Sample collection of Cryptotermes domesticus

The C. domesticus specimens used in this study were provided by the National Institute of Ecology. The samples were collected from an apartment in the Seoul metropolitan area, South Korea, specifically from the interior of a sliding door frame infested with termites (Fig. 1). The collected specimens were preserved in alcohol (>95% EtOH) for both morphological and molecular identification of colony members (Lee et al., 2024). The samples were stored under refrigerated conditions until further analysis. For genetic extraction, the abdomen of two soldier termites and three worker termites was excised, and extracted from the remaining body parts.

DNA extraction and sequencing

Genomic DNA was extracted from collected C. domesticus specimens using the Clear-S Quick DNA Extraction Kit (IVT-3002; Invirustech, Gwangju, Korea), following the manufacturer’s protocol. The extracted genomic DNA underwent whole-genome sequencing (WGS) for nucleotide sequence analysis. WGS was performed using the Illumina NovaSeq platform with 150 bp libraries, and the sequencing data were processed using a bioinformatics pipeline developed by Invirustech (Gwangju, Korea).

Primer design for gene amplification and selection

To develop species-specific primers for amplification, this study utilized WGS data to identify candidate target sequences. Primers were designed using Primer3 software (Untergasser et al., 2012) to generate five primer-probe sets per species (Table 1). These primers were specifically designed to amplify unique genetic regions to ensure species specificity. During the design process, primer characteristics were optimized by considering melting temperature (Tm), GC content, and the potential for secondary structure formation.

qPCR assays were performed using the purified genomic DNA extracted from two species as templates. The qPCR reactions were set up in a final volume of 20 µL, which consisted of 10 µL EzAmp^TM HS qPCR 2X Master Mix (SYBR Green, Low Rox), 3 µL of genomic DNA (1 ng/µL), 0.5 µL of each primer (10 pmol), and 6 µL of nuclease-free water. The thermal cycling conditions included an initial denaturation at 95°C for 5 minutes, followed by 45 cycles of denaturation at 95°C for 10 seconds and annealing/extension at 60°C for 30 seconds. To determine the melting temperatures of the primers, a melting curve analysis was performed, with temperature increments of 0.5°C/second from 60°C to 95°C. From the tested primer sets, two were selected based on their robust amplification efficiency and absence of non-specific amplification in closely related species.

Whole-genome sequencing

WGS was performed using the Illumina NovaSeq platform with 150 bp libraries. Paired-end sequencing was applied to obtain high-quality sequence data, which produced reads of 150 bp in length. The sequencing process yielded a total of 285,523,378 raw reads, with quality scores of Q20 (97.31%) and Q30 (92.93%) and a GC content of 39.88%. WGS data for other termite species were obtained from the Sequence Read Archive (SRA) database of NCBI GenBank. Based on the GenomeScope2 analysis, the total genome size of C. domesticus was estimated to be approximately 1.11-1.12 Gb, with repetitive sequences accounting for approximately 37.0-37.4% (~413-415 Mb) of the genome.

Development and selection of species-specific primers

To enhance the detection accuracy of C. domesticus, a TaqMan-based probe assay with high specificity and sensitivity was employed. Initially, genomic DNA was extracted from C. domesticus specimens, followed by WGS to identify species-specific genetic regions. Based on this information, a total of five primer sets were designed and evaluated for amplification efficiency and specificity.

To validate the specificity of the designed primers, qPCR analysis was performed control groups of native and non-native termite species found in South Korea. The control group consisted of the native Reticulitermes speratus, Reticulitermes kanmonensis, and the alien termite species C. gestroi, which has been confirmed to have been introduced into the country.

The validation results showed that all primer sets exhibited high amplification efficiency; however, clear single bands were observed only for Cdomes set 1, set 2, and set 4 (Fig. 2). Notably, Cdomes set 4 caused amplification in other native termite species, indicating a potential for non-specific amplification. In contrast, Cdomes set 1 and set 2 produced sharp bands exclusively in C. domesticus, with no non-specific amplification observed in R. speratus, R. kanmonensis, or C. gestroi. Based on these results, Cdomes set 1 and set 2 were selected as the optimal primer sets for species-specific detection of C. domesticus.

Locus verification

For species-specific detection, it is crucial to select genetic loci that are similar to those of other species but can be clearly distinguished, to ensure the design of accurate primers. Furthermore, to maintain the reliability of the detection method over time, the target gene region must exhibit high conservation across generations and be resistant to mutations (Yan & Slinger, 2011).

In this study, the target genetic loci of the designed primers were evaluated. Due to limited genomic research on C. domesticus, direct verification of the target loci was challenging. Therefore, BLASTx analysis was performed, revealing that the gene sequences showed high amino acid sequence similarity with protein-coding genes from various insect species (Table 2). Based on these results, the selected target gene in this study is likely to be a protein-coding gene.

To reach a more definitive conclusion, further WGS of C. domesticus is necessary. Future genomic studies to verify the function and conservation of the target gene could significantly enhance the reliability of the qPCR-based detection method developed in this study.

Specificity and sensitivity of species-specific primers

In qPCR, the cycle threshold (Ct) value serves as an indicator of the initial quantity of target DNA, with lower Ct values indicating higher amplification sensitivity and the ability to detect even low concentrations of target DNA (Azeem et al., 2023). To evaluate the sensitivity of the species-specific primers, the Ct values for each primer set were measured, and the primer set with the lowest Ct value was ultimately selected as the species-specific marker (Park et al., 2013).

A comparison of the Ct values for the two candidate primer sets (Cdomes set 1 and Cdomes set 2; Fig. 3, Table 3) revealed that Cdomes set 1 showed a lower Ct value (30.804) than Cdomes set 2 (32.100), indicating higher sensitivity in C. domesticus. Furthermore, PCR gel electrophoresis analysis (Fig. 2) showed that Cdomes set 2 generated a faint band in C. gestroi, a non-native termite species confirmed to have been introduced into South Korea, suggesting potential for misidentification under certain conditions. In contrast, Cdomes set 1 produced clearer bands, with no non-specific bands observed in other non-native termite species introduced into South Korea or in native termite species. Based on these results, Cdomes set 1 was selected as the optimal primer set for species-specific detection of C. domesticus.