CN113621731A - Method for tracing and identifying origin of Guizhou blueberry honey - Google Patents
Method for tracing and identifying origin of Guizhou blueberry honey Download PDFInfo
- Publication number
- CN113621731A CN113621731A CN202110989346.9A CN202110989346A CN113621731A CN 113621731 A CN113621731 A CN 113621731A CN 202110989346 A CN202110989346 A CN 202110989346A CN 113621731 A CN113621731 A CN 113621731A
- Authority
- CN
- China
- Prior art keywords
- honey
- blueberry
- guizhou
- pollen
- detected
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 235000012907 honey Nutrition 0.000 title claims abstract description 279
- 240000000851 Vaccinium corymbosum Species 0.000 title claims abstract description 175
- 235000003095 Vaccinium corymbosum Nutrition 0.000 title claims abstract description 174
- 235000017537 Vaccinium myrtillus Nutrition 0.000 title claims abstract description 174
- 235000021014 blueberries Nutrition 0.000 title claims abstract description 174
- 238000000034 method Methods 0.000 title claims abstract description 40
- 241000196324 Embryophyta Species 0.000 claims abstract description 91
- 239000000523 sample Substances 0.000 claims abstract description 45
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 25
- 230000003321 amplification Effects 0.000 claims abstract description 24
- 238000001514 detection method Methods 0.000 claims abstract description 13
- 238000003753 real-time PCR Methods 0.000 claims description 20
- 241000256844 Apis mellifera Species 0.000 claims description 19
- 241000894007 species Species 0.000 claims description 10
- 238000012408 PCR amplification Methods 0.000 claims description 8
- 241000257303 Hymenoptera Species 0.000 claims description 6
- 239000012634 fragment Substances 0.000 claims description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 210000003763 chloroplast Anatomy 0.000 claims description 4
- 238000004062 sedimentation Methods 0.000 claims description 3
- 238000002955 isolation Methods 0.000 claims description 2
- 108020004414 DNA Proteins 0.000 description 66
- 238000005516 engineering process Methods 0.000 description 13
- 238000011529 RT qPCR Methods 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 8
- 239000000203 mixture Substances 0.000 description 8
- 238000004519 manufacturing process Methods 0.000 description 7
- 239000000843 powder Substances 0.000 description 5
- 238000012163 sequencing technique Methods 0.000 description 5
- 241000256843 Apis mellifera ligustica Species 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000003752 polymerase chain reaction Methods 0.000 description 4
- 238000003908 quality control method Methods 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- PXFBZOLANLWPMH-UHFFFAOYSA-N 16-Epiaffinine Natural products C1C(C2=CC=CC=C2N2)=C2C(=O)CC2C(=CC)CN(C)C1C2CO PXFBZOLANLWPMH-UHFFFAOYSA-N 0.000 description 3
- 241000256846 Apis cerana Species 0.000 description 3
- 241000208838 Asteraceae Species 0.000 description 3
- 241000608847 Gnaphalium Species 0.000 description 3
- 240000005323 Hoya carnosa Species 0.000 description 3
- 235000010654 Melissa officinalis Nutrition 0.000 description 3
- 238000000137 annealing Methods 0.000 description 3
- 238000012165 high-throughput sequencing Methods 0.000 description 3
- 235000015763 Artemisia ludoviciana Nutrition 0.000 description 2
- 230000004544 DNA amplification Effects 0.000 description 2
- 241000208421 Ericaceae Species 0.000 description 2
- 108020005120 Plant DNA Proteins 0.000 description 2
- 235000012511 Vaccinium Nutrition 0.000 description 2
- 241000736767 Vaccinium Species 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 108091093088 Amplicon Proteins 0.000 description 1
- 241000358063 Apis cerana cerana Species 0.000 description 1
- 241001061264 Astragalus Species 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- 244000035851 Chrysanthemum leucanthemum Species 0.000 description 1
- 235000008495 Chrysanthemum leucanthemum Nutrition 0.000 description 1
- 241000207199 Citrus Species 0.000 description 1
- 241000218691 Cupressaceae Species 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 240000007472 Leucaena leucocephala Species 0.000 description 1
- 235000010643 Leucaena leucocephala Nutrition 0.000 description 1
- 244000183278 Nephelium litchi Species 0.000 description 1
- 235000015742 Nephelium litchi Nutrition 0.000 description 1
- 241000218641 Pinaceae Species 0.000 description 1
- 235000005205 Pinus Nutrition 0.000 description 1
- 241000218602 Pinus <genus> Species 0.000 description 1
- 235000011613 Pinus brutia Nutrition 0.000 description 1
- 235000011609 Pinus massoniana Nutrition 0.000 description 1
- 241000018650 Pinus massoniana Species 0.000 description 1
- 241000736211 Platycladus Species 0.000 description 1
- 240000002924 Platycladus orientalis Species 0.000 description 1
- 244000128206 Pyracantha coccinea Species 0.000 description 1
- 241001295692 Pyracantha fortuneana Species 0.000 description 1
- REFJWTPEDVJJIY-UHFFFAOYSA-N Quercetin Chemical compound C=1C(O)=CC(O)=C(C(C=2O)=O)C=1OC=2C1=CC=C(O)C(O)=C1 REFJWTPEDVJJIY-UHFFFAOYSA-N 0.000 description 1
- 235000004789 Rosa xanthina Nutrition 0.000 description 1
- 241000220222 Rosaceae Species 0.000 description 1
- 241000208292 Solanaceae Species 0.000 description 1
- 235000002634 Solanum Nutrition 0.000 description 1
- 241000207763 Solanum Species 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 240000003243 Thuja occidentalis Species 0.000 description 1
- 235000008109 Thuja occidentalis Nutrition 0.000 description 1
- 240000007313 Tilia cordata Species 0.000 description 1
- 240000008424 Vaccinium ashei Species 0.000 description 1
- 235000013468 Vaccinium ashei Nutrition 0.000 description 1
- 244000063464 Vitex agnus-castus Species 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 235000006533 astragalus Nutrition 0.000 description 1
- 230000010138 bee pollination Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 235000009347 chasteberry Nutrition 0.000 description 1
- 235000020971 citrus fruits Nutrition 0.000 description 1
- 238000010835 comparative analysis Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 229930003935 flavonoid Natural products 0.000 description 1
- 235000017173 flavonoids Nutrition 0.000 description 1
- -1 flavonoids compounds Chemical class 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 230000007760 free radical scavenging Effects 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 235000021049 nutrient content Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 235000009048 phenolic acids Nutrition 0.000 description 1
- 150000007965 phenolic acids Chemical class 0.000 description 1
- 239000012804 pollen sample Substances 0.000 description 1
- 235000012015 potatoes Nutrition 0.000 description 1
- 230000001915 proofreading effect Effects 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000013441 quality evaluation Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000001953 sensory effect Effects 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 210000004233 talus Anatomy 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
Abstract
The invention relates to the technical field of honey detection, in particular to a method for tracing and identifying the producing area of Guizhou blueberry honey. The method provided by the invention comprises the following steps: establishing a Guizhou blueberry honey producing area plant background database, separating different types of plant pollen in Guizhou blueberry honey, extracting DNA from each plant pollen, respectively carrying out DNA bar code amplification, comparing obtained DNA bar code sequences in the producing area plant background database, determining the honey source plant type of the Guizhou blueberry honey, and calculating the content percentage of DNA bar code amplification products of each honey source plant pollen in all honey source plant pollen DNA bar code amplification products; the method comprises the steps of taking blueberries as main honey source plants, designing specific primers and probes according to DNA barcode sequences of the blueberries, determining DNA content threshold values of blueberry pollen in Guizhou blueberry honey, and identifying honey to be detected or detecting authenticity and maturity of the honey to be detected according to the threshold values.
Description
Technical Field
The invention relates to the technical field of honey detection, in particular to a method for tracing and identifying the producing area of Guizhou blueberry honey.
Background
The honey is prepared from Apis cerana cerana cerana or Apis mellifera, and is collected in 2020 edition of Chinese pharmacopoeia. The honey is a natural product, not a standardized product, and the sensory and physicochemical indexes such as character characteristics, nutritional ingredients, smell, color and the like of the honey are closely related to the production place, season and production mode, especially honey source plant varieties. The honey collected by bees is the important source of various elements in honey, and the honey from different plant sources and geographical sources has different nutrient content, so the difference of economic value is large. The honey produced in various regions of the world is different, the safety and the standardization of the honey product used by various countries are ensured by the honey product standards used by various countries, and the quality grade further provides a purchasing decision reference for consumers.
The honey powder source plant has wide Chinese regions and various climate types, the honey powder source plant resources are rich and widely distributed, the distribution of the honey source plants has certain regional characteristics, different regions have different honey source plants, and honey with local characteristics is formed after the honey is collected by bees, so that the honey powder can become geographical marking products and original place protection products of various regions. Many countries and regions have established origin protection, geographical identification and traditional special protection systems for famous honey. Related systems are established in countries and regions such as European Union, and geographic marking products and original place protection products in specific regions are protected.
The Guizhou is located in the southwest part of China, belongs to the mountain area of subtropical plateau, has warm and humid climate, various landform types and complex surface composition substances and soil types. The unique geographical position, the mountain-water forest field lake grass ecosystem and the south karst hill achievement make the Guizhou rich and unique biodiversity. The Guizhou southeast area is one of main production areas for planting blueberries in China, and the blueberry variety mainly planted in Majiang county in Guizhou is the rabbit-eye blueberry. Bee pollination is beneficial to fruit setting and fruit setting of the blueberries, the yield and the quality of the blueberries are obviously improved, and meanwhile, the blueberry honey with unique taste can be produced. About 1.82kg of blueberry honey can be collected by bees in each group during the blooming period of blueberries from 3 to 4 months per year. The Guizhou blueberry honey is light amber in color, unique in flavor, high in acidity, easy to crystallize and the like, contains 15 phenolic acids and flavonoids compounds, has the highest quercetin content, and has a certain free radical scavenging capacity, but the authenticity identification and the origin tracing of the Guizhou blueberry honey cannot be realized by the indexes.
DNA barcode (DNA barcoding) technology of biotechnology and DNA macro barcode (DNAmeta-barcoding) technology based on high throughput sequencing technology can rapidly and accurately identify each DNA molecular sequence in honey-source plants and honey, more accurately obtain mixed pollen identification results, be used for deducing plant sources and geographical sources in honey, and be used for deducing geographical sources of plants regardless of plant compositions of single flowers and multiple flowers.
The effective honey tracing and identifying technology and the honey maturity quality control technology are developed, brands of famous and high-quality commodities can be prevented from being utilized, high profits can be obtained by means of the brand of the famous and high-quality commodities, honey varieties are confused, the honey market is disturbed, a basis is provided for honey tracing and quality evaluation, and the method has important significance for developing local characteristic honey industry.
Disclosure of Invention
The invention aims to provide a method for tracing and identifying the producing area of Guizhou blueberry honey.
In order to achieve the purpose, the invention adopts a DNA bar code technology to establish a Guizhou blueberry honey producing area plant background database, adopts a DNA macro bar code technology based on a high-throughput sequencing technology to quickly and accurately identify the DNA bar code sequence of each pollen in Guizhou blueberry honey so as to determine the pollen type in the Guizhou blueberry honey, determines the DNA proportion of main and auxiliary honey powder source plants in the honey, and uses the Guizhou blueberry honey producing area plant background database to determine the regional characteristics of honey source plants so as to carry out qualitative detection on the Guizhou blueberry honey. The method comprises the steps of counting pollen of main honey source plant blueberries through microscopic examination, extracting DNA, carrying out PCR amplification sequencing to obtain a specific fragment, designing a specific primer/TaqMan probe for qPCR amplification aiming at the specific fragment to obtain a Ct threshold value and a corresponding pollen quantity threshold value for judging Guizhou blueberry honey, and carrying out identification, authenticity and quality control detection on the Guizhou blueberry honey so as to carry out quantitative detection on the Guizhou blueberry honey.
Specifically, the invention provides the following technical scheme:
the invention provides a method for tracing and identifying the origin of Guizhou blueberry honey, which comprises the following steps:
establishing a plant background database of a producing area of Guizhou blueberry honey;
separating different kinds of plant pollen in Guizhou blueberry honey, extracting DNA from each plant pollen, respectively carrying out DNA bar code amplification, comparing obtained DNA bar code sequences in a plant background database of a producing area, determining the honey source plant kinds of the Guizhou blueberry honey, and calculating the content percentage of DNA bar code amplification products of various honey source plant pollen in all honey source plant pollen DNA bar code amplification products;
the method comprises the steps of taking blueberries as main honey source plants, designing specific primers and probes aiming at DNA barcode sequences of blueberry pollen, carrying out PCR amplification by using the specific primers and the probes, determining a DNA content threshold of the blueberry pollen in Guizhou blueberry honey, and identifying honey to be detected or detecting authenticity and maturity of the honey to be detected according to the threshold.
In the above method, the establishing of the background database includes: collecting plant samples with the radius of 5 kilometers by taking a Guizhou blueberry honey bee field as a center, obtaining DNA barcode sequences of the plant samples, and identifying the species of the plant samples according to the DNA barcode sequences.
Wherein the plant sample comprises flowers and leaves. The collection time of the plant sample covers the whole period from the collection of nectar by the bees to the maturity of honey brewing.
The method for establishing the plant background database of the production area of the Guizhou blueberry honey can better ensure the completeness and integrity of the plant background database of the production area.
For DNA barcodes of plants, the present invention selects a chloroplast trnL gene fragment.
The invention discovers that the accuracy of tracing and identifying the honey producing area can be better ensured by taking the chloroplast trnL gene segment as the DNA bar code of the honey producing area plant.
Preferably, the amplification primer used for the trnL gene segment is shown as SEQ ID NO. 1-2.
According to the method, the honey of the origin serving as a geographical marking product and a product for protecting the origin is subjected to source tracing and quality control research based on a DNA technology, and the Guizhou blueberry honey is defined according to the qualitative and quantitative analysis result so as to ensure the accuracy of the origin and the controllable quality of the honey. The method for tracing and identifying the producing area of the Guizhou blueberry honey comprises the steps of firstly constructing a plant background database of the producing area of the Guizhou blueberry honey, and establishing a plant background database as perfect as possible by collecting plants within 5 kilometers of the radius of a bee field and covering the whole honey brewing period in the collection time so as to meet the requirement that when a DNA bar code technology and a DNA macro-bar code technology are used, an obtained unknown target is compared with an identified target in a reference database to acquire species information. The method of the invention uses DNA bar code (trnL sequence) technology on the basis of traditional sporopollen, and realizes the rapid identification of species through sequencing and comparative analysis of plant DNA bar codes.
In the method, the separation of the plant pollen in the Guizhou blueberry honey comprises the following steps: after the Guizhou blueberry honey is mixed with water, the mixed pollen is separated according to different sedimentation rates of different pollen, and then the pollen is separated through microscope observation to obtain single-kind pollen.
In the invention, the determination of the plant pollen type contained in the honey requires the separation of pollen: separating mixed pollen according to the principles of different pollen volumes, different weights and different sedimentation rates, observing the uniqueness of the pollen under a microscope, respectively extracting DNA from a single pollen liquid, and then performing PCR amplification. The invention discovers that a sample of mixed pollen can be separated into dozens or even hundreds of samples of single pollen species according to the pollen mixing degree. The step of separating mixed pollen in honey is not seen in the prior art, but the invention finds that the step is essential to tracing the origin of honey. Without pollen isolation, differences in the copy number of template DNA or the concentration of DNA between species may result in over-amplification of some species and lower amplification of other species. The pollen separation can prevent the amplicons of other groups in the sample from covering certain specific groups which cannot be amplified, so that the DNA of each species in the sample can be amplified, and the accuracy of later-stage analysis of the composition of the honey-source plants is ensured.
In the method, the identification method of the honey to be detected is as follows: and performing fluorescent quantitative PCR by using the DNA of the honey to be detected as a template and using the specific primers and the probe, if the Ct value of the fluorescent quantitative PCR is not higher than the threshold, judging that the honey to be detected is Guizhou blueberry honey, and if the Ct value of the fluorescent quantitative PCR is higher than the threshold, judging that the honey to be detected is non-Guizhou blueberry honey or adulterated honey of Guizhou blueberry honey.
Further, the invention provides a specific primer probe set for identifying Guizhou blueberry honey, wherein the sequence of a specific primer is shown as SEQ ID NO.3-4, and the sequence of a probe is shown as SEQ ID NO. 5.
The invention also provides a kit containing the specific primer probe set. The kit can be used for identification, authenticity and maturity detection of Guizhou blueberry honey.
The invention provides any one of the following applications of the specific primer probe set or the specific primer kit:
(1) the application in identifying Guizhou blueberry honey;
(2) the application in detecting the authenticity of the Guizhou blueberry honey is disclosed;
(3) the application of the blueberry honey in Guizhou to detecting the maturity of the blueberry honey in Guizhou is disclosed.
The invention provides a method for identifying or detecting authenticity of Guizhou blueberry honey, which comprises the following steps: and (2) carrying out fluorescence quantitative PCR by using DNA of the honey to be detected as a template and using a specific primer with a sequence shown as SEQ ID NO.3-4 and a probe with a sequence shown as SEQ ID NO.5, if the Ct value of the fluorescence quantitative PCR of every 15.0g of the honey to be detected is 24-29, judging that the honey to be detected is Guizhou blueberry honey, and if the Ct value of the fluorescence quantitative PCR of every 15.0g of the honey to be detected is more than 29, judging that the honey to be detected is non-Guizhou blueberry honey or adulterated honey of the Guizhou blueberry honey.
In the method, the threshold value of the number of the main honey source plant pollen contained in the Guizhou blueberry honey can be calculated according to the relation between the DNA concentration of the pollen and the Ct value and the relation between the DNA concentration of the pollen and the number of the pollen, and the honey to be detected can be identified according to the threshold value of the number of the main honey source plant pollen.
As another embodiment, the present invention provides a method for identifying or detecting authenticity of a guizhou blueberry honey, comprising: detecting the number of blueberry pollen in the honey to be detected, if the number of the blueberry pollen in every 15.0g of the honey to be detected is 1800 grains, judging that the honey to be detected is Guizhou blueberry honey, and if the number of the blueberry pollen in every 15.0g of the honey to be detected is less than 300 grains, judging that the honey to be detected is non-Guizhou blueberry honey or adulterated honey of Guizhou blueberry honey.
The invention provides a method for detecting the maturity of apis cerana blueberry honey in Guizhou, which comprises the following steps: taking DNA of honey to be detected as a template, carrying out fluorescence quantitative PCR by using a specific primer with a sequence shown as SEQ ID NO.3-4 and a probe with a sequence shown as SEQ ID NO.5, judging the honey to be sealed and ripe medium-bee blueberry honey if the Ct value of each 15.0g of honey to be detected is 24-25, and judging the honey to be unsealed and ripe medium-bee blueberry honey if the Ct value of each 15.0g of honey to be detected is 26-29;
as another embodiment, the invention provides a method for detecting maturity of apis cerana blueberry honey in guizhou, which comprises the following steps: the method comprises the steps of detecting the number of blueberry pollen in honey to be detected, judging to be mature Chinese bee blueberry honey with a sealed cover if the number of blueberry pollen in every 15.0g of honey to be detected is 1000-1800 grains, and judging to be mature Chinese bee blueberry honey without a sealed cover if the number of blueberry pollen in every 15.0g of honey to be detected is less than 1000 grains.
In the above method, the reaction procedure of the fluorescent quantitative PCR is as follows: 94 ℃ for 3 min; 92 deg.C, 30s, 60 deg.C, 50s, 72 deg.C, 30s, 72 deg.C, 2min, 40 cycles.
The invention has the technical effects that: the method provided by the invention determines the regional characteristics, species identification and plant composition of the Guizhou blueberry honey by a qualitative and quantitative detection method combining a DNA bar code technology and sporopollen science. The specific primers and the probes provided by the invention are combined with fluorescent quantitative PCR (polymerase chain reaction) to realize the identification of the Guizhou blueberry honey and the authenticity and quality control detection of the Guizhou blueberry honey. The method can be used as an important means for distinguishing the Guizhou blueberry honey region, honey source and quality, is used for distinguishing the Guizhou blueberry honey from other honey with different types, different production places and different qualities, and provides an effective method for standardizing the Guizhou blueberry honey market, developing geographical sign products and region characteristic honey.
Drawings
Fig. 1 is a microscopic examination of the blueberry pollen in example 2 of the present invention, wherein the arrow indicates the blueberry pollen.
FIG. 2 is an amplification curve of fluorescent quantitative PCR amplification using specific primers and TaqMan probes in example 3 of the present invention, wherein 1: a blueberry trnL sequence; 2-26: 25 plant trnL sequence 27: blank control.
FIG. 3 is an amplification curve of DNA of blueberry pollen with different concentrations in example 3 of the present invention.
FIG. 4 is a qPCR amplification curve for 13 honeys of example 3 of the present invention.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1 establishment of plant background database of Guizhou blueberry Honey producing area
Plant samples (including flowers and leaves) with the radius of 5 kilometers in each bee field of a blueberry plantation in the Majiang region of Guizhou are collected in the blueberry flowering period, and the collection time covers the whole period from honey collection of bees to honey brewing maturity.
Extracting genome DNA of each plant sample, and performing PCR amplification on trnL barcode segments in chloroplast genomes by using primers shown in SEQ ID NO. 1-2.
The primer sequences are as follows:
SEQ ID NO.1(F 5’-3’)：CGAAATCGGTAGACGCTACG；
SEQ ID NO.2(R 5’-3’)：GGGGATAGAGGGACTTGAAC。
the PCR amplification reaction procedure was as follows:
at 92 deg.C for 3 min; at 92 deg.C, 30s, 58 deg.C, 30s, 72 deg.C, 45s, 35 cycles; 72 deg.C, 5 min.
And performing DNA sequencing, sequence proofreading and splicing on the PCR product after removing the primers and the low-quality area, and finally obtaining the DNA barcode sequence of each sample. And judging the accuracy of each sequence by adopting a BLAST method, performing multi-sequence comparison by adopting MEGA X software, comparing variation sites of trnL sequences, identifying plant species by comparing GeneBank data, and constructing a background database of trnL barcode segments of the plants in the Guizhou blueberry honey producing areas by using each DNA barcode sequence obtained from all plant samples and corresponding plant species information, wherein the results are shown in Table 1.
TABLE 1 Guizhou Majiang blueberry Honey producing area plant background library
Note: and √ denotes a measurement.
Example 2 Honey-derived plant composition detection of Guizhou blueberry Honey
1. Separation of pollen in Guizhou blueberry honey, extraction of genome DNA and PCR amplification
1.0g of honey sample was weighed into a 5ml centrifuge tube and sampled in parallel at least 3 times. Adding water for dilution, separating mixed pollen according to different pollen volumes, weights and settling speeds, and observing the pollen singularity under a light microscope, wherein the mixed pollen sample can be separated into more than one hundred samples, and the microscopic form of the blueberry pollen is shown in figure 1 (400 times optical microscope). After DNA is extracted from a single pollen liquid, primers with sequences shown as SEQ ID NO.1-2 are respectively used for PCR amplification.
Detecting each PCR product by 1% agarose gel electrophoresis, performing high-throughput sequencing on the amplified product after cutting and recovering gel by adopting a MiSeq sequencing platform, performing systematic analysis on the sequencing result by using Qiame 2 and Abys software, comparing the sequencing result in the plant background database of honey producing areas established in the embodiment 1, determining the type of honey plants, and calculating the content percentage of the DNA barcode amplified products of various honey plant pollens in the total DNA barcode amplified yield of all the honey plant pollens.
The DNA barcode sequence in the Guizhou Chinese bee blueberry honey is amplified, sequenced and compared and analyzed, and the result shows that the honey-source plants with the highest proportion in the honey collected by the Chinese bee are blueberries of the vaccinium genus of the Ericaceae family, next, the biota orientalis of the biotaceae family, the potatoes of the solanum genus of the Solanaceae family and the plants of the multi-genus of the Compositae family, and 13 plants of the 15 genera of the families are present in the proportion of more than 1 percent, and the details are shown in Table 2.
TABLE 2 Honey source plant composition of honeybee blueberry
The DNA barcode sequence in the Italian bee blueberry honey of Guizhou is amplified, sequenced and compared and analyzed, and the result shows that the honey-source plants with the highest proportion in the Italian bee honey are blueberries of the Vaccinium genus of the Ericaceae family, plants of multiple genera of the Compositae family, pyracantha fortuneana of the pyracantha genus of the Rosaceae family, arborvitae of the Platycladus genus of the Cupressaceae family and masson pine of the Pinus genus of the Pinaceae family, and 14 plants of 17 genera of the 14 families with DNA proportion of > 1% are contained, and the detailed table is 3.
TABLE 3 Italian bee blueberry Honey source plant composition
The unique plant composition in Guizhou blueberry honey is based on the unique geographical environment and the unique plant of the production place, and other regions have no reproducibility. The affine cudweed of the compositae affine cudweed plant is used as an important auxiliary honey powder source in blueberry honey, and the nutrition and the drug effect of the affine cudweed herb honey can improve the effect of Guizhou blueberry honey.
Example 3 identification, authenticity and maturity evaluation of Guizhou blueberry Honey
1. Specific primer, probe design and fluorescent quantitative PCR method
According to the DNA proportion of various nectariferous plants determined in the example 2, with the DNA barcode sequence (trnL sequence) of the pollen of the plant with the highest DNA proportion, namely the blueberry (main nectariferous plant), as a target sequence, the background database of the honey producing area plant constructed in the step 1 is used for finding out a trnL target differential fragment region of the blueberry pollen, and a specific Primer and a specific TaqMan probe are designed and synthesized by using Primer Premier 5.0 Primer design software.
The specific primer sequences are as follows:
SEQ ID NO.3(F，5’-3’)：GGCAAACAAATAAAGATTCC；
SEQ ID NO.4(R，5’-3’)：TCGACGAAAGAATTCCTA。
the specific TaqMan probe sequence is as follows:
SEQ ID NO.5(P，5’-3’)：FAM-TCTACCAACGCAGTCAACTCCAT-BHQ1。
according to a fluorescent quantitative PCR reaction system shown in Table 4, annealing temperature optimization is carried out on a specific primer and a Taqman probe: set 8 sets of annealing temperatures: 56. 57, 58, 59, 60, 61, 62, 63 ℃. The annealing temperature was determined to be 60 ℃ from the Ct value of qPCR and the results of the amplification curve.
The optimized qPCR reaction condition is 94 ℃ and 3 min; 92 deg.C, 30s, 60 deg.C, 50s, 72 deg.C, 30s, 72 deg.C, 2min, 40 cycles. Each sample was done 3 replicates.
TABLE 4 TaqMan fluorescent quantitation PCR reaction System
To verify the specificity of the designed primers and probes, 26 plants from the background pool were selected and plant DNA was extracted using DP320 plant genomic DNA extraction kit. The specificity of the specific primers and the Taqman probe was verified, and the results are shown in FIG. 2. The concentration of the blueberry pollen DNA is 18.3 ng/mu L, the blueberry pollen DNA is detected by a blueberry honey TaqMan probe specificity qPCR detection method, an amplification curve exists, the reaction is positive, the Ct value is 28.60, and the amplification curve is S-shaped fluorescence; the other 25 plants and the blank control are all negative reactions, no fluorescence value is increased, and no specific fluorescence amplification curve is seen, which indicates that the designed blueberry specific primer and the TaqMan probe have stronger specificity, the primer and the probe only have specificity to blueberry pollen, an amplification curve can be generated, and other plants in a background library are not amplified.
2. Establishment of a Standard Curve
Diluting blueberry pollen with water, counting with a blood counting chamber under a light microscope, and repeating for 5 times. Extracting DNA from pollen liquid with the same amount of pollen, measuring the concentration of the DNA, diluting by a four-time gradient to obtain the amount of pollen and the concentration of DNA (0.07-66.5 ng/. mu.L) of each liquid, performing qPCR according to the reaction system shown in Table 4 and the optimized reaction program by using the DNA as a template, and collecting fluorescence signals at 60 ℃ to obtain a pollen DNA amplification curve, wherein the result is shown in FIG. 3.
In a linear range, a linear equation is obtained by regression of Lg value and Ct value of pollen DNA concentration; meanwhile, the pollen DNA concentration value is taken as an X axis, and the pollen number is taken as a Y axis, so that a linear regression equation is obtained. And calculating the corresponding pollen grain number by using the Ct value amplified by qPCR by taking the pollen DNA concentration as an intermediate transformation relation. The experiment was repeated 3 times, with 3 replicates for each concentration, with the following results:
the functional relationship between the Lg value and the Ct value of the obtained DNA concentration of the blueberry polleny＝-5.6205x+65.285(R20.9950) and the functional relationship between the DNA concentration of blueberry pollen and the number of pollen, y, 77.107x-6.1772 (R)20.9987) and taking the DNA concentration of the blueberry pollen as an intermediate transformation relation to obtain a relation formula between the number of the blueberry pollen and the Ct value:
in the formula: p is the number of pollen contained in the sample, and A is the Ct value obtained by the pollen DNA amplification of the sample. Since lg concentration unit pg/ml and pollen DNA concentration unit ng/uL, the result is multiplied by 10-6Corresponding to the number of pollen.
3. Determination of authenticity threshold and maturity threshold of Guizhou blueberry honey
200 honey samples of various grades or brands of single variety/all-flower honey are purchased from labels of different manufacturers and bee farmers in various regions or are self-reported, and the honey samples comprise 13 honey varieties of single flower honey and 3 all-flower honey (10 samples respectively) such as Guizhou blueberry honey (75 samples), Canadian blueberry honey (10 samples), linden honey (11 samples), date honey (11 samples), citrus honey (10 samples), lychee honey (11 samples), astragalus honey (10 samples), vitex honey (11 samples), acacia honey (11 samples) and wild chrysanthemum honey (10 samples). The results of the tests on the partial honey samples are shown in fig. 4 and table 5. The detection results of 200 samples are counted, and the results show that the Ct values of 15.0g of Guizhou blueberry honey fluorescence quantitative PCR are all less than 29, the Ct values of qPCR amplification curves of other varieties of honey including Canadian blueberry honey are all greater than 30, the honey can be obviously distinguished from the Guizhou blueberry honey, and the honey is judged to be non-Guizhou blueberry honey.
The authenticity index of the Guizhou blueberry honey thus determined is defined as follows: the Ct value of 15.0g of blueberry honey is 24-29, the number of blueberry pollen is 300-1800 grains, the Ct value of other honey samples except the blueberry honey is more than 29, amplification is not generated when the Ct is less than or equal to 29, and the blueberry honey can be obviously distinguished from Guizhou blueberry honey.
Wherein the Ct value of 15.0g of sealed mature honey is 24-25, and the Ct value of unsealed mature honey or non-mature honey is 26-29. The mature Chinese bee honey (Chinese bee capped blueberry honey) and the non-mature Chinese bee honey (Chinese bee uncapped blueberry honey) in the Guizhou blueberry honey can be clearly distinguished, but the Italian bee capped blueberry honey and the Chinese bee uncapped blueberry honey cannot be distinguished.
The maturity index of the apis cerana blueberry honey in Guizhou thus determined is defined as follows: the Ct value of 15.0g of sealed mature honey is 24-25, the number of blueberry pollen is 1000-1800 grains, the Ct value of unsealed honey or non-medium honey is 26-29, and the number of blueberry pollen is less than 1000 grains.
TABLE 5 identification and quality determination of blueberry Honey
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
<110> bee institute of Chinese academy of agricultural sciences
Guizhou Institute of modern agricultural development
<120> origin tracing and identifying method for Guizhou blueberry honey
<130> KHP211117640.3
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
<210> 4
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
<210> 5
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
tctaccaacg cagtcaactc cat 23
Claims (10)
1. A method for tracing and identifying the origin of Guizhou blueberry honey is characterized by comprising the following steps:
establishing a plant background database of a Guizhou blueberry honey producing area;
separating different kinds of plant pollen in Guizhou blueberry honey, extracting DNA from each plant pollen, respectively carrying out DNA bar code amplification, comparing obtained DNA bar code sequences in a plant background database of a producing area, determining the honey source plant kinds of the Guizhou blueberry honey, and calculating the content percentage of DNA bar code amplification products of each honey source plant pollen in all honey source plant pollen DNA bar code amplification products;
the method comprises the steps of taking blueberries as main honey source plants, designing specific primers and probes aiming at DNA barcode sequences of blueberry pollen, carrying out PCR amplification by using the specific primers and the probes, determining a DNA content threshold of the blueberry pollen in Guizhou blueberry honey, and identifying honey to be detected or detecting authenticity and maturity of the honey to be detected according to the threshold.
2. The method of claim 1, wherein the establishing of the context database comprises: collecting plant samples with a radius of 5 kilometers by taking a Guizhou blueberry honey bee field as a center, obtaining DNA barcode sequences of the plant samples, and identifying the species of each plant sample according to the DNA barcode sequences;
preferably, the plant sample comprises flowers and leaves;
more preferably, the time of collection of the plant sample covers the whole period from the collection of nectar by the bees to the maturity of honey brewing.
3. The method of claim 1 or 2, wherein the DNA barcode sequence is a chloroplast trnL gene fragment;
preferably, the sequence of the amplification primer used for the trnL gene fragment is shown in SEQ ID NO. 1-2.
4. The method as claimed in any one of claims 1 to 3, wherein the isolation of plant pollen in Guizhou blueberry honey comprises: after the Guizhou blueberry honey is mixed with water, the mixed pollen is separated according to different sedimentation rates of different pollen, and then the pollen is separated through microscope observation to obtain single-kind pollen.
5. The method according to any one of claims 1 to 4, characterized in that DNA of honey to be detected is used as a template, the specific primers and the probe are used for carrying out fluorescence quantitative PCR, if the Ct value of the fluorescence quantitative PCR is not higher than the threshold, the honey to be detected is judged to be Guizhou blueberry honey, and if the Ct value of the fluorescence quantitative PCR is higher than the threshold, the honey to be detected is judged to be non-Guizhou blueberry honey or adulterated honey of Guizhou blueberry honey.
6. The specific primer probe set for identifying the Guizhou blueberry honey is characterized in that the sequence of the specific primer is shown as SEQ ID NO.3-4, and the sequence of the probe is shown as SEQ ID NO. 5.
7. A kit comprising a set of specific primer probes according to claim 6.
8. Use of a specific primer probe set according to claim 6 or a kit according to claim 7 for any of the following:
(1) the application in identifying Guizhou blueberry honey;
(2) the application in detecting the authenticity of the Guizhou blueberry honey is disclosed;
(3) the application of the blueberry honey in Guizhou to detecting the maturity of the blueberry honey in Guizhou is disclosed.
9. The identification or authenticity detection method of Guizhou blueberry honey is characterized in that DNA of honey to be detected is used as a template, specific primers with sequences shown as SEQ ID NO.3-4 and probes with sequences shown as SEQ ID NO.5 are used for carrying out fluorescence quantitative PCR, if the Ct value of the fluorescence quantitative PCR of each 15.0g of the honey to be detected is 24-29, the honey to be detected is judged to be Guizhou blueberry honey, and if the Ct value of the fluorescence quantitative PCR of each 15.0g of the honey to be detected is larger than 29, the honey to be detected is judged to be non-Guizhou blueberry honey or adulterated honey of Guizhou blueberry honey;
or detecting the number of blueberry pollen in the honey to be detected, if the number of the blueberry pollen in every 15.0g of the honey to be detected is 1800 grains in 300-grain, judging that the honey to be detected is Guizhou blueberry honey, and if the number of the blueberry pollen in every 15.0g of the honey to be detected is less than 300 grains in 15.0g of the honey to be detected, judging that the honey to be detected is non-Guizhou blueberry honey or adulterated honey of the Guizhou blueberry honey.
10. The maturity detection method of Guizhou Zhongfeng blueberry honey is characterized in that DNA of honey to be detected is used as a template, a specific primer with a sequence shown as SEQ ID NO.3-4 and a probe with a sequence shown as SEQ ID NO.5 are used for carrying out fluorescence quantitative PCR, if the Ct value of each 15.0g of honey to be detected is 24-25, the honey is judged to be well-sealed blueberry honey, and if the Ct value of each 15.0g of honey to be detected is 26-29, the honey is judged to be well-unsealed blueberry honey;
or detecting the number of blueberry pollen in the honey to be detected, if the number of blueberry pollen in every 15.0g of the honey to be detected is 1800 pieces, judging the honey to be the mature Chinese bee blueberry honey, and if the number of blueberry pollen in every 15.0g of the honey to be detected is less than 1000 pieces, judging the honey to be the mature Chinese bee blueberry honey which is not sealed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110989346.9A CN113621731A (en) | 2021-08-26 | 2021-08-26 | Method for tracing and identifying origin of Guizhou blueberry honey |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110989346.9A CN113621731A (en) | 2021-08-26 | 2021-08-26 | Method for tracing and identifying origin of Guizhou blueberry honey |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113621731A true CN113621731A (en) | 2021-11-09 |
Family
ID=78387925
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110989346.9A Pending CN113621731A (en) | 2021-08-26 | 2021-08-26 | Method for tracing and identifying origin of Guizhou blueberry honey |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113621731A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110331194A (en) * | 2019-06-28 | 2019-10-15 | 西安医学院 | A method of affine cudweed kind is identified using psbA-trnH sequence |
| CN113699220A (en) * | 2021-08-26 | 2021-11-26 | 中国农业科学院蜜蜂研究所 | Method for identifying honey and tracing honey producing area by regional plant sources |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20120124654A (en) * | 2011-05-04 | 2012-11-14 | 대한민국(농촌진흥청장) | Analysis of the purity of honey, using pollen mixed in honey |
| WO2014171767A1 (en) * | 2013-04-17 | 2014-10-23 | 주식회사 제놀루션 | Method for determining authenticity of honey using dna detection |
| CN113699220A (en) * | 2021-08-26 | 2021-11-26 | 中国农业科学院蜜蜂研究所 | Method for identifying honey and tracing honey producing area by regional plant sources |
| CN114088848A (en) * | 2022-01-20 | 2022-02-25 | 中国农业科学院蜜蜂研究所 | Method for identifying origin of honey production area and application |
-
2021
- 2021-08-26 CN CN202110989346.9A patent/CN113621731A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20120124654A (en) * | 2011-05-04 | 2012-11-14 | 대한민국(농촌진흥청장) | Analysis of the purity of honey, using pollen mixed in honey |
| WO2014171767A1 (en) * | 2013-04-17 | 2014-10-23 | 주식회사 제놀루션 | Method for determining authenticity of honey using dna detection |
| CN113699220A (en) * | 2021-08-26 | 2021-11-26 | 中国农业科学院蜜蜂研究所 | Method for identifying honey and tracing honey producing area by regional plant sources |
| CN114088848A (en) * | 2022-01-20 | 2022-02-25 | 中国农业科学院蜜蜂研究所 | Method for identifying origin of honey production area and application |
Non-Patent Citations (5)
| Title |
|---|
| "A DNA barcoding approach to identify plant species in multiflower honey", FOOD CHEMISTRY, vol. 170, 23 August 2014 (2014-08-23), pages 308 - 315 * |
| ALICE VALENTINI: "DNA Barcoding for Honey Biodiversity", DIVERSITY, no. 2, 19 April 2010 (2010-04-19), pages 610 - 617 * |
| 岳锦萍: "蜂蜜的主要成分及其鉴别技术", 食品安全质量检测学报, vol. 9, no. 19, 31 October 2018 (2018-10-31), pages 5138 - 5145 * |
| 张得钧;元王涛;马明呈;刘明成;: "青海五裂茶子叶绿体DNAtrnL-F序列的遗传变异研究", 基因组学与应用生物学, no. 06, 28 December 2014 (2014-12-28), pages 1319 - 1323 * |
| 张致豪: "贵州蓝莓蜜 TaqMan 探针 实时荧光 PCR 检测方法的建立与应用", 现代食品科技, vol. 40, no. 3, 6 December 2023 (2023-12-06), pages 1 - 11 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110331194A (en) * | 2019-06-28 | 2019-10-15 | 西安医学院 | A method of affine cudweed kind is identified using psbA-trnH sequence |
| CN110331194B (en) * | 2019-06-28 | 2023-03-31 | 西安医学院 | Method for identifying affine cudweed variety by utilizing psbA-trnH sequence |
| CN113699220A (en) * | 2021-08-26 | 2021-11-26 | 中国农业科学院蜜蜂研究所 | Method for identifying honey and tracing honey producing area by regional plant sources |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Liao et al. | Using SSR to evaluate the genetic diversity of potato cultivars from Yunnan province (SW China) | |
| CN113621731A (en) | Method for tracing and identifying origin of Guizhou blueberry honey | |
| CN113699220A (en) | Method for identifying honey and tracing honey producing area by regional plant sources | |
| HERAWATI et al. | Variation analysis of three Banyumas local salak cultivars (Salacca zalacca) based on leaf anatomy and genetic diversity | |
| Priolli et al. | Genetic diversity among Brazilian soybean cultivars based on SSR loci and pedigree data | |
| CN108359738B (en) | Torreya sinensis EST-SSR primer and variety fingerprint construction method | |
| CN111349717B (en) | SSR (simple sequence repeat) markers of sweet cherry stock resources and fingerprint spectrum database thereof | |
| CN110878376B (en) | SSR molecular marker primer for identifying dendrobium huoshanense and application thereof | |
| KR102163233B1 (en) | SSR primer set for discriminating Agaricus bisporus cultivar Sae Jeong, Sae-Ah, Seolgang and uses thereof | |
| JP5763315B2 (en) | Identification method of domestic and foreign rice or tissues derived from it, or their processed products | |
| CN106544438A (en) | A kind of application of genetic marker in purpleapricot identification | |
| Natawijaya et al. | Genetic structure and diversity between and within African and American oil palm species based on microsatellite markers | |
| KR102335806B1 (en) | Molecular marker based on chloroplast genome sequence for discriminating Zizyphus jujuba 'SanJo' cultivar and uses thereof | |
| CN113584204B (en) | DNA fragment related to kernel yield of camellia seeds, SNP molecular marker closely linked with DNA fragment and application of DNA fragment | |
| CN111394499B (en) | Nucleic acid composition for screening high-anthocyanin tea trees, application of nucleic acid composition and method for breeding high-anthocyanin tea trees | |
| Güçlü et al. | Genetic characterization of Turkish commercial opium poppy (Papaver somniferum L.) cultivars using ISSR and SSR markers | |
| KR101699518B1 (en) | Primer set for discrimination of a ginseng cultivar Gumpoong and a landrace Hwangsook and uses thereof | |
| He et al. | Construction of Fingerprint of Michelia Germplasm by Fluorescent SSR Markers | |
| CN112111591B (en) | Pinus bungeana EST-SSR primer and application thereof in population genetic diversity analysis | |
| CN112176088B (en) | SSR primer group for distinguishing litchi varieties and application thereof | |
| KR102412793B1 (en) | SNP genetic markers and primer sets for discriminating domestic wheat cultivar and uses thereof | |
| CN112266972B (en) | SSR molecular marker primer group for identifying longan varieties and application thereof | |
| CN113981124B (en) | Sakura SSR molecular marker primer and application thereof in identification of 42 sakura varieties | |
| CN112921114B (en) | DNA sequence marker for identifying hemp plant sex and detection primer thereof | |
| TWI447227B (en) | Method and kit for identifying phalaenopsis varieties |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |