by loading 1 μl sample of stock DNA. It was later diluted to prepare the working solution of 50 ng/μl concentration using the NFW (Nuclease Free Water) for further PCR based assays.

For molecular identification of S. oryzae, genomic DNA of the insects was amplified in vitro in thermocyclers using already reported and available specific primers from the literature. Initially, four different primers (SOF1-SOR1, SOF2-SOR2, SOF3-SOR3, and SOF4-SOR4) were assessed for S. oryzae, that amplified the cytochrome oxidase (COX) gene of S. oryzae ( Table 1 ). These primers were then used to amplify COI gene from extracted DNA of adult insects isolated from infested wheat grain samples.

The PCR amplification was performed in a total reaction volume of 10 µL, comprising 1 µL of template DNA, 1 µL of primer mix (containing equimolar concentrations of both forward and reverse primers, each at 10µM concentration), 5 µL of GoTaq^® G2 Green Master Mix (Promega), and 3 µL of nuclease-free water (NFW). The thermocycling conditions included an initial denaturation step at 95°C for 5 minutes, followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at the primer-specific annealing temperature (Ta) for 30 seconds, and extension at 72°C for 1 minute. A final extension was carried out at 72°C for 10 minutes, and the amplified products were subsequently held at 4°C. Reactions were conducted using a Q Cycler 96 thermal cycler (Hain Life Sciences, UK).

The resulting PCR products were resolved by electrophoresis on a 2% agarose gel prepared in 1× TBE (Tris-Borate-EDTA) buffer and stained with ethidium bromide at a concentration of 0.5 mg/mL. Electrophoresis was carried out at 90 V for 45 minutes. DNA bands were visualized under ultraviolet (UV) illumination, and fragment sizes were estimated by comparing the banding patterns to expected sizes based on the primer specifications.

The sequencing of the amplified PCR products was conducted to confirm the identity of the target insect pest species by analyzing the nucleotide composition of the cytochrome oxidase (COX) gene region. This process was carried out through a commercial sequencing service provided by Eurofins Genomics India Pvt. Ltd. The resulting amplicons were subjected to Sanger sequencing to generate precise DNA sequences of the targeted COX region. To verify the identity of the amplified sequences and ensure species-specific amplification, the obtained sequences were analyzed using the Basic Local Alignment Search Tool (BLAST) available on the National Center for Biotechnology Information (NCBI) website (https://www.ncbi.nlm.nih.gov/tools/primer-blast), using default parameters. The sequences were compared with publicly available COX gene sequences in the NCBI database to confirm their alignment with the cytochrome oxidase gene of S. oryzae and to check for any homology with unrelated species. This comparative analysis helped validate the specificity and accuracy of the amplified region. Species-specific primers for S. oryzae were designed by aligning COX gene sequences from S. oryzae and other insect species to identify conserved and unique regions. The alignment was performed using MEGA 11 (Molecular Evolutionary Genetics Analysis) software. The primary objective of this alignment was to identify conserved motifs within the COX gene suitable for designing primers capable of selectively amplifying S. oryzae DNA without cross-reactivity with non-target species. This strategy facilitated the development of new primers from conserved regions that allowed for precise and extended amplification of the COX gene, which in turn enabled reliable species-level identification following sequencing.

The newly designed forward and reverse primers were synthesized and procured from Eurofins Genomics India Pvt. Ltd. These primers were then used in subsequent PCR assays for species-specific amplification and molecular identification of S. oryzae in wheat grain samples.

The PCR amplification was carried out in a total reaction volume of 25 µL, prepared by combining 1 µL of template DNA (at a concentration of 50 ng/µL), 2 µL of custom-designed primer mix (comprising equal volumes of forward and reverse primers, each at 10 µM concentration), 12.5 µL of GoTaq^® G2 Green Master Mix (Promega), and 9.5 µL of nuclease-free water (NFW). A no-template control (NTC), containing all reaction components except the DNA template, was included to detect any contamination or nonspecific amplification. PCR amplification was performed using a Q Cycler 96 thermal cycler (Hain Life Sciences, UK). The thermocycling conditions included an initial denaturation at 95°C for 5 minutes, followed by 35 amplification cycles consisting of denaturation at 94°C for 30 seconds, primer annealing at 56°C for 30 seconds, and extension at 72°C for 1 minute. The final extension was carried out at 72°C for 10 minutes, after which the reaction mixtures were held at 4°C until further analysis. To confirm the amplification of the target cytochrome oxidase (COX) gene region, the PCR products were subjected to agarose gel electrophoresis. A 2% agarose gel was prepared in 1× TBE (Tris-Borate-EDTA) buffer and stained with ethidium bromide at a final concentration of 0.5 mg/mL. The gel was run at a constant voltage of 90 V for 45 minutes. The resulting DNA bands were visualized under ultraviolet (UV) light using a gel documentation system. Band sizes were estimated by comparing the migration pattern of the PCR amplicons to a standard 100 bp DNA ladder (Bangalore Genie, India), which served as the molecular weight reference. The presence of specific bands at expected sizes confirmed successful amplification of the target COX gene fragment.

To verify the specificity of the designed primers, genomic DNA was also extracted from eight distinct unrelated species, including R. dominica; Tribolium castaneum; Tribolium confusum; Callosobruchus chinensis; Oryzaephilus surinamensis; Lasioderma serricorne; Corcyra cephalonica; and aphid, Raphalosiphum maidis using the previously described method.

Specificity of the designed primers towards S. oryzae was confirmed in standard PCR reaction as mentioned above by amplifying the designed primers using pure DNA of S. oryzae adults collected in eight different lots from the local market, contaminated and uncontaminated wheat grains and eight different unrelated insect species. NTC was kept for the experiment to check the specificity of the reaction. The PCR reaction cocktail, master mixture, thermal cycler profile, and electrophoretic conditions were similar as described earlier. This assay was replicated twice for confirmation.

The sensitivity of the primers was evaluated using real-time PCR (qPCR). To detect S. oryzae contamination, DNA samples were extracted from both contaminated and uncontaminated wheat grains. This approach enabled quantitative assessment of S. oryzae infestation. Serial dilutions of the DNA were prepared, ranging from 10 ng/μl to 0.1 pg/μl, to assess the sensitivity of the primers. These DNA dilutions were used as templates for the qPCR experiments. Additionally, a NTC and DNA from healthy, uncontaminated grains were included to ensure specificity of the reactions. Pure S. oryzae DNA served as the positive control, while the NTC acted as the negative control. The DNA-binding fluorescent dye SYBR Green, in combination with the primers, provided high detection specificity during qPCR.

The qPCR reaction was carried out in a total volume of 20 µl, containing 1 µl of template DNA, 2 µl of the designed primer mix (equal volumes of forward and reverse primers), 10 µl of SYBR Green dye (Promega GoTaq^® G2 Green), and 7 µl of nuclease-free water (NFW). The reaction was conducted without DNA template for the NTC. The amplification protocol followed the same parameters as the conventional PCR experiments, and the reactions were performed using a Q Cycler 96 thermal cycler (Hain Life Sciences, UK). Cycle threshold (Ct) values were obtained for each DNA dilution. These results were validated by performing a melting curve analysis and constructing a standard curve. The efficiency of the qPCR was calculated by plotting Ct values against the logarithmic scale of DNA concentrations (in g) and determining the regression equation from the resulting graph.

Concerning the amount and quality of DNA analysis using the BioDrop Touch PC + Spectrophotometer (BioDrop, Cambridge shire, UK), the majority of positive control DNA samples of test insect fell between 500 and 800 ng/μl, whereas the DNA extracted from grain samples fell between 800 and 2 μg/μl.

Based on the multiple sequence alignment of S. oryzae accessions with sequences of other wheat-infesting insects available in the NCBI database, conducted using nucleotide BLAST and MEGA 11 software, two distinct primer sets were designed ( Table 2 ). These two sets of forward and reverse primer pairs (KNSoCox1F1/KNSoCox1R1 and KNSoCox2F1/KNSoCox2R1) lie within the COI and COII region of S. oryzae, respectively.

In order to assess the KNSoCox1F1/KNSoCox1R1 and KNSoCox2F1/KNSoCox2R1 primer pairs efficacy, the genomic DNA extracted from eight distinct isolates of S. oryzae collected from the local market, the genomic DNA of contaminated and uncontaminated wheat grains, and the genomic DNA of eight distinct unrelated species ( Table 3 ) were used as templates for the PCR assay. NTC was kept for the confirmation.

The chances of cross-species amplification for the developed PCR assay designed to detect S. oryzae infestation were negated by PCR-based amplicon generation ( Figures 1 , 2 ). It showed that each set of the designed primers produced only a single band of 176 bp and 248 bp from the DNA of S. oryzae and contaminated grains, but not from the other 8 unrelated insect species, uncontaminated grains and NTC.

The specificity of the designed primers for S. oryzae DNA and S. oryzae-infested wheat grains was assessed using a standard PCR reaction. S. oryzae DNA served as the positive control, and DNA with no template was used as the negative control. The results were verified through agarose gel electrophoresis, which displayed the amplified PCR products. The findings showed that primer 1 (KNSoCox1F1/KNSoCox1R1) targeting the COX I region and primer 2 (KNSoCox2F1/KNSoCox2R1) targeting the COX II region of S. oryzae DNA specifically bound at 176 bp and 248 bp, respectively, for both S. oryzae and contaminated wheat grains. No bands were observed for DNA extracted from uncontaminated grains or from eight other unrelated insect species ( Figures 1 , 2 ). Consequently, these primers did not amplify DNA from insect-free grains or other insects. Additionally, the assay was repeated twice, and no significant variation in the results was found.

Therefore, the two primers that were found specific to S. oryzae were further tested through quantitative PCR (qPCR) to check the sensitivity of the primers by using different dilutions.

Real-time PCR was used to detect S. oryzae grain contamination using DNA samples isolated from contaminated and uncontaminated wheat grains. This approach makes it possible to assess the presence of S. oryzae infestation quantitatively. It makes it possible to even detect the S. oryzae DNA sample having infestation level of one insect per 10 kg of contaminated wheat grains for both the primers (Primer 1- KNSoCox1F1/KNSoCox1R1 and Primer 2- KNSoCox2F1/KNSoCox2R1). Ct (Cycle threshold) values were obtained for all these samples (Primer 1 & 2: Table 3 ). The Ct is defined as the cycle at which PCR enters the exponential phase and the fluorescence emission exceeds the threshold limit. The results were confirmed using standard curve (Primer 1: Figure 3 ; Primer 2: Figure 4 ) and melting curve (Primer 1: Figure 5 ; Primer 2: Figure 6 ) analysis. The efficiency of the qPCR was computed by graphing Ct values against -log (DNA concentration in g) and figuring out the regression equation.