**1. Introduction**

Honey is a sweet natural product produced by honey bees using the nectar, secretions of living parts, or honeydew of plants [1,2]. Due to the broader geographical distribution, *Apis mellifera* honey (AMH), *A. cerana* honey (ACH), and *A. dorsata* honey (ADH) are the three dominant types of honey in the Asian market. Giant honeybee (*Apis dorsata* F.) is distributed throughout South and Southeast Asia and China [3]. The colonies of *A. dorsata* are generally found in rainforests or on the cliffs, but they also can be occasionally found in building ledges of urban areas. Even though *A. dorsata* is not domesticated and cannot be maintained for honey harvest or pollination purposes, it plays an important role in the pollination of tropical rainforest plants and local crops [4–6]. Due to the fact that *A. dorsata* is considered the most defensive honeybee compared to other *Apis* spp., ADH is harvested by highly motivated experts (so-called honey hunters) [7]. Furthermore, ADH contains the highest concentration of phenolic compounds and flavonoids compared to other honeys, thus exhibiting high DPPH (2,2-diphenyl-1-picrylhydrazy) free radical-scavenging activity, FRAP (Ferric reducing-antioxidant power assay) values and the lowest AEAC (Ascorbic acid Equivalent Antioxidant Capacity) values, as well. This indicates that ADH has strong

**Citation:** Mohamadzade Namin, S.; Yeasmin, F.; Choi, H.W.; Jung, C. DNA-Based Method for Traceability and Authentication of *Apis cerana* and *A. dorsata* Honey (Hymenoptera: Apidae), Using the *NADH dehydrogenase 2* Gene. *Foods* **2022**, *11*, 9285. https://doi.org/10.3390/ foods11070928

 Academic Editor: Alessandra Bendini

Received: 28 February 2022 Accepted: 22 March 2022 Published: 23 March 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

antioxidant properties and medical values [8,9]. Asian honeybee (*A. cerana* F.) is widespread in South, South East, and Eastern Asia from Afghanistan to Far East Russia and Japan [10]. It is one of the domesticated honeybees; however, due to its lower productivity compared to *A*. *mellifera*, most beekeepers prefer *A. mellifera* over *A. cerana* [11]. In addition, high interspecific competition between *A. mellifera* and *A. cerana* on the same niche resulted in the decline of *A. cerana* colonies in many countries such as China and Korea in the last decades [11–13]. This led to lower production of ADH and ACH compared to AMH.

Although honey is one of the most widely consumed natural products, it is one of the most counterfeited food products in the market [14]. Due to the fast growth of the human population and the rising demands toward the consumption of organic and local products, the entomological origin of honey has been taken into consideration. Therefore, the market price of ADH and ACH is several times higher than AMH. This situation makes ADH and ACH vulnerable to adulteration problems, either by mislabeling (claiming the false geographical, botanical, or entomological origin of honey) or by mixing (overfeeding the bees with sugars, adding sweeteners or syrups, and dilution with cheaper honey) in order to increase the economic profit [2,3,15–19].Thus, it is important to develop rapid, reliable, and cost-effective identification methods for the entomological origin of honey to solve the adulteration problem in the market.

Molecular detection of the entomological origin of honey by using the set of specific primers is regarded as a rapid, accurate, and suitable tool for the identification of the origin of animal products and processed foods [20–23]. Considering the method of processing honey by honeybees, the bee cells can remain inside of the honey. Given the opportunity to extract bee DNA from honey, it is possible to use it for the identification of the entomological origin of honey. Compared to the other identification methods for entomological origin of honey, such as SDS-PAGE or chemical-based methods [12,24,25], the DNA-based method is more precise, quick, and suitable for analysis of a large sample size [26]. Recently, several studies were conducted using DNA-based methods to identify the entomological origin of honey. Zhang et al. [26] developed a gDNA-based method for the identification of two different major honeys, AMH and ACH, in the market. Two sets of primers were designed to amplify *Major royal jelly protein 2* (*MRJP2*) gene, resulting in the different sizes of PCR product in the gel electrophoresis, making it useful to discriminate ACH from AMH. In addition, it is also possible to identify the honey samples through Real-Time PCR based on their melting temperature analysis [26].

Mitochondrial DNA (mtDNA) is present in most cells with high copy numbers. It is characterized by a high genetic variation between related species but a low intraspecific variation [27–29]. Therefore, it is suitable to use mtDNA for taxonomic and phylogenetic analysis. Targeting the *cytochrome oxidase I* (*COI*) gene of mtDNA, Kim et al. [30] designed species-specific primers to differentiate ACH and AMH. PCR with designed primer sets produced amplicons with a length of 133bp and 178 bp for *A. mellifera* and *A. cerana*, respectively. Although the size of the amplicons is distinguishable and even applicable for relatively old honey samples, Zhang et al. [26] reported the designed primers for *A. mellifera* was not species-specific as they made the same length of band from ACH-originated DNA extracts in China. Soares et al. [31] developed species-specific primers to amplify the intergenic region of tRNAleu-cox2, enabling the detection of *A. cerana* DNA using PCR. In addition, they discriminated ACH and AMH using high-resolution melting curve analysis targeting the 16S rRNA gene, making it possible to detect the entomological origin of ACH. However, the lack of species-specific primer designed for mtDNA of *A. mellifera* makes it difficult to use it for adulteration studies. The only species-specific primer set that is available to detect *A. mellifera* is provided by Zhang et al. [26]. However, the size of the PCR product (~560 bp) is largely applied to relatively old honey samples due to DNA degradation, and it is important to design species-specific primers targeting smaller regions. On the other hand, there is no species-specific primer available for reliable and cost-effective identification of the entomological origin of ADH.

In this study, we aimed to develop a rapid and accurate PCR-based method to recognize the entomological origin of ADH, ACH, and AMH. This method can also be applied to discriminate between pure and adulterated honey. We also aimed to provide speciesspecific primers targeting smaller parts of mtDNA to avoid the negative effect of possible DNA degradation, which may happen during the storage of honey. In this study, three species-specific primers for ADH, ACH, and AMH were designed to amplify the short part of the *NADH dehydrogenase 2* (*ND2*) region of the mtDNA. Our experiment suggests that species-specific primer sets targeting *ND2* not only successfully distinguished ADH, ACH,s and AMH, but also detected mixed 1% AMH from ADH or ACH. Additionally, several honey samples from different countries were used to evaluate the accuracy of the developed method.

### **2. Material and Methods**

### *2.1. Schematic Overview of the Experimental Design*

In this study, 3 species-specific primer sets were designed to test the traceability of the entomological origin of honey. The specificity and sensitivity of the primers were tested first with the DNA extracts from honeybees with different geographical origins. Then, the DNA extracts from artificially mixed honey samples were used to evaluate the applicability of using designed primer sets in honey authentication. Subsequently, the developed method was used to check the entomological origin of honey provided by honey hunters and beekeepers (Figure 1).

**Figure 1.** Schematic overview of the experimental design.

### *2.2. Designing Species-Specific Primers*

*NADH dehydrogenase 2* (*ND2*) region of mitochondrial DNA was used as a target area. The complete mitochondrial genome sequence of *A. cerana*, *A. dorsata* and, *A. mellifera* were obtained from NCBI (Table S2) and used for designing species-specific primers using OLIGO 7 primer analysis software (Table 1). The Primer-BLAST tool was initially used to determine primer specificity (http://www.ncbi.nlm.nih.gov/tools/primer-blast accessed on 25 March 2020). Designed primers were synthesized by Macrogen (Daejan, Korea).


**Table 1.** Specific-primers for honey identification, nucleotide sequence, primer length, and the expected length of PCR product.

#### *2.3. Evaluate the Specificity and Sensitivity of Designed Primer Sets Using Bee DNA*

### - DNA extraction from honeybees

DNA extracts from adult or larvae samples of honeybees of *A. cerana* (5 adults from Nepal, 5 adults from Korea), *A. dorsata* (5 larvae from Thailand, 5 adults from Nepal) and *A. mellifera* (5 adults from Nepal, 5 adults from Korea) were used to test the specificity and sensitivity of the primer sets. Bee DNA was extracted from the left hind leg of adult honeybee or head and thorax part of larvae using DNeasy blood and tissue kit (Qiagen, Hilden, Germany) following manufacturer's instruction.

### - Specificity and sensitivitytest of designed primers

The DNA extracts from honeybees with different geographical origins were used to examine the specificity of the designed species-specific primers. The PCR procedure was carried out in a 20 μL reaction volume mixture containing 100 ng of template DNA and 1μL of each primer (10 pmole/μL) using AccuPower PCR PreMix (Bioneer, Daejan, Korea). The thermal cycling procedure contained an initial pre-denaturation at 95 ◦C for 5 min, and 35 cycles of 95 ◦C for 30 s, 52 ◦C for 30 s, 72 ◦C for 40 s, and a final extension of 72 ◦C for 5 min in a BIOER thermal cycler. 7 μL of PCR products were analyzed using 2.5% agarose gel in TAE buffer and the bands were visualized by EcoDye (BIOFACT) and gel document system (GSD-200D).

To evaluate the detection limit of the species-specific primers, DNA extracts from different bee samples were serially diluted by 10-fold (100 to 0.01 ng/μL) and used for PCR analysis.


DNA extract from honeybees were used to evaluate the possibility of using real-time PCR-based detection of adulteration of ACH and ADH. The real-time PCR was carried out using 10 μL of 2X Real-Time PCR Master Mix (BioFACT) including SYBR Green I, 100 ng of DNA template and 1 μL of each primer (10 pmole/μL) in 20 μL of total reaction mixture. PCR cycling was as follows: 95 ◦C for 15 min, following 35 cycles of 95 ◦C for 30 s, 52 ◦C for 30 s and 72 ◦C for 40 s. For analyzing melt curve, real-time PCR products were denatured at 95 ◦C for 15 s, annealed at 52 ◦C for 1 min then followed by melting curve ranging from 52 to 95 ◦C with temperature increments of 0.3 ◦C every 20 s. The data of real-time PCR and melt curve analysis were processed using FQD-96a V1.0.13 software (BIOER, Hangzhou, China).

### *2.4. PCR-Based Sensitivity Test of Honey Samples*


Three pure honey samples (ACH and AMH from Korea and ADH from Thailand) were used to test the sensitivity of species-specific primers to detect honey adulteration. AMH was mixed with ACH or ADH in different proportions (100:0, 99:1, 95:5, 50:50, 20:180, 0:100), and then used for DNA extraction.

### - DNA extraction from honey samples

To extract DNA from honey samples, 40 mL distilled water was added to 15 g of honey, incubated at 45 ◦C for 30 min, vortexed and centrifuged at 15,000 rpm for 30 min. The supernatant was discarded, and the pellet was dissolved in 1 mL of distilled water and centrifuged at 15,000 rpm for 15 min. The supernatant was discarded, and the pellet was used for DNA extraction using DNeasy mericon Food Kit (Qiagen, Hilden, Germany) following manufacturer's protocol. The concentration and purity of the DNA extracts were evaluated using Nano Drop spectrophotometer (Life Real). Extracted DNA was used for the subsequent PCR analysis.

### - Polymerase chain reaction (PCR)

For sensitivity (or adulteration) test, 2 rounds of PCR were performed with same primer sets. In the first round of PCR, 100 ng of DNA was used as a template DNA following the same protocol described above. Then, 5 μL of PCR product was used as a template DNA for the second round of PCR with the same protocol described above. A total of 7 μL of final PCR products were analyzed in 2.5% agarose gel. In addition, PCR using DNA extracts from honey samples (10 with ACH and 5 with ADH labels) was conducted for honey adulteration test using the species-specific primers. Two rounds of PCR were performed as described above. To confirm the amplified DNA sequence, PCR products were analyzed in 2.5% agarose gel, purified and sequenced by Macrogen (Daejan, Korea) using an ABI 3130xl capillary automated.

### *2.5. Adulteration Analysis of the Honey Samples*


The purity of 20 honey samples (10 ACH, 5 ADH and 5 AMH) from different localities (Nepal, Thailand and Korea) were evaluated. ACH samples were provided by beekeepers from Nepal (*n* = 2), Thailand (*n* = 5) and Korea (*n* = 3). ADH samples were provided by honey hunters from Thailand (*n* = 5). AMH samples were harvested directly by beekeepers from Korea (*n* = 5) (Table S1). All samples were collected in 2020. Honey samples were stored at −20 ◦C and 4 ◦C prior to DNA extraction, respectively.


DNA was extracted from all honey samples using the method that was described before. PCR using DNA extracts from honey samples (10 with ACH and 5 with ADH labels) was conducted to check honey adulteration using the species-specific primers. There were 100 ng of DNA used for the first PCR and 5 μL of PCR product used as a template DNA in the second PCR following the procedure described before. PCR products were analyzed in 2.5% agarose gel and sequenced by Macrogen (Daejan, Korea) using an ABI 3130xl capillary automated. All sequences were generated in both directions and the forward and reverse sequences were assembled in BIOEDIT v7.0.5.2 (Hall, 1999) to produce a consensus sequence for each sample and the assembled sequences generated in this study were used to confirm the identification through DNA barcoding and have been deposited in GenBank under accession numbers MW660861-MW660880.


From melting curve analysis, melting temperatures for 3 species of honey bees were compared by one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test. *p* values less than 0.05 were considered to be statistically significant. The statistical analysis was conducted using The R project software version 4.0.5 [32].
