A method for quickly identifying the bee species origin of honey from six species of honey bees

Abstract

The present invention provides a method for quickly identifying the bee species sources of six bee honeys of the genus Apis. It is achieved by the following steps: (1) collecting honeys from different bee species; (2) extracting genomic DNA of honey samples; (3) amplifying partial fragments of the mitochondrial 16S rRNA gene of the honey bee using universal primers, and the target fragment length is 470-479bp; (4) using a single restriction endonuclease AseI to quickly digest the above PCR product; (5) the digestion product is subjected to 4% agarose gel electrophoresis and then subjected to chromatographic analysis in a fluorescent imaging system. The present invention adopts PCR-RFLP method, which is simple, fast, reliable and highly specific. It can simultaneously identify the honey produced by six kinds of bees (Apis mellifera, Apis cerana, black large bees, large bees, small bees and black small bees), and has great application and promotion value.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biological detection, and relates to a method for rapidly identifying the bee species sources of honey of six bee species of the genus Apis, and is a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) detection method for distinguishing honey produced by different bee species. Technical Background

[0002] Honey bees (Apis spp.) are not only pollinators of crops, but also produce honey and other bee products. The Chinese honey bees (Apis cerana cerana, referred to as Chinese honey bees) and the Italian honey bees (Apis mellifera ligustica, referred to as Italian honey bees) are the representative bee species of the oriental honey bees and the western honey bees, respectively. They are the two largest bee species raised in China and the most widely raised. According to statistics, the annual honey production of the Chinese honey bees is 50,000 to 100,000 tons, and the annual honey production of the Italian honey bees is about 400,000 tons. However, the black honey bees (Apislaboriosa), giant honey bees (Apis dorsata), small honey bees (Apis florea) and black small honey bees (Apisandreniformis) are not artificially domesticated and are in a wild state. The honey they produce is collectively called wild honey. In China, the black honey bees are distributed in southern Tibet, western and southern Yunnan, and southern Guangxi. The giant honey bees are mainly distributed in southern Yunnan, southern Guangxi and Hainan Province. The small honey bees are distributed in southern Yunnan and southern Guangxi, while the black small honey bees are only distributed in southern Yunnan.

[0003] The quality characteristics and nutritional functions of honey are closely related to the nectar plants, bee species, geographical origin, climate, etc. Scarcity makes things valuable, and because wild honey has high biological activity, low yield and difficulty in obtaining, the price of wild bee honey continues to rise. With people's pursuit of high-end honey and wild honey, the current market price of Chinese bee honey is 80-200 yuan / 500g, Italian bee honey is 30-50 yuan / 500g, black big bee honey (cliff honey or rock honey) and big bee honey (big hanging honey) are 150-400 yuan / 500g, small bee honey and black small bee honey are often called small hanging honey, and the price is 100-300 yuan / 500g. Driven by profit, the phenomenon of using low-priced Italian bee honey to impersonate or mix with high-priced bee honey has also emerged.

[0004] At present, the methods for identifying bee species in honey are mainly based on proteomics (Cai Dongmei. Identification and Quantification of Animal-derived Proteins in Honey Based on Liquid Chromatography Tandem Mass Spectrometry [D]. Chinese Academy of Agricultural Sciences, 2021.) and metabolomics methods (such as a method for distinguishing Chinese bee honey from Italian bee honey with application number CN202011120746.8; WANG X, ROGERS KM, LI Y, et al. Untargeted and Targeted Discrimination of Honey Collected by Apis cerana and Apis mellifera Based on Volatiles Using HS-GC-IMS and HS-SPME-GC–MS [J]. Journal of Agricultural and Food Chemistry, 2019, 67(43): 12144-12152; WANG X, LI Y, CHEN L, et al. Analytical Strategies for LC-MS-Based Untargeted and Targeted Metabolomics Approaches Reveal the Entomological Origins of Honey[J].Journal of Agricultural and Food Chemistry,2022,70(4):1358-1366.). These two omics methods often rely on liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry, which are not only costly but also have high requirements on instrument performance and operators, and have limitations in practical applications.

[0005] DNA is a macromolecule that contains all the genetic information of an organism. Technologies based on polymerase chain reaction (PCR) play an important role in food traceability. At present, the methods for identifying the origin of honey bee species by relying on PCR technology include designing bee species-specific primers (such as the PCR method and application for identifying Chinese bee honey and Italian bee honey with application number CN202111079089.1; ZHANG YZ, WANG S, CHEN YF, et al. Authentication of Apis cerana Honey and Apis mellifera Honey Based on Major Royal Jelly Protein 2Gene[J]. Molecules, 2019, 24(2): 289; MOHAMADZADE NAMIN S, YEASMIN F, CHOI HW, et al. DNA-Based Method for Traceability and Authentication of Apis cerana and A.dorsata Honey(Hymenoptera:Apidae), Using the NADH dehydrogenase 2Gene[J]. Foods, 2022, 11(7): 928.), real-time fluorescence quantitative PCR combined with melting curve analysis (SOARES S, GRAZINA L, MAFRA I, et al. Novel diagnostic tools for Asian (Apis cerana) and European (Apis mellifera) honey authentication [J]. Food Research International, 2018, 105: 686-693.) and DNA barcode technology (KEK SP, CHIN NL, TAN SW, et al. Molecular Identification of Honey Entomological Origin Based on Bee Mitochondrial 16S rRNA and COI Gene Sequences [J]. Food Control, 2017, 78: 150-159.). Bee species-specific primers can only identify the target bee species and are prone to false negatives. Both real-time fluorescence quantitative PCR and DNA barcode technology have high requirements for instruments and are expensive.In addition, many studies focused on identifying honey from Chinese bees and Italian bees, and a small number of studies focused on honey from large bees and small bees. However, there is currently no method that can simultaneously identify the honeys of the six major bee species in my country. Therefore, there is an urgent need for a simple, fast and low-cost method that can simultaneously identify the honeys of the six major bee species. Summary of the invention

[0006] The invention aims to provide a method for quickly identifying the bee species sources of honey of six bee species of the genus Apis.

[0007] To achieve the above object, the present invention is implemented through the following technical solutions:

[0008] (1) Extracting genomic DNA from honey samples;

[0009] (2) using 16S rRNA-F (SEQ ID NO.1) and 16S rRNA-R (SEQ ID NO.2) as primers and the genomic DNA obtained in step (1) as a template, performing PCR amplification to obtain a 16S rRNA gene fragment with a product size of 470-479 bp;

[0010] (3) using AseI restriction endonuclease to digest the 16S rRNA gene fragment obtained in step (2);

[0011] (4) Perform agarose gel electrophoresis on the enzyme cleavage products, and photograph and analyze the electrophoresis results.

[0012] Furthermore, in step (1), the pretreatment before extracting DNA from the honey sample is to first dilute the honey with distilled water, vortex to mix, incubate at 45°C for 10 minutes, centrifuge at 10,000 r / min for 30 minutes, and discard the supernatant; then suspend the precipitate in ultrapure water, centrifuge at 12,000 r / min for 15 minutes, discard the supernatant, and store the precipitate at -80°C until DNA extraction.

[0013] Furthermore, in step (2), the PCR amplification program parameters are set as follows: 94°C, 2 min; followed by 30 cycles of amplification: 94°C, 30 s; 50°C, 30 s; 72°C, 30 s; and finally 72°C for extension for 5 min.

[0014] Furthermore, in step (3), the conditions for AseI digestion of the 16S rRNA gene fragment obtained in step (2) are 37° C. for 2 hours.

[0015] Furthermore, in step (4), the electrophoresis result having both 414bp and 60bp bands is Italian bee honey, the electrophoresis result having both 219bp, 202bp and 54bp bands is Chinese bee honey, the electrophoresis result having both 217bp, 125bp, 60bp, 38bp and 30bp bands is black honey bee honey, the electrophoresis result having both 175bp, 128bp, 60bp and 46bp bands is big bee honey, the electrophoresis result having both 249bp, 165bp and 60bp bands is small bee honey, the electrophoresis result having both 250bp and 229bp bands is black small bee honey.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention can quickly and simultaneously distinguish honey produced by six species of bees of the genus Apis (Apis mellifera, Apis cerana, black giant bee, giant bee, miniature bee and black miniature bee), can overcome the problems of false negative results easily caused by bee species-specific primers and high instrument requirements and high costs of technologies such as DNA barcode sequencing, etc., has the advantages of simplicity, economy and high specificity, and provides a new method for identifying the bee species source of honey. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The following are gel electrophoresis images of PCR products from six bee species.

[0019] Figure 2 This is the gel electrophoresis diagram of PCR products from honey of six bee species.

[0020] Figure 3 This is the gel electrophoresis diagram of the enzyme digestion products of six bees.

[0021] Figure 4 This is the gel electrophoresis diagram of the enzymatic digestion products of honey from six types of bees. DETAILED DESCRIPTION

[0022] The present invention will be further described below in conjunction with the accompanying drawings. The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional technical means in the art, and the reagents used are all analytically pure.

[0023] Example 1: PCR-RFLP analysis of six bee species and their honey

[0024] 1. Preparation and preservation of samples

[0025] (1) The chest muscles of six species of bees (Apis mellifera, Apis cerana, Apis mellifera, Apis mellifera, Apis mellifera and Apis mellifera) were placed in a 1.5 mL centrifuge tube, and the muscles were crushed using a tissue grinder. The crushed products were stored at -80°C for later use;

[0026] (2) Take 15 g of honey from each of the six bees, add 30 mL of distilled water to dissolve and dilute, incubate in a 45°C water bath for 10 min, vortex and centrifuge at 10,000 r / min for 30 min, and discard the supernatant. Then, suspend the precipitate in 1 mL of ultrapure water, centrifuge at 12,000 r / min for 15 min, discard the supernatant, and store the precipitate at -80°C until DNA extraction.

[0027] 2. DNA Extraction

[0028] The total DNA of honey bee tissues and honey sample precipitates was extracted using a blood / cell / tissue genomic DNA extraction kit (Beijing Tiangen Biochemical Technology Co., Ltd.). The DNA quality and concentration were determined by Nano Drop 2000 ultra-micro-volume UV spectrophotometer.

[0029] 3. Primer Design

[0030] MEGA 7 software was used to compare the mitochondrial 16S rRNA gene sequences of six bees submitted to GeneBank, and the conservative sequences of the 16S rRNA genes of the six bees were screened. Then, Primer Premier 5.0 software or other primer design software was used to design universal primers. After a large number of experiments for optimization, the primer DNA sequences finally obtained were: 16SrRNA-F (SEQ ID NO.1): 5'-TGACTTACGTCGATTTGAAC-3', 16S rRNA-R (SEQ ID NO.2): 5'-GACTGTACAAAGGTAGCATAAT-3'. The expected amplified fragment size of the Chinese honey bee is 475p, and its nucleotide sequence is shown in SEQ ID No.3. The expected amplified fragment size of the Italian honey bee is 474bp, and its nucleotide sequence is shown in SEQ ID No.4. The expected amplified fragment size of the black honey bee is 470bp, and its nucleotide sequence is shown in SEQ ID No.5. The expected amplified fragment size of the giant honey bee is 473bp, and its nucleotide sequence is shown in SEQ ID No.6. The expected amplified fragment size of the honey bee is 474 bp, and its nucleotide sequence is shown in SEQ ID No. 7. The expected amplified fragment size of the black honey bee is 479 bp, and its nucleotide sequence is shown in SEQ ID No. 8. Primer synthesis can be completed by a company with primer synthesis services. The primers used in this example were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0031] 4. PCR reaction

[0032] The upstream and downstream primers were diluted to a concentration of 10 μM, and PCR amplification was performed using the DNA obtained in step 2 as a template. The total reaction system for PCR amplification was 20 μL, including 2 μL DNA template, 0.5 μL each of the upstream and downstream primers (10 μM), 10 μL 2×Taq PCR StarMix, and 7 μL ddH2O.

[0033] The PCR reaction program was as follows: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 30 s, 30 cycles; and final extension at 72°C for 5 min.

[0034] 5. Detection of PCR amplification products

[0035] Weigh agarose according to the mass concentration of 20g / L, add 1×TAE buffer and 1×nucleic acid dye, heat to dissolve, and prepare a 2% agarose solution. After cooling slightly, pour it into the gel casting machine equipped with a gel tray, insert the sample grid, and let it stand at room temperature until the gel is completely solidified. Vertically pull out the sample grid and place the gel and its tray into the electrophoresis tank filled with 1×TAE buffer. Take 5μL PCR product and mix it with 1μL 6×Loading Buffer and add it to the gel spotting well, and add 5μL DNA molecular weight standard (100~1500bp) to one of the spotting wells. Turn on the power and perform electrophoresis at 5V / cm~10V / cm for 30~45min before detection. After the electrophoresis is completed, take out the agarose gel and observe and record it in the gel imager.

[0036] 6. Enzyme Digestion Analysis

[0037] The 16S rRNA PCR product was digested, AseI was added to the PCR product, reacted at 37°C for 2 hours, and then incubated at 65°C for 20 minutes to inactivate AseI.

[0038] 7. Detection of enzyme digestion products

[0039] Weigh agarose according to the mass concentration of 40g / L, add 1×TAE buffer and 1×nucleic acid dye, heat to dissolve, and prepare a 4% agarose solution. After cooling slightly, pour it into the gel casting machine equipped with a gel tray, insert the sample grid, and let it stand at room temperature until the gel is completely solidified. Vertically pull out the sample grid and place the gel and its tray into the electrophoresis tank filled with 1×TAE buffer. Take 10μL of enzyme digestion product and mix it with 2μL 6×Loading Buffer and add it to the gel spotting well, and add 5μL DNA molecular weight standard (50~500bp) to one of the spotting wells. Turn on the power and perform electrophoresis at 5V / cm~10V / cm for 30~45min before detection. After the electrophoresis is completed, take out the agarose gel and place it on the gel imager for imaging. Determine the size of the amplified band according to the DNA molecular weight standard.

[0040] 8. Results Analysis

[0041] The results of PCR amplification of the extracted genomic DNA of six bees using the universal primers designed in the present invention are as follows: Figure 1 As shown in the figure, M: RealBand DNA Marker (100-1500bp); AM1, AM2: Italian bees; AC1, AC2: Chinese bees; AL1, AL2: black bees; AD1, AD2: big bees; AF1, AF2: small bees; AA1, AA2: small black bees. The results of PCR amplification of the genomic DNA of the six kinds of honey extracted by the universal primers designed in the present invention are shown as follows: Figure 2 As shown in the figure, M: RealBand DNA Marker (100-1500bp); AMH1, AMH2: Italian bee honey; ACH1, ACH2: Chinese bee honey; ALH1, ALH2: black giant bee honey; ADH1, ADH2: giant bee honey; AFH1, AFH2: honey bee honey; AAH1, AAH2: black giant bee honey.

[0042] The electrophoresis results of the PCR amplification products of six bee species after digestion with AseI restriction endonuclease are as follows: Figure 3 As shown in the figure, M: 50bp DNA Marker (50-500bp); AM1, AM2: Italian bees; AC1, AC2: Chinese bees; AL1, AL2: black bees; AD1, AD2: big bees; AF1, AF2: small bees; AA1, AA2: small black bees. The electrophoresis results of the PCR amplification products of six bee honeys digested with AseI restriction endonuclease are shown in the figure. Figure 4As shown in the figure, M: 50bp DNA Marker (50-500bp); AMH1, AMH2: Italian bee honey; ACH1, ACH2: Chinese bee honey; ALH1, ALH2: black giant bee honey; ADH1, ADH2: giant bee honey; AFH1, AFH2: honey bee honey; AAH1, AAH2: black giant bee honey.

[0043] The results showed that the universal primers designed for the six bee species in this study successfully amplified the 16S rRNA gene fragments of the six bee species and their honey, proving the effectiveness and specificity of the designed universal primers. From the results of AseI digestion, it can be seen that the amplified products of the six species of bees and their honeys formed bee species-specific band patterns after digestion, among which Italian bees / Italian bees honey had 2 electrophoresis bands (414bp and 60bp), Chinese bees / Chinese bees honey had 3 electrophoresis bands (219bp, 202bp and 54bp), black big bees / black big bees honey had 5 electrophoresis bands (217bp, 125bp, 60bp, 38bp and 30bp), big bees / big bees honey had 4 electrophoresis bands (175bp, 128bp, 60bp and 46bp), small bees / small bees honey had 3 electrophoresis bands (249bp, 165bp and 60bp), and black small bees / black small bees honey had 2 electrophoresis bands (250bp and 229bp).

[0044] The invention can simultaneously identify the bee species sources of honey of six bee species of the genus Apis using only a pair of primers and a restriction endonuclease, has the characteristics of rapidity, economy, convenience and high accuracy, and has great application and promotion value.

[0045] Although the above embodiments have described the present invention in detail, the protection scope of the present invention is not limited thereto, and modifications can be made to the scheme of the present invention without deviating from the core, all of which belong to the protection scope of the present invention.

Claims

1. A method for quickly identifying the bee species sources of honey from six species of bees, characterized in that: This is accomplished by the following steps: (1) Extracting genomic DNA from honey; (2) using SEQ ID No. 1 and SEQ ID No. 2 as primer sequences and the genomic DNA obtained in step (1) as a template, performing PCR amplification to obtain a 16S rRNA gene fragment with a size of 470 to 479 bp; (3) using restriction endonuclease AseI to digest the 16S rRNA gene fragment obtained in step (2), and performing 4% agarose gel electrophoresis, and photographing and analyzing the electrophoresis results. The electrophoresis analysis results are as follows: honey with 414 bp and 60 bp bands is Italian honey, honey with 219 bp, 202 bp and 54 bp bands is Chinese honey, honey with 217 bp, 125 bp, 60 bp, 38 bp and 30 bp bands is black honey, honey with 175 bp, 128 bp, 60 bp and 46 bp bands is big honey, honey with 249 bp, 165 bp and 60 bp bands is small honey, and honey with 250 bp and 229 bp bands is black small honey; The six types of bees are Italian bees, Chinese bees, black giant bees, giant bees, small bees and black giant bees.

2. The method according to claim 1, characterized in that: In step (1), the pretreatment for extracting honey DNA is to dissolve and dilute 15 g of honey in 30 mL of distilled water, incubate in a 45°C water bath for 10 min, vortex and oscillate, centrifuge at 10,000 r / min for 30 min, discard the supernatant, then suspend the precipitate in 1 mL of ultrapure water, centrifuge at 12,000 r / min for 15 min, discard the supernatant, and store the precipitate at -80°C until DNA extraction.

3. The method according to claim 1, characterized in that In step (2), the PCR amplification program parameters were set as follows: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 30 s, 30 cycles; final extension at 72°C for 5 min.

4. The method according to claim 1, characterized in that In step (3), the method of using AseI to digest the 16SrRNA gene fragment obtained in step (2) is as follows: adding restriction endonuclease AseI, reacting at 37°C for 2 hours, and then incubating at 65°C for 20 minutes to inactivate AseI.

5. The method of claim 1 is used for rapidly identifying the bee species sources of honey of six species of bees of the genus Apis.

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