CN109825618A - A kind of dual PCR detection method detecting Bee venoms and bee larva bacillus - Google Patents
A kind of dual PCR detection method detecting Bee venoms and bee larva bacillus Download PDFInfo
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Abstract
The present invention provides a kind of dual PCR detection method for detecting Bee venoms and bee larva bacillus, for Bee venoms in honeybee and bee product and the detection of bee larva bacillus.The method of the present invention has high degree of specificity to the detections of Bee venoms and bee larva bacillus, between each other and with other honeybee pathogenic bacteria no cross reactions, and it is reproducible；The sensitivity for detecting two kinds of pathogenic bacteria can reach about 1 pg/ μ L；Whole operation does not need expensive instrument and reagent, and once-through operation can detect two kinds of cause of diseases simultaneously, and total overall reaction is completed within 2 hours.The present invention provides new technological means for the quick detection of Bee venoms in honeybee and bee product and bee larva bacillus, for bee colony healthy and safe in protectorate and the sound development of bee product international trade is promoted to have important practical significance and practical application value.
Description
Technical field
The present invention relates to a kind of dual PCR detection methods for detecting Bee venoms and bee larva bacillus, belong to
In field of biotechnology, Bee venoms and the detection of bee larva bacillus suitable for honeybee and bee product.
Background technique
Honeybee American foul brood (American foulbrood, AFB) and European foul brood (European
Foulbrood, EFB) it is two kinds of bacillary, acute, devastating infectious diseases that can cause bee larva death, honeybee can be caused young
Large quantities of death of worm eventually lead to the destruction of entire bee colony, endanger huge.The Ministry of Agriculture and State Administration of Quality Supervision, Inspection and Quarantine's joint are formulated newest
The two is classified as two class infectious diseases by " People's Republic of China (PRC) enter the territory animal quarantine epidemic disease register ".World Organization for Animal Health
(OIE) it is also included in quarantine register, is classified as inlet and outlet honeybee and bee product quarantine object.
American foul brood is to endanger one of the main bacterial disease of bee larva growth at present, and it is numerous that less serious case reduces bee colony
Power is grown and acquires, severe one leads to the destruction of bee colony.Disease propagation is rapid, it is serious to cause harm, and is difficult to cure after generation, it is difficult to root
It removing, and often recurs, the bee larva of especially 1 age in days is very sensitive to bacillus larvae, once infection will soon
It is dead.
European foul brood is a kind of pernicious infectious disease of bee larva, propagates fast, harm greatly, larva can be caused large quantities of
It is dead.Diseased bee colonies cannot normal gathering honey and breeding, bee colony, which weakens, is even annihilated.By epidemiological survey, currently without hair
Existing any honeybee has resistance, Eastern bee more easy infection than apis mellifera, especially middle bee morbidity to European foul brood
It is even more serious.
The pathogen of American foul brood is bee larva bacillus (Paenibacillus Larvae, PL), is
A kind of gram-positive bacterium of spore form.The gemma of generation has 7 layers of structure to surround, and this special tectonic makes larva gemma
The gemma of bacillus has especially strong vitality, has extremely strong resistance to heat, chemical substance etc., in the severe ring such as high temperature drying
It can at least survive 35 years under border.It can survive 4 weeks -6 weeks under the gemma sunlight irradiation being suspended in honey.Bacillus larvae is this
The resistance superpower to external environment makes him that can survive in honey, royal jelly, propolis and Bee Pollen for a long time.Honeybee Europe
Continent foulbrood is caused by Bee venoms (Melissococcus pluton, MP), which is a kind of gram
Positive bacteria, but have dyeing property unstability, it sometimes may be Gram-negative bacteria, amphimicrobian.Bee venoms pair
Extraneous poor environment has especially strong resistance, can survive in honeycomb or honey 1 year or so, can under room temperature and drying condition
Survival 17 months, can also survive in honey, royal jelly, propolis and Bee Pollen, can be propagated by honeybee and bee product.
In recent years, as the international trade of honeybee and bee product gradually increases, thus bring honeybee epidemic disease safety problem
It is increasingly subject to each side's concern.
China is few to the research of bee disease, except to the important epidemic disease biology of honeybee, epidemiology and bee colony disease diagnosis side
Face is carried out outside some researchs, and is almost blank for the detection research of bee class cause of disease in bee product.It is total according to former national quality inspection
The requirement of office, will reinforce the detection of bee class cause of disease in bee product from now on, especially to honeybee American foul brood and Europe children
Worm decaying disease reinforces quarantine, passes through the incoming country of product to prevent bee disease.
For the detection of import bee product, it is concentrated mainly on medicament residue, production and processing health and the quality such as adulterated at present
The problem of secure context, especially controls strictly the artificial pollution in antibiotic residue and process of manufacture, and for bee
The bee class cause of disease that may be carried in product lacks enough attention.Due to using honeybee the control of antibiotic, some sanitary pipes
The bad apiary of manage bar part leads to bee class pestilence by poor management, and some cause of diseases can pass through the products such as honey
It is propagated, this just increases the incoming risk of cause of disease.Simultaneously because bee product import volume is increasing in recent years, wherein quite
A part is unprocessed honey raw material, and it is higher to propagate a possibility that bee is sick.Therefore, it is highly desirable to implement import bee product
The detection of bee class epidemic disease, works out effective detection method.
So the present invention establishes a kind of double PCR detection side for detecting Bee venoms and bee larva bacillus
Method can detect the most important pathogen of both honeybees simultaneously, applied to the quick detection of honeybee and bee product, for protection
The sound development of domestic apiculture prevents epidemic disease incoming, the development of bee product international trade is promoted to have important practical significance.
Summary of the invention
It is an object of the invention to a kind of double PCR detection sides for detecting Bee venoms and bee larva bacillus
Method has sensitivity high, specific for Bee venoms in honeybee and bee product and the detection of bee larva bacillus
By force, economic and convenient feature can satisfy current honeybee disease diagnosis and inlet and outlet bee product quarantine demand.
In order to achieve the object of the present invention, technical scheme is as follows:
A kind of double PCR primer detecting Bee venoms and bee larva bacillus, including for expanding honeycomb honeybee
Upstream primer MP-F, the downstream primer MP-R of coccus specific fragment and the upstream for expanding bee larva bacillus specific fragment are drawn
Object PL-F, downstream primer PL-R, each primer nucleotide sequences are as follows:
Upstream primer MP-F:5 '-AGAAGAGTGGCGGACGGGTGA -3 '；
Downstream primer MP-R:5 '-ACCGTCACGAGGAAAACAGTTACTC -3 '；
Upstream primer PL-F:5 '-GCCAAGGAAGAACGGCCAGGG -3 '；
Downstream primer PL-R:5 '-CCACCTCTGCGTGCTGGCAA -3 '.
Another object of the present invention is to provide one kind to be based on above-mentioned detection Bee venoms and bee larva gemma bar
The detection method of bacterium double PCR primer, comprising the following steps:
(1) double PCR reaction system configures: 2 × PCR Master Mix 12.5 μ L, 10 μm of ol/L upstream and downstream primer MP-F
With each 0.5 μ L of MP-R, 10 μm of ol/L upstream and downstream primer PL-F and PL-R 2 μ L of each 0.75 μ L, sample to be tested DNA, then plus
Enter 8.0 μ L of deionized water, makes to react 25.0 μ L of total volume.
(2) double PCR response procedures: 95 DEG C of 3 min of initial denaturation；95 DEG C of denaturation 45 s, 64 DEG C of 30 s of annealing,
72 DEG C of 45 s of denaturation, totally 35 recycle；72 DEG C of 5 min of extension, 4 DEG C of heat preservations.
(3) pcr amplification product tests and analyzes and determines: taking 8 μ L pcr amplification products, 0.01% GelRed core is being added
On 1.5% Ago-Gel of acid dye after electrophoresis, result judgement is carried out after detecting pcr amplification product on gel imaging system:
When the band for being 401 bp containing size in the double PCR amplified production, then the sample to be tested contains honeycomb honeybee
Coccus；
When the band for being 706 bp containing size in the double PCR amplified production, then the sample to be tested contains bee larva
Bacillus；
When the band in the double PCR amplified production simultaneously containing band and 706 bp that size is 401 bp, then it is described to
Sample contains Bee venoms and bee larva bacillus simultaneously；
When in the double PCR amplified production do not contain size be 401 bp band and without containing size be 706 bp item
Band, then the sample to be tested without containing Bee venoms and does not contain bee larva bacillus.
The invention has the following advantages that
(1) good stability and specificity: system of the present invention compares analysis by a large amount of Blast, has screened honeycomb honeybee ball
Bacterium and highly conserved, the special gene order of bee larva bacillus；By after being compared to multiple groups primer select two
To best primer；The specificity of reaction is further increased by adjusting annealing temperature, this method is to Bee venoms and honey
The detection of larva of bee bacillus have high degree of specificity, between each other and with other honeybee pathogenic bacteria no cross reactions, in addition
Also there is preferable repeatability；
(2) high sensitivity: the present invention further promotes its sensitivity by the multiple optimization to primer ratio and annealing temperature,
The sensitivity for detecting two kinds of target genes can reach 1 pg/ μ L, and synchronism is good；
(3) double check, time saving and energy saving, testing cost is low: Bee venoms and bee larva bacillus as honeybee most
Important is also the most common pathogenic bacteria, is not easily distinguishable according to clinical symptoms, needs to identify by cause of disease, can be with one using the present invention
Two kinds of pathogens of secondary detection, total overall reaction is completed within 2 hours, time saving and energy saving.Meanwhile valuableness is not needed using the present invention
Fluorescent PCR instrument does not need synthesis expensive probe and corresponding reagent yet, and testing cost is low, easy to operate, is conducive to department, base
Laboratory applications are promoted.
Detailed description of the invention
Fig. 1 is the Bee venoms specific primer Gradient annealing temperature PCR amplification rear electrophoresis result of embodiment 1.Its
Middle M:100bp Ladder DNA Marker；1~8 annealing temperature is respectively 50 DEG C, 51 DEG C, 53 DEG C, 55.9 DEG C, 59.3 DEG C,
62.1℃、64.1℃、65℃。
Fig. 2 is the bee larva bacillus specific primer Gradient annealing temperature PCR amplification rear electrophoresis knot of embodiment 1
Fruit.Wherein M:100bp Ladder DNA Marker；1~8 annealing temperature is respectively 50 DEG C, 51 DEG C, 53 DEG C, 55.9 DEG C, 59.3
℃、62.1℃、64.1℃、65℃。
Fig. 3 is the primer ratio of the Bee venoms of embodiment 2 and the dual PCR detection method of bee larva bacillus
Example and annealing temperature optimum results.Primer concentration: 10 μm of ol/L upstream and downstream primer MP-F and MP-R 1~20 are 0.5 μ L；
10 μm of ol/L upstream and downstream primer PL-F and PL-R:1~5 are respectively respectively 0.75 μ L of 0.6 μ L, 6~10,11~15 difference
It is respectively 1.0 μ L for 0.9 μ L, 16~20.Annealing temperature: 1,6,11,16 be 58 DEG C；2,7,12,17 be 60 DEG C；3,8,13,18
It is 62 DEG C；4,9,14,19 be 64 DEG C；5,10,15,20 be 66 DEG C.
Fig. 4 is the specificity of the Bee venoms of embodiment 4 and the dual PCR detection method of bee larva bacillus
Measurement result.Wherein 1: Bee venoms；2: Bee venoms+Ascosphaera apis；3: Bee venoms+honeybee pair wound
Cold bacillus；4: Bee venoms+Aspergillus flavus；5: Bee venoms+Eastern bee microsporidian；6: Bee venoms
+ bee larva bacillus；7: Bee venoms+bee larva bacillus mode bacterium；8: bee larva bacillus；9:
Bee larva bacillus+Ascosphaera apis；10: bee larva bacillus+bee paratyphus bacillus；11: bee larva bud
Spore bacillus+Aspergillus flavus；12: bee larva bacillus+Eastern bee microsporidian.
Fig. 5 is the sensitivity of the Bee venoms of embodiment 5 and the dual PCR detection method of bee larva bacillus
Measurement result.Wherein M:100bp Ladder DNA Marker I；1~8 Bee venoms and bee larva bacillus mould
Plate DNA concentration be respectively 1 μ g/ μ L, 100 ng/ μ L, 10 ng/ μ L, 1 ng/ μ L, 100 pg/ μ L, 10 pg/ μ L, 1 pg/ μ L,
0.1 pg/μL。
Specific embodiment
In order to which the present invention is furture elucidated rather than the limitation present invention, it is illustrated with reference to embodiments.Following implementations
Experimental method described in example is unless otherwise specified conventional method；Honeycomb honeybee ball is removed in the reagent and biomaterial
The bacterial strains such as bacterium, bee larva bacillus are outer by specialized agency's offer, commercially obtain unless otherwise specified.
Embodiment 1:
A kind of double PCR primer detecting Bee venoms and bee larva bacillus, including for expanding honeycomb honeybee
Upstream primer MP-F, the downstream primer MP-R of coccus specific fragment and the upstream for expanding bee larva bacillus specific fragment are drawn
Object PL-F, downstream primer PL-R, each primer nucleotide sequences are as follows:
Upstream primer MP-F:5 '-AGAAGAGTGGCGGACGGGTGA -3 '；
Downstream primer MP-R:5 '-ACCGTCACGAGGAAAACAGTTACTC -3 '；
Upstream primer PL-F:5 '-GCCAAGGAAGAACGGCCAGGG -3 '；
Downstream primer PL-R:5 '-CCACCTCTGCGTGCTGGCAA -3 '.
As depicted in figs. 1 and 2, in 50 DEG C~65 DEG C of annealing temperature, the two all has good expanding effect.
Embodiment 2:
The primer ratio and annealing temperature of Bee venoms and the dual PCR detection method of bee larva bacillus optimization knot
Fruit:
(1) double PCR reaction system configures: 2 × PCR Master Mix 12.5 μ L, 10 μm of ol/L upstream and downstream primer MP-F
With each 0.5 μ L of MP-R, 10 μm of ol/L upstream and downstream primer PL-F and PL-R each the μ L of 0.60 μ L~0.75, sample to be tested DNA 2
μ L, then supplementing deionized water makes to react 25.0 μ L of total volume.
(2) double PCR response procedures: 95 DEG C of 3 min of initial denaturation；95 DEG C of 45 s of denaturation, 58 DEG C~66 DEG C are moved back
Fire 30 s, 72 DEG C of 45 s of denaturation, totally 35 recycle；72 DEG C of 5 min of extension, 4 DEG C of heat preservations.
(3) optimum results: primer ratio and annealing temperature optimum results move back as shown in figure 3, when primer ratio is 1: 1.5
When fiery temperature is 64 DEG C, the two expanding effect is consistent, and electrophoretic band is also brighter, so Bee venoms in reaction system
Primer amount with bee larva bacillus is respectively 0.5 μ L and 0.75 μ L, and optimum annealing temperature is 64 DEG C.
Embodiment 3:
Utilize the detection of the double PCR primer optimization of detection Bee venoms provided by the invention and bee larva bacillus
Method, comprising the following steps:
(1) double PCR reaction system configures: 2 × PCR Master Mix 12.5 μ L, 10 μm of ol/L upstream and downstream primer MP-F
With each 0.5 μ L of MP-R, 10 μm of ol/L upstream and downstream primer PL-F and PL-R 2 μ L of each 0.75 μ L, sample to be tested DNA, then plus
Enter 8.0 μ L of deionized water, makes to react 25.0 μ L of total volume.
(2) double PCR response procedures: 95 DEG C of 3 min of initial denaturation；95 DEG C of denaturation 45 s, 64 DEG C of 30 s of annealing,
72 DEG C of 45 s of denaturation, totally 35 recycle；72 DEG C of 5 min of extension, 4 DEG C of heat preservations.
(3) pcr amplification product tests and analyzes and determines: taking 8 μ L pcr amplification products, 0.01% GelRed core is being added
On 1.5% Ago-Gel of acid dye after electrophoresis, result judgement is carried out after detecting pcr amplification product on gel imaging system:
If the band for being 401 bp containing size in the double PCR amplified production, the sample to be tested contain honeycomb honeybee
Coccus；
If the band for being 706 bp containing size in the double PCR amplified production, the sample to be tested contain bee larva
Bacillus；
If not containing the band that size is 401 bp and the item for being 706 bp without containing size in the double PCR amplified production
Band, then the sample to be tested without containing Bee venoms and does not contain bee larva bacillus.
Embodiment 4:
The specific assay of Bee venoms and the dual PCR detection method of bee larva bacillus:
Respectively with 4 kinds of common honeybees such as Ascosphaera apis, bee paratyphus bacillus, Aspergillus flavus and Eastern bee microsporidian
Pathogen (helminth) be sample, according to a conventional method extract DNA after respectively with Bee venoms or bee larva bacillus
Collocation sample-adding carries out PCR reaction and result judgement in the method for embodiment 3, as shown in Figure 4, only Bee venoms and honeybee
Bacillus larvae (mode bacterium) generates the purpose band of specificity, illustrates that this kit has stronger specificity.
Embodiment 5:
The measurement of the sensitivity of Bee venoms and the dual PCR detection method of bee larva bacillus:
Respectively using the plasmid containing Bee venoms and bee larva bacillus purpose segment as template, carries out 10 times and be incremented by
It is added in reaction system after being diluted to corresponding concentration, PCR reaction and result judgement is carried out in the method for embodiment 3, by Fig. 5
It is found that Bee venoms and bee larva bacillus 1pg/ μ L template be still after 1.5% agarose gel electrophoresis for PCR product
Apparent DNA band can be amplified, it was confirmed that the sensitivity of 2 targets can reach in dual PCR detection method of the invention
To 1 pg/ μ L, there is preferable sensitivity.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, is all covered by the present invention.
SEQUENCE LISTING
<110>Inspection & Quarantine Technology Center of Fujian Entry-Exit Inspection & Quar
<120>a kind of dual PCR detection method for detecting Bee venoms and bee larva bacillus
<130> 4
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213>artificial sequence
<400> 1
agaagagtgg cggacgggtg a 21
<210> 2
<211> 25
<212> DNA
<213>artificial sequence
<400> 2
accgtcacga ggaaaacagt tactc 25
<210> 3
<211> 21
<212> DNA
<213>artificial sequence
<400> 3
gccaaggaag aacggccagg g 21
<210> 4
<211> 20
<212> DNA
<213>artificial sequence
<400> 4
ccacctctgc gtgctggcaa 20
Claims (2)
1. a kind of double PCR primer for detecting Bee venoms and bee larva bacillus, it is characterised in that: including being used for
Upstream primer MP-F, downstream primer MP-R and the amplification bee larva bacillus for expanding Bee venoms specific fragment are special
Upstream primer PL-F, the downstream primer PL-R of segment, each primer nucleotide sequences are as follows:
Upstream primer MP-F:5 '-AGAAGAGTGGCGGACGGGTGA -3 '；
Downstream primer MP-R:5 '-ACCGTCACGAGGAAAACAGTTACTC -3 '；
Upstream primer PL-F:5 '-GCCAAGGAAGAACGGCCAGGG -3 '；
Downstream primer PL-R:5 '-CCACCTCTGCGTGCTGGCAA -3 '.
2. a kind of dual PCR detection method for detecting Bee venoms and bee larva bacillus, which is characterized in that including
Following steps:
(1) double PCR reaction system configures: 12.5 μ L, 10 μm of ol/L of 2 × PCR Master Mix are described in claim 1
Upstream and downstream primer MP-F and MP-R each 0.5 μ L, 10 μm of ol/L upstream and downstream primer PL-F and PL-R described in claim 1 is each
Then 8.0 μ L of deionized water is added in 2 μ L of 0.75 μ L, sample to be tested DNA, make to react 25.0 μ L of total volume；
(2) double PCR response procedures: 95 DEG C of 3 min of initial denaturation；95 DEG C of denaturation 45 s, 64 DEG C of annealing 30 s, 72 DEG C
45 s are denaturalized, totally 35 circulations；72 DEG C of 5 min of extension, 4 DEG C of heat preservations；
(3) pcr amplification product tests and analyzes and determines: taking 8 μ L pcr amplification products, 0.01% GelRed nucleic acid dye is being added
On 1.5% Ago-Gel of material after electrophoresis, result judgement is carried out after detecting pcr amplification product on gel imaging system:
When the band for being 401 bp containing size in the double PCR amplified production, then the sample to be tested contains honeycomb honeybee
Coccus；
When the band for being 706 bp containing size in the double PCR amplified production, then the sample to be tested contains bee larva
Bacillus；
When the band in the double PCR amplified production simultaneously containing band and 706 bp that size is 401 bp, then it is described to
Sample contains Bee venoms and bee larva bacillus simultaneously.
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