**4. Discussion**

Although previous attempts based on DNA barcoding of 16S rRNA and *COI* genes were helpful to inspect mislabeling [33], it was not functional to detect honey adulteration. In spite of the availability of species-specific primers to differentiate ACH from AMH [30], the primers developed by Kim et al. [30] were only applicable to honey originated from Korea but failed to differentiate ACH and AMH originated from China [26]. Since Soares et al. [31] only developed species-specific primers (AC1-F/AC1-R) to amplify

111 bp of tRNAlux-cox2 intergenic region of *A. cerana* mtDNA, the new *A. mellifera* speciesspecific primers were needed for the adulteration test. In addition, although AC1-F/AC1-R primers were useful in the discrimination of ACH from AMH, unlike ACF2/ACR2, they also amplified the non-specific band from ADH DNA extract, suggesting AC1-F/AC1-R was not enough to distinguish ACH from ADH (Supplementary Figure S1). In this study, we provided not only the first species-specific primer set to identify ADH, but also two new species-specific primer sets to identify ACH and AMH. Notably, our newly designed primers successfully amplified specific bands only from the targeted DNA sample and were able to discriminate both ACH and ADH from AMH, and vice versa, thus providing new DNA-based assay for testing entomological origin of honey (refer to Figure 4A,B).

Zhang et al. [26] developed a gDNA-based method for the identification of two different major honeys from domesticated honeybees in the market. Two sets of primers (C-F/C-R for *A. cerana* and M-F/M-R for *A. mellifera*) were designed to amplify the Major royal jelly protein 2 (MRJP2) gene, resulting in the different size of PCR product in the gel electrophoresis, making it useful to discriminate ACH from AMH. In addition, it is possible to identify the honey samples through real-time PCR-based Tm analysis. Although the predicted size of the PCR product was 212 bp for *A. cerana* and 393 bp for *A. mellifera*, but the length of amplicons for *A. mellifera* was 560 bp from the PCR, as the primers were designed based on complementary DNA (cDNA) without an intron. ACH and AMH samples were distinguishable using C-F/C-R and M-F/M-R primer sets; however, 560 bp tends to be long for accurate honey identification and adulteration test with relatively old honey samples, which can possibly have DNA degradation problems. Honey is a complex matrix, and its phenolic/H2O2 induced oxidative stress would lead the DNA that remained inside honey to be easily degraded as storage time increased [31,34–36]. Notably, Schnell et al. [37] reported the diminished rate of successful amplification of amplicon size larger than 380 bp and in the fragmented DNA, thus the amplification of short amplicons is preferable [38]. Thus our new species-specific primer sets with amplicon size ranging 224~377 bp (refer to Figures 2 and 3) would provide a better chance to successfully examine the old honey samples. Although the speed of degradation of DNA inside honey is not well understood and very difficult to predict accurately as different honey have different biochemical compositions, DNA degradation problem needs to be considered while examining the entomological origin of honey via DNA-based assay.

Real-time PCR-based identification of the entomological origin of honey was successfully developed previously to discriminate ACH and AMH using species-specific primers [26,31]. Refer to the Figure 5, the primer sets developed in the present study can be used to differentiate the entomological origin of three different types of honey. Although ADH and AMH can be simply differentiated using melting curve analysis, this method should be applied to differentiate ACH and AMH with caution due to the close Tm of the amplicons. Tm-based identification method is quick and accurate without the requirement of the gel electrophoresis step. Thus, it will provide a possible high-throughput analyses method for the identification of the origin of honey.

The cost of conducting analysis for one honey sample using the combination of two-round PCR and subsequent gel electrophoresis using the methodology described in current research is 6.4\$ per honey sample (DNA extraction kit, PCR master mix, agarose powder, ladder, TBE buffer, staining dye and primer cost), however, these expenses for authentication analysis using Real-Time PCR technic is about 5.2\$ (DNA extraction, master mix, and primer cost). The electricity and labor cost required to run the equipment have not been considered in our calculation. Although the cost of the authentication analysis per sample is slightly lower using Real-Time PCR, it is more expensive to establish such facilities in comparison to the conventional PCR method. Furthermore, according to our calculations, the duration of analysis using Real-Time PCR is slightly longer (~35 min) than conventional PCR.
