**3. Results**

### *3.1. Specificity Test of Species-Specific Primers*

The DNA extracts from different honeybees with different geographical origins were used to examine the specificity of the designed species-specific primers. Each primer

set successfully amplified *ND2* region from the DNA samples extracted from *A. cerana*, *A. dorsata,* and *A. mellifera* with an amplicon size of 224, 302, and 377 bp, respectively (Figure 2). None of the non-specific DNA amplification was observed with tested primer sets, suggesting these three species-specific primers can be successfully used to distinguish the origin of the honeybee at the DNA level.

**Figure 2.** Agarose gel electrophoresis of PCR products amplified from DNA extracted of honeybees with species-specific primers. Bee DNA (Lanes 1–3: *A. cerana* DNA, Lanes 4–6: *A. dorsata* DNA, Lanes 7–9: *A. mellifera* DNA) Primers (Lanes 1, 4 and 7: *A. cerana* specific primers AC-F/AC-R, Lanes 2, 5 and 8: *A. dorsata* specific primers AD-F/AD-R, Lanes 3, 6 and 9: *A. mellifera* specific primers AM-F/AM-R), M: 100 bp ladder.

#### *3.2. Sensitivity Test of Primers Using Bee and Honey DNA*

To evaluate the sensitivity of PCR-based assay, DNA samples from 3 different bees (*A. cerana*, *A. dorsata* and *A. mellifera*) were serially diluted (100 to 0.01 ng/μL) and used for PCR. The PCR condition using primer sets showed that all primer sets are able to amplify the specific bands (Figure 3). From the *A. dorsata* DNA, the AD-F/AD-R primer set successfully amplified the band with the expected size (302 bp) (Figure 3A). The intensity of characteristic bands was gradually raised as the concentration of DNA template increased, and the band could be visible when the DNA template was as low as 0.1 ng. From the *A*. *mellifera* (Figure 3B) and *A. cerana* DNA, similarly, AM-F/AM-R and AC-F/AC-R primer sets were also able to amplify specific bands with a detection limit of total 0.1 ng template DNA in the PCR reaction. This suggests that our species-specific primers can be used to detect the origin of honeybee samples with a low amount of DNA.

In other to test the ability to detect the target DNA among pure and adulterated honey samples, AMH was mixed with either ADH or ACH in different proportions. DNA was extracted from pure and mixed honey and used for the subsequent PCR analysis. Importantly, although the same amount of DNA (100 ng) from honey and bee samples was used for PCR, we were not able to detect the specific band from the first round of PCR with DNA from honey, unlike with DNA from bees (Figures 2 and 3). This is likely due to actual amount of bee DNA being lower in DNA extracted from honey, as the honey sample contains biological tissues of other organisms (e.g., plant, microorganism, and other insect tissues). Thus, we performed another round of PCR by using 5 μL of PCR product as a template for analyzing the honey samples. In the second round of PCR, AC-F/AC-R and AD-F/AD-R primer sets produced a single band at the expected size with DNA from 100% ACD (Figure 4A, lane 1) and 100% ADH (Figure 4B, lane 1), respectively. On the contrary, the AM-F/AM-R primer set failed to amplify the band from the DNA extracted from 100% ACH (Figure 4A, lane 2) or 100% ADH (Figure 4B, lane 2). Neither AC-F/AC-R nor AD-F/AD-R primer sets amplified the specific bands from DNA extracted from 100% AMH (Figure 4A,B, lane 11).

**Figure 3.** Sensitivity test of the designed species-specific primers using serially diluted DNA extract of *A. dorsata* (**A**), *A. mellifera* (**B**), and *A. cerana* (**C**). Lane M, DNA marker; Lane 1, 100 ng; lane 2, 10 ng; lane 3, 1 ng; lane 4, 0.1 ng; lane 5, 0.01 ng; lane 6, negative control.

**Figure 4.** Adulteration test with artificially mixed honey samples. PCR products amplified from DNA extracted either from mixtures of ACH and AMH (**A**) or ADH and AMH (**B**) were analyzed by DNA gel electrophoresis. The proportions of AMH inside either ACH or ADH and usage of species-specific primer sets are shown. AC-F/AC-R, *A. cerana* specific primers; AD-F/AD-R, *A. dorsata* specific primers; AM-F/AM-R, *A. mellifera* specific primers; M, 1 kb ladder.

DNA form ACH and ADH with different concentrations of AMH were also tested with species-specific primers (Figure 4A,B, lanes 3–12). In both conditions, the speciesspecific band of for *A. mellifera* was visible when the concentration of AMH was as low as 1%. The intensity of *A. mellifera* species-specific band gradually increased when the DNA from mixed honey with a proportion of 1 to 50% AMH were used and remained constant up to 100% AMH. To examine the possibility of using species-specific primer sets in the practical adulteration assay, 20 honey samples labeled as ADH, ACH, and AMH from different localities were tested. Analysis of the sequences of PCR products indicated that the primer sets are specific enough to detect the entomological origin of honey from different geographical localities.

### *3.3. Melting Curve Analysis by Real-Time PCR*

To evaluate the possibility of use of melting curve analysis for detecting ACH or ADH adulteration, a real-time PCR experiment was conducted using the same PCR condition and primer sets and DNA extracted from honeybees. The result was confirmed using agarose gel electrophoresis and sequencing. Melting curve analysis of real-time PCR products demonstrated two distinct curves allowing the discrimination of *A. dorsata* from *A. mellifera* (Figure 5A) and of ADH from AMH. The melting temperature (Tm) of amplicons generated from *A. dorsata* (69.2 ± 0.1 ◦C) was distinct from the *A. mellifera* (72.4 ± 0.1 ◦C); hence, the detection of Tm could be an alternative method to detect the origin of ADH in addition to standard PCR method. Melting curve analyses of PCR products between *A. cerana* and *A. mellifera* were also performed (Figure 5B). Tm of amplicons of *A. cerana* (71.9 ± 0.2 ◦C) was distinct from *A. mellifera* (72.4 ± 0.1 ◦C) but very similar; hence, the use of Tm for distinguishing *A. cerana* and *A. mellifera* need more caution. The results of one-way ANOVA indicated that there was a significant difference between the Tm values of all three species (F value = 325.2, *p*-value < 0.001).

**Figure 5.** Conventional melting curves obtained by real-time PCR amplification targeting *ND2* region of mtDNA using DNA extracts from honeybees. ( **A**) *A. dorsata* (Tm = 69.2 ± 0.1) and *A. mellifera* (Tm = 72.4 ± 0.1). (**B**) *A. cerana* (Tm = 71.9 ± 0.2) and *A. mellifera* (Tm = 72.4 ± 0.1).
