CN113862374B - PCR method for identifying Chinese bee honey and Italian bee honey and application - Google Patents
PCR method for identifying Chinese bee honey and Italian bee honey and application Download PDFInfo
- Publication number
- CN113862374B CN113862374B CN202111079089.1A CN202111079089A CN113862374B CN 113862374 B CN113862374 B CN 113862374B CN 202111079089 A CN202111079089 A CN 202111079089A CN 113862374 B CN113862374 B CN 113862374B
- Authority
- CN
- China
- Prior art keywords
- honey
- bee
- italian
- chinese
- bee honey
- 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.)
- Active
Links
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
-
- 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
- C12Q1/686—Polymerase chain reaction [PCR]
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a PCR method for identifying Chinese bee honey and Italian bee honey and application thereof, belonging to the technical field of animal source component molecular detection. The invention identifies Chinese bee honey and Chinese bee honey according to the specific band difference of Chinese bee and Chinese bee genome DNA. After the primer pair is adopted for carrying out PCR amplification treatment, the specific band appears at 194bp of the Chinese bee honey, the specific band appears at 330bp of the Italian bee honey, and the mixed honey sample of the Chinese bee honey and the Italian bee honey can amplify two specific bands of 194bp and 330bp at the same time, so that the Chinese bee honey and the Italian bee honey can be rapidly and accurately identified. The invention has the characteristics of simplicity, convenience, rapidness, strong specificity, lower cost and wide applicability.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a nucleic acid sequence for identifying Chinese bee honey and Italian bee honey, a method and application thereof.
Background
Honey is a widely eaten natural health food and has been popular with consumers since ancient times. Honey contains various trace components in addition to abundant sugars, including free amino acids, proteins, phenolic compounds, minerals, vitamins, organic acids, and the like (Ouchemoukh et al 2005, da Silva et al 2016); meanwhile, the honey also plays a role in anti-inflammatory and antioxidant aspects. Thus, honey is favored by more and more consumers.
China is a large country for raising bees, and beekeeping has become an important component of modern agriculture in China. The bee cultivation amount, the bee product yield and the export amount in China are all in the first place in the world. Statistics data in 2018 of China society of bee-keeping shows that 47.9 ten thousand tons of honey, 3000 tons of royal jelly, 6000 tons of beeswax, 4000 tons of bee pollen and 350 tons of propolis are produced in China. Chinese bees (Apis cerana cerana), chinese bees for short, italian bees (Apis mellifera ligustica) for short, and Italian bees for short, wherein the annual honey production of Chinese bees is 5-10 ten thousand tons, and the annual honey production of Italian bees is about 40 ten thousand tons (Zhang Yanzheng. Research on the authenticity of sources of Chinese honey and Italian bees honey [ D ]. Hangzhou: zhejiang university, 2019.). Based on the low yield of medium honey and the preference of partial consumers for traditional native honey, the price of medium honey is several times that of medium honey, and the phenomenon of medium honey sales caused by mixing or pseudo-mixing of medium honey also exists in the market.
The current method for identifying Chinese bee honey and Italian bee honey mainly focuses on the difference of protein components in honey (for example, a method for identifying Chinese bee honey and Italian bee honey with application number CN201510999246.9, a method for identifying Chinese bee honey and Italian bee honey based on royal jelly main protein components with application number CN 201610545277.1) or the difference of organic components in honey (for example, a method for identifying Chinese bee honey and Italian bee honey with application number CN201810008004.2, application of indole-3-methyl acetate with application number CN202010197053.2 in identifying Chinese bee honey and Italian bee honey, and a method for distinguishing Chinese bee honey and Italian bee honey with application number CN 202011120746.8). There is also a method for identifying Chinese bee honey and Italian bee honey by liquid chromatography-mass spectrometry (such as a method for identifying Chinese bee honey and Italian bee honey with application number of CN 202010198016.3) in the identification technology. However, the time for distinguishing the Chinese bee honey from the Italian bee honey by the protein detection is long, the liquid chromatography-mass spectrometry has high requirements on instruments, and operators require professional technicians, so that the method has limitations in practical application.
With the development of modern molecular biology, techniques based on the Polymerase Chain Reaction (PCR) play an important role in identifying animal origin of products. There have been studies to identify species sources of honey by mitochondrial DNA (Kim, c. -k.; lee, d.c.; choi, s. -h.detection of Korean Native Honey and European Honey by Using Duplex Polymerase Chain Reaction and Immunochromatographic assay.korean j.food sci.anim.resource.2017, 37, 599.) but mitochondrial DNA copy numbers are higher, content in different tissues is different, and stability is poor. The animal nuclear genome DNA sequence has high species specificity, avoids nonspecific amplification, and is more suitable for PCR qualitative and quantitative analysis. Aiming at nuclear genome DNA, identification of Chinese bee honey and Italian bee honey by designing primers through royal jelly main protein 2 gene (Zhang Yanzheng. Research on the source authenticity of Chinese bee honey and Italian bee honey bee species [ D ]. Hangzhou: zhejiang university, 2019.) is also studied in China, and double PCR and fluorescence quantitative PCR are adopted to identify Chinese bee honey and Italian bee honey. The double PCR method can identify the mixing of 1% of Chinese bee honey, but the method has poor detection effect on Chinese bee honey contained in mixed honey, and does not provide the sensitivity of Chinese bee primers, so that false negative can be caused in practical application; the developed fluorescent quantitative PCR does not clearly indicate the identification sensitivity and is easily limited by the equipment and the cost is high. Therefore, there is a need for a PCR method with high specificity, high sensitivity and low cost to identify Chinese bee honey and Italian bee honey.
Disclosure of Invention
In order to solve the technical problems, the invention provides a nucleotide sequence and a PCR method for identifying Chinese honey and honey.
In order to achieve the above purpose, the invention adopts the following technical scheme:
nucleotide sequences for identifying apis cerana honey and apis cerana honey, comprising a primer pair for simultaneously amplifying the sequence shown in SEQ ID No.1 and the sequence shown in SEQ ID No. 2.
The nucleotide sequence as described above preferably comprises a primer pair for detecting Chinese bee honey as shown in SEQ ID No.3 and SEQ ID No.4 and a primer pair for detecting Italian bee honey as shown in SEQ ID No.5 and SEQ ID No. 6.
A test kit for identifying chinese bee honey and chinese bee honey comprising a nucleotide sequence as described above.
The detection kit as described above preferably further comprises 2 x Taq DNA MasterMix.
A PCR method for identifying Chinese bee honey and honey of a apis cerana, comprising the following steps:
s1, extracting genome DNA of honey,
s2, carrying out PCR amplification on the extracted honey genome DNA by adopting primer sequences shown as SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 and SEQ ID No. 6;
and S3, carrying out electrophoresis on the amplified product, and judging a result.
In the above PCR method, preferably, in the step S1, the pretreatment before extracting the DNA of the honey is to dilute the honey by adding sterile water, shaking and mixing uniformly, incubating for 10min at 40 ℃, centrifuging for 30min at 7000r/m, and discarding the supernatant; the precipitate was suspended in ultrapure water, centrifuged at 13000r/min for 20min, the supernatant was discarded, and the precipitate was retained for DNA extraction.
In the PCR method as described above, preferably, in step S2, the final concentration of the primer in each primer pair in the reaction system for PCR amplification is 0.25. Mu. Mol/L, and the reaction procedure is as follows: initial pre-denaturation at 94 ℃ for 2min; denaturation at 94℃for 30s, annealing at 58℃for 30s, elongation at 72℃for 30s,35 cycles; extending at 72℃for 10min.
As described above, in the PCR method, preferably, in the step S3, if a specific band of 194bp size appears in the electrophoresis result, it is indicated that the medium bee honey is contained, if a specific band of 330bp size appears, it is indicated that the medium bee honey is contained, and if both bands of 194bp and 330bp exist, it is indicated that the medium bee honey is mixed with the intentional bee honey.
The PCR method is applied to the identification of Chinese bee honey and Italian bee honey.
The invention has the beneficial effects that:
the invention provides a method for identifying Chinese bee honey and Italian bee honey, which is used for identifying the Chinese bee honey and the Italian bee honey according to the specific band difference of Chinese bee and Italian bee genome DNA. The specific primer PCR amplification treatment is adopted to amplify the honey sample, wherein the specific band appears at 194bp of the Chinese bee honey, the specific band appears at 330bp of the Chinese bee honey, and the mixed honey sample of the Chinese bee honey and the Chinese bee honey can amplify the two specific bands of 194bp and 330 bp. The invention achieves the identification purpose by distinguishing the specific bands of different honeys, and can rapidly and accurately identify the Chinese honey and the Italian honey. The invention has the characteristics of simplicity, convenience, rapidness, strong specificity, lower cost and wide applicability.
The PCR method for identifying the Chinese bee honey and the Italian bee honey can effectively identify the result that the Chinese bee honey is doped with at least 1% of Italian bee honey, and meanwhile, the method directly compares the whole genome of the Chinese bee and the Italian bee to obtain a specific nucleotide sequence, so that a false negative result is avoided.
The PCR method for identifying the Chinese bee honey and the Italian bee honey only needs a common PCR instrument, a gel electrophoresis instrument and a gel imaging system or an ultraviolet transmission instrument, and the detection process only needs 3 hours, and the required reagent is simple and easy to obtain, so that the method has the advantages of simple instrument and equipment requirements, short time consumption and low cost, and is suitable for popularization.
Drawings
FIG. 1 shows the result of amplification of genomic DNA of Zhongfeng and Italian bees.
FIG. 2 shows the result of amplification of DNA extract of honey of Zhongfeng and Yifeng.
FIG. 3 shows the amplification results of the genomic DNA template of the medium bees at different concentrations.
FIG. 4 shows the results of amplification of DNA extracts of medium honey at various concentrations.
FIG. 5 shows the amplification results of the genomic DNA templates of the Ipomoea batatas at different concentrations.
FIG. 6 shows the amplification results of DNA extracts of honey at different concentrations.
FIG. 7 shows the results of double PCR amplification of mixed genomic DNA.
FIG. 8 shows the results of double PCR amplification of the mixed honey DNA extract.
FIG. 9 shows the results of amplification of different primer pairs of Chinese bees.
FIG. 10 shows the results of amplification of different bee primer pairs.
Detailed Description
The following examples serve to further illustrate the invention but are not to be construed as limiting the invention. Modifications and substitutions made to the invention without departing from the spirit and nature of the invention are intended to be within the scope of the invention.
Unless otherwise indicated, all technical means used in the examples are conventional means well known to those skilled in the art, and unless otherwise specified, all reagents used in the present invention are of analytical purity or above.
Example 1: specific detection of Chinese bees and Italian bees
1 preparation and preservation of samples
1.1 sampling
Collecting one bee and one Italian bee, and storing at-80deg.C; collecting Mel and Mel respectively 15g, and storing at 4deg.C.
Treatment of bees: freezing bee sample, washing with double distilled water, removing water with absorbent paper, placing head and chest in 1.5mL centrifuge tube, grinding with liquid nitrogen or crushing with tissue grinder, and storing the crushed product at-80deg.C;
treatment of honey: diluting 15g of honey in 50mL of ultrapure water, incubating for 10min at 42 ℃, centrifuging for 30min at 7000r/min after vortex oscillation, discarding supernatant, suspending the precipitate in 1mL of purified water, centrifuging for 20min at 13000r/min, discarding supernatant, and storing the precipitate at-80 ℃.
1.2 DNA extraction
DNA template preparation uses the usual phenol-chloroform crude extraction method or other well-known extraction methods with the same efficacy, which are all reported usual methods for extracting DNA from the above samples.
2 primer design
Comparing the genome DNA sequences of the apis cerana and apis cerana, and screening to obtain the specific nucleotide sequences for identifying apis cerana and apis cerana. Primers were designed according to the screened specific nucleotide sequences of Zhongfeng and Italian bees using Primer Premier 5.0 software or other Primer design software. Through a plurality of experiments, the sequence of the finally obtained primer DNA is shown in the table 1, wherein the genome amplification of Hua Mifeng (Apis cerana cerana Fabricius) in the pair of the primer C-F and the C-R is expected to have the amplification fragment size of 194bp, the nucleotide sequence of the primer is shown as SEQ ID No.1, the genome amplification of Italian bee (Apis mellifera ligustica) in the pair of the primer M-F and the M-R is expected to have the amplification fragment size of 330bp, and the nucleotide sequence of the primer is shown as SEQ ID No. 2. The primer synthesis is completed by a company having a primer synthesis service, and the primers used in this example are synthesized by the marmor family.
TABLE 1 Chinese bee PCR amplification primers and Italian bee PCR amplification primers designed according to the present invention
Experiments show that the primer has strong specificity, is specifically expressed in the corresponding species, and has no target fragment amplification in non-native species. The primers are respectively extended to 3 'and 5' to form primers with one base and two bases or modified forms, and experiments prove that the specific amplification can be still carried out; the primers shown in Table 1 are optimal from the viewpoints of economy and comprehensive effects.
3 PCR detection
3.1 PCR reaction
Preparing the upstream and downstream primers in the table 1 into primers with the concentration of 10 mu mol/L, carrying out PCR amplification by taking the DNA extracted in the step 1.2 as a template, adopting the sequences of the primers shown as SEQ ID No.3 and SEQ ID No.4 in the PCR amplification of the Chinese bee honey, and adopting the sequences shown as SEQ ID No.5 and SEQ ID No.6 in the PCR amplification of the Chinese bee honey, and adding the sequences of the primers into a PCR reaction system according to the use amount and the concentration of the upstream and downstream primers, wherein the use amount and the concentration are shown in the table 2;
TABLE 2 reaction System for PCR amplification according to the invention
The PCR reaction procedure was: initial pre-denaturation at 94 ℃ for 2min; denaturation at 94℃for 30s, annealing at 58℃for 30s, elongation at 72℃for 30s,35 cycles; extending at 72℃for 10min.
3.2A blank control was set up at the same time as the PCR reaction, with double distilled water as the blank control for the PCR reaction.
3.3 amplifying by taking DNA extracted from Chinese bees and Italian bees as templates and DNA extracted from two kinds of honey as templates.
Detection of 4 PCR amplified products
Agarose is weighed according to the mass concentration of 20g/L, added into 1 xTAE buffer solution, heated and dissolved to prepare 2% agarose solution, after being cooled slightly, poured into an electrophoresis plate, inserted into a comb plate, solidified into gel at room temperature, and then placed into the 1 xTAE buffer solution, and the comb plate is gently pulled vertically upwards. Mixing 5 mu L of PCR product with 1 mu L of 6 XRNA/DNA Loading Buffer, adding into gel sample application holes, adding DNA molecular weight standard into one of the sample application holes, and detecting after electrophoresis for 30-45 min under the condition of 2-5V/cm by switching on a power supply.
After electrophoresis, taking out agarose gel, and placing on a gel imager or an ultraviolet transmission instrument for imaging. And judging the size of the amplified band according to the molecular weight standard of the DNA, and archiving an electronic file formed by the electrophoresis result or photographing by a photographing system.
5 analysis of results
The results of amplification using genomic DNA extracted from Zhongyi bees are shown in FIG. 1, where M: is BM5000DNA Marker; l1: chinese bee genome DNA and Chinese bee primers C-F and C-R; l2: italian bee genome DNA and Chinese bee primers C-F and C-R; l3: blank control and Chinese bee primers C-F and C-R; l4: chinese bee genome DNA and Italian bee primers M-F and M-R; l5: italian bee genome DNA and Italian bee primers M-F and M-R; l6: blank and Italian primers M-F and M-R. The result of the amplification of DNA extracted from Chinese and Italian bee honey is shown in FIG. 2, wherein L1: chinese honey DNA and Chinese honey primers C-F and C-R; l2: honey DNA and Chinese bee primers C-F and C-R; l3: blank control and Chinese bee primers C-F and C-R; l4: chinese honey DNA and Italian bee primers M-F and M-R; l5: honey DNA and honey primers M-F and M-R; l6: blank and Italian primers M-F and M-R. The result shows that the method uses the specific primers and the intention bee primers of the Chinese bees and the DNA and the intention bee DNA of the Chinese bees as templates to carry out PCR amplification reaction, thereby well verifying the effectiveness and the specificity of the specific primers and the intention bee primers of the Chinese bees. It can be seen that both the Chinese bee primer and the Italian bee primer can amplify fragments with specific sizes of corresponding species well, and target bands are amplified in non-native species.
Example 2: sensitivity test
The middle bee-specific primers and the honey specific primers were amplified under the conditions of example 1 using 100ng, 10ng, 1ng, 0.1ng, 0.01ng, 0.001ng of the middle bee genomic DNA, the middle bee DNA extract and the honey DNA extract as templates, respectively. The results are shown in FIGS. 3-6, wherein FIG. 3 shows the results of amplification of the genomic DNA templates of the bees at different concentrations; FIG. 4 shows the results of amplification of DNA extracts of medium honey at various concentrations; FIG. 5 shows amplification results of the genomic DNA templates of Ipomoea batatas at different concentrations; FIG. 6 shows the amplification results of DNA extracts of honey at different concentrations. The numbering in lanes is as follows: m: is BM5000DNA Marker; l1:100ng; l2:10ng; l3:1ng; l4:0.1ng; l5:0.01ng; l6:0.001ng; l7: blank control. The result shows that 0.1ng of template is amplified, and the amplified fragment of the specific primer has better sensitivity. Example 3: detecting the mixing of Chinese bee honey with Italian bee honey
The invention is to identify the bee honey and detect the adulteration thereof, and judge whether the DNA of the bee is existed or not through the PCR amplification of the bee-intention primer, thereby judging whether the bee honey is adulterated or not.
1 preparation of Mixed samples
1.1. Mixed genomic DNA preparation
The extracted genomic DNA of Chinese bees and Italian bees is diluted to the same concentration, and mixed according to the proportion of the Italian bees genomic DNA of 0%, 1%, 5%, 10%, 30%, 50%, 80% and 100%, so as to prepare a mixed genomic DNA sample.
1.2. Preparation of mixed honey DNA extract
The method comprises the steps of preparing mixed honey samples with different proportions by using Chinese bee honey and Chinese bee honey, wherein the Chinese bee honey comprises the following proportions in sequence: 0%, 1%, 5%, 10%, 30%, 50%, 80% and 100%. The mixed honey DNA was extracted using the method of example 1, to obtain a mixed honey DNA extract.
The 2 PCR and electrophoresis method were the same as in example 1.
The results of the 3-gel electrophoresis are shown in FIGS. 7 and 8.
4 analysis of results
Gel imaging obtained by double PCR amplification of mixed genomic DNA is shown in FIG. 7, gel imaging obtained by double PCR amplification of mixed honey DNA extract is shown in FIG. 8, and the numbers in lanes are as follows: m: is BM5000DNA Marker; l1: the proportion of the Italian bee template is 0 percent; l2: the proportion of the Italian bee template is 1%; l3: 5% of a Italian bee template; l4: the proportion of the Italian bee template is 10%; l5: 30% of a template of Italian bees; l6: 50% of a template of Italian bees; l7: 80% of a template of Italian bees; l8: the proportion of the Italian bee template is 100 percent. It can be seen that a specific band of 330bp was observed at the time of mixing 1% of the template DNA of Ipomoea batatas; and as the proportion of the template DNA of the Ipomoea batatas rises, the brightness of the specific band of the Ipomoea batatas gradually increases, and the brightness of the specific band (194 bp) of the Chinese bees gradually decreases. The sensitivity of the method for identifying the true and false of the honey in the invention can reach 1%.
Example 3 screening of specific primers for bees and Italian bees
The different pairs of Chinese bee primers and Italian bee primer were used as shown in Table 3, and the Chinese bee genomic DNA and Italian bee genomic DNA were used as templates, respectively, and amplified under the conditions of example 1, followed by electrophoresis detection. The results are shown in FIGS. 9-10, where FIG. 9 shows the amplification results for different Chinese bee primer pairs; FIG. 10 shows the amplification results of different bee primer pairs. The numbering in the lanes of FIG. 9 is as follows: m: is BM5000DNA Marker; l1: chinese bee primers C1-F, C1-R and Chinese bee genome DNA; l2: chinese bee primers C1-F, C1-R and Italian bee genome DNA; l3: chinese bee primer C1-F, C1-R and blank control; l4: chinese bee primers C-F, C-R and Chinese bee genome DNA; l5: chinese bee primers C-F, C-R and Italian bee genome DNA; l6: chinese bee primer C-F was used with C-R and blank. The numbering in the lanes of FIG. 10 is as follows: m: is BM5000DNA Marker; l1: italian bee primer M-F, M-R and Chinese bee genome DNA; l2: italian bee primer M-F, M-R and Italian bee genome DNA; l3: italian bee primer M-F and M-R and blank control; l4: italian bee primers M1-F, M1-R and Chinese bee genome DNA; l5: italian bee primers M1-F and M1-R and Italian bee genome DNA; l6: italian bee primer M1-F and M1-R and blank control; l7: italian bee primers M2-F, M2-R and Chinese bee genome DNA; l8: italian bee primers M2-F and M2-R and Italian bee genome DNA; l9: italian bee primer M2-F was used with M2-R and blank.
TABLE 3 Chinese bee PCR amplification primers and Italian bee PCR amplification primers used in this comparative example
The result shows that although the Chinese bee primers C1-F and C1-R can amplify Chinese bee specific bands, under the same operation condition, the amplified bands have lower brightness than the bands amplified by the primer pairs C-F and C-R provided by the invention, which indicates that the primers provided by the invention have the best effect; the results of the Italian bee primer are similar, and the effect of the primer pair M1-F and M1-R and the effect of the primer pair M2-F and M2-R are inferior to those of the primer pair M-F and M-R provided by the invention; comprehensively considering, the primer pair provided by the invention has an optimal effect.
Sequence listing
<110> university of agriculture in China
<120> PCR method for identifying Chinese bee honey and Italian bee honey and application thereof
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 194
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
ccgagcattt atttgtctcc gagatttatg acagtttatt gaaaaaaata gtttccactt 60
ctattaagtt taaacagaag atgatattac gaacaagaag attaccgttt tcgagaagaa 120
tttaaaattc aacatttaaa attgtatatt tttattctaa ttctaaagat aatatcgttg 180
acccaatggc gatt 194
<210> 2
<211> 330
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
tggcggtaaa ggacgatgat tgacgactgc aaagaaacta tcttgccgtg cccgggagcc 60
atattatgac gtattcatgt gagcctttat atacaagtga aattcatatc cttagaaaat 120
ttcttcttaa ttgaattgga aaaaatcaca atggagcatg ataataatca ataataatat 180
tatgtatttg caataatagt aactcaaatt agaagagaat tcctcagatt aaattaatgc 240
ataaagatat caagataccg acaatctgga attaatcagc atatacttaa attttttcga 300
actagtatta cgttgatcgc agttgagtca 330
<210> 3
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
ccgagcattt atttgtct 18
<210> 4
<211> 16
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
aatcgccatt gggtca 16
<210> 5
<211> 14
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
tggcggtaaa ggac 14
<210> 6
<211> 16
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
tgactcaact gcgatc 16
<210> 7
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
ccgagcattt atttgtct 18
<210> 8
<211> 16
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
ttaatcgcca ttgggt 16
<210> 9
<211> 16
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
atctaatggc ggtaaa 16
<210> 10
<211> 16
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
aactgcgatc aacgta 16
<210> 11
<211> 17
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
aaatcacaat ggagcat 17
<210> 12
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
ttagcctagt tagaacagaa 20
Claims (8)
1. The primer for identifying the Chinese bee honey and the Italian bee honey is characterized in that the primer pair for detecting the Chinese bee honey has the sequences shown as SEQ ID No.3 and SEQ ID No. 4;
and a primer pair for detecting the honey of the Italian bees, wherein the sequences of the primer pair are shown as SEQ ID No.5 and SEQ ID No. 6.
2. A test kit for identifying chinese bee honey and chinese bee honey, comprising the primer of claim 1.
3. The test kit of claim 2, further comprising 2 x Taq DNA MasterMix.
4. The PCR method for identifying the Chinese bee honey and the Italian bee honey is characterized by comprising the following steps of:
s1, extracting genome DNA of honey,
s2, carrying out PCR amplification on the extracted honey genome DNA by adopting primers shown as SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 and SEQ ID No. 6;
s3, electrophoresis is carried out on the amplified product, and a result is judged;
in step S3, if a specific band with the size of 194bp appears in the electrophoresis result, the specific band with the size of 330bp appears in the electrophoresis result, the specific band is indicated to contain Chinese bee honey.
5. The PCR method according to claim 4, wherein in step S1, the pretreatment before extracting the DNA of the honey is to dilute the honey by adding sterile water, shaking and mixing, incubating for 10min at 40 ℃, centrifuging for 30min at 7000r/m, and discarding the supernatant; the precipitate was suspended in ultrapure water, centrifuged at 13000r/min for 20min, the supernatant was discarded, and the precipitate was retained for DNA extraction.
6. The method according to claim 4, wherein in step S2, the final concentration of the primer pair in the reaction system for PCR amplification is 0.25. Mu. Mol/L.
7. The method according to claim 4, wherein in step S2, the PCR reaction procedure is as follows: initial pre-denaturation at 94 ℃ for 2min; denaturation at 94℃for 30s, annealing at 58℃for 30s, elongation at 72℃for 30s,35 cycles; extending at 72℃for 10min.
8. Use of the primer according to claim 1 for the identification of apis cerana honey and apis cerana honey.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111079089.1A CN113862374B (en) | 2021-09-15 | 2021-09-15 | PCR method for identifying Chinese bee honey and Italian bee honey and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111079089.1A CN113862374B (en) | 2021-09-15 | 2021-09-15 | PCR method for identifying Chinese bee honey and Italian bee honey and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113862374A CN113862374A (en) | 2021-12-31 |
| CN113862374B true CN113862374B (en) | 2023-06-13 |
Family
ID=78995887
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111079089.1A Active CN113862374B (en) | 2021-09-15 | 2021-09-15 | PCR method for identifying Chinese bee honey and Italian bee honey and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113862374B (en) |
-
2021
- 2021-09-15 CN CN202111079089.1A patent/CN113862374B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN113862374A (en) | 2021-12-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102676514B (en) | Single nucleotide polymorphism (SNP) mark relevant with milk production traits of Chinese Holstein dairy cattle and application thereof | |
| CN108165647B (en) | Molecular marking method for identifying non-heading Chinese cabbage Suzhou green, dwarf yellow and Wuta-tsai | |
| CN112080576A (en) | SSR molecular marker for distinguishing and identifying apocarya from pecan, Dabie pecan and Hunan pecan and application thereof | |
| CN113862374B (en) | PCR method for identifying Chinese bee honey and Italian bee honey and application | |
| CN105861657B (en) | One kind CNV segment relevant to mastitis for milk cows resistance and its application | |
| CN110029172B (en) | Double PCR detection kit for equine and donkey-derived components | |
| CN104988240B (en) | Differentiate the method for bee colony Higher production royal jelly character using SNP marker rs16287910 | |
| CN108060208B (en) | Method for identifying snake bile | |
| CN107574257B (en) | Core SSR primer and kit for identifying pea variety and purity | |
| CN106957915B (en) | Primer and kit for detecting apple tree/pear tree rot pathogen and quantitative detection method | |
| CN114525355A (en) | Method for identifying authenticity of Vicia species and special SSR primer combination thereof | |
| KR100487995B1 (en) | Early selection analysis method for gene of pigs with quality meat | |
| CN111500773B (en) | Fluorescent quantitative RT-PCR primer, probe and kit for identification of serotype of epidemic hemorrhagic disease virus | |
| CN105734144A (en) | Real-time fluorescent PCR (polymerase chain reaction) specific detection system of gadus morhua and application | |
| KR101253939B1 (en) | Bio-markers for detecting tissue-specific expression in bovine's muscle regions, primer sets for detecting the bio-markers and methods of identification of bovines' muscle regions using the same | |
| CN114561479B (en) | Primer for identifying iron-shell shrimp individuals in macrobrachium rosenbergii and application of primer | |
| JP2010011764A (en) | Method for judging kind of raw material tea of green tea extract | |
| CN111944919B (en) | Banana fusarium wilt tropical No.4 small species visual detection technology system capable of being operated in field and at normal temperature | |
| CN113755607B (en) | Universal primer for specifically identifying donkey, horse and mule source components in donkey-hide gelatin and raw materials thereof, detection method and application | |
| CN111334605B (en) | Specific primer, probe, kit and method for detecting sugarcane components | |
| CN110317895B (en) | LAMP primer group for detecting sweet potato source components and application thereof | |
| CN105734142A (en) | Real-time fluorescent PCR (polymerase chain reaction) specific detection system of acipenser ruthenus and application | |
| CN112359131A (en) | LAMP primer group for detecting barley stripe disease and application thereof | |
| Soumiya et al. | Evaluation of Easy and Economic Protocols for High Quality Genomic DNA Extraction and Sex Determination in Papaya Using SCAR Markers | |
| Anitha et al. | Genetic Diversity Analysis of Castor (Ricinus communis L.) Varieties Using RAPD Markers |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |