CN106282322A - A kind of Laboratory Animal Resource quality quick monitoring method - Google Patents
A kind of Laboratory Animal Resource quality quick monitoring method Download PDFInfo
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- CN106282322A CN106282322A CN201510281845.7A CN201510281845A CN106282322A CN 106282322 A CN106282322 A CN 106282322A CN 201510281845 A CN201510281845 A CN 201510281845A CN 106282322 A CN106282322 A CN 106282322A
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- 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
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- 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
- C12Q2600/16—Primer sets for multiplex assays
Abstract
The present invention relates to a kind of Laboratory Animal Resource quality quick monitoring method.Disclose a kind of can the method for objective microbe infection conditions in specificity identification Laboratory Animal Resource, screening obtains the extraordinary PCR primer of specificity, has detection sensitivity and accuracy.
Description
Technical field
The invention belongs to field of biology, more particularly it relates to an Laboratory Animal Resource quality
Quick monitoring method.
Background technology
The application of Laboratory Animal Resource (embryo, sperm etc.) Refrigeration Technique has many advantages.In Medical Biology
In research, needing the laboratory animal of various strain, maintaining animal strains in isolator needs to spend in a large number
Man power and material, cost dearly.Animal resources as will be temporarily do not needed carry out freezing, it is possible to long
Phase preserves, not only safety but also can save man power and material.Animal resources transport is used to replace live animal transport,
Not only facilitate but also transmission of disease can be reduced, it is simple to the mutual exchange of international laboratory animal strain.Additionally,
Valuable hereditary material can be preserved, set up gene bank, prevent the genetic drift of laboratory animal.
If in Laboratory Animal Resource with pathogenic microorganism, can to the quality of sexual cell etc., preservation and
Effects of being impregnated etc. produce impact, and may cause also carrying these cause of diseases micro-in the laboratory animal finally obtained
Biology, finally affects Quality of Experimental Animals.And pathogenic microorganism can cause animal epidemic disease in laboratory animal
, even there is death in sick performance, makes animal reproduction rate decline and animal development is bad, affect animal self
Stability and reactivity, even cause Amphixenosis, have a strong impact on zooperal being normally carried out,
And the health and safety of keeper and experimenter is threatened.
Having known together at present, Laboratory Animal Resource (embryo, sperm etc.) refrigeration operation is not the most aseptic technology,
In the transportation of animal, in the refrigerating process of resource, including the reagent needed for freezing and laboratory
Environment may all can cause in Laboratory Animal Resource micro-with cause of disease relatively common in some daily life
Biological.And the main freezing medium that laboratory is used is liquid nitrogen (-196 DEG C), content of microorganisms in liquid nitrogen
The lowest, but, storing and assigning process by microbiological contamination, thus can become freezing bud
Spore, yeast, antibacterial and virus have effective culture medium.In the microbial quality of laboratory animal controls,
A lot of pathogenic microorganisms must be excluded, but at present to these pathogenic microorganisms in Laboratory Animal Resource
The most strictly detect control, so it is (micro-to set up Laboratory Animal Resource (embryo, sperm an etc.) quality
Biological) quick monitoring method is up to standard with the microbial quality ensureing Laboratory Animal Resource.
Staphylococcus aureus, Klebsiella pneumonia, beta hemolytic streptococcus are to compare in laboratory animal
Pathogenic microorganism more typically, they are widely present in air and water, be humans and animals body surface, skin,
Hair, nose, oral cavity, gastral common bacteria, be also conditionality pathogenic bacterium.
National Standard of the People's Republic of China GB14922.2-2011 regulation staphylococcus aureus detection uses
The method that isolated culture combines with biochemical identification.Suspicious specimen is taked to be inoculated in high salt mannitol (SP)
Culture medium, puts 37 DEG C and cultivates 18-24h.About 1mm, projection, the bacterium of yellow is formed in SP culture medium
Fall, the culturing gene S. aureus fermentation mannitol of periphery of bacterial colonies and red yellowing.Picking
Single bacterium colony is transferred in blood agar plate, cultivates 18-24h for 37 DEG C, forms white or golden yellow, protruding, circle
Shape, the group that opaque, smooth surface, surrounding have β zone of hemolysis.After Gram’s staining, microscopy is observed,
It is gram-positive cocci, is arranged in Fructus Vitis viniferae spherical, without pod membrane and spore.When meeting result above,
Make plasma-coagulase experiment.Set up known clotting of plasma enzyme positive and negative Staphylococcus aureus simultaneously
Bacterium and nutrient broth medium are as comparison.But when all there is coagulated mass in known positive strain and strain to be checked,
Can determine whether that bacterial strain to be checked is staphylococcus aureus.
National Standard of the People's Republic of China GB14926.13-2001 regulation Klebsiella pneumonia uses and divides
The method combined with biochemical identification from culture method.Suspicious specimen is taked to be inoculated in cholate sulfur breast agar (DHL)
Culture medium, puts 37 DEG C and cultivates 18-24h.Form pale pink in DHL culture medium, swell greatly, smooth wet
Profit, in mucus shape, adjacent bacterium colony is easily fused into extract sample, and the pull-out of direct inoculation pin picking moment is longer
Silk.After Gram’s staining, microscopy is observed, and it is Gram-negative brevibacterium, single, one-tenth pair or one-tenth short chain
Arrangement, has pod membrane, without spore.Picking list bacterium colony is transferred in potassium iodide or triple sugariron (TSI) agar culture medium
Cultivating 18-24h for 36 ± 1 DEG C, inclined-plane produces acid, and bottom produces acid aerogenesis, or only produces acid not aerogenesis, and hydrogen sulfide is cloudy
Property.VP tests the positive, and nitrate reduction test is positive, and MR is negative, and Xi Mengshi citrate utilizes real
Testing the positive, the malonate test positive, urease-positive, E.C. 4.1.1.18 is positive, ornithine decarboxylation
Enzyme is negative, utilizes glucose and lactose, and indole is negative, and semi-solid dynamic test is negative.All meet
The every testing result person in city, can determine whether that bacterial strain to be checked is Klebsiella pneumonia.
National Standard of the People's Republic of China GB14926.13-2001 regulation beta hemolytic streptococcus uses
The method that isolated culture combines with biochemical identification.Suspicious specimen is taked to be inoculated in blood agar culture-medium,
Put 37 DEG C and cultivate 18-24h.Forming about 1mm on blood agar culture-medium, canescence is circular, protruding, and half
Transparent or opaque, smooth surface, around there are 2-4mm clear-cut, water white transparency zone of hemolysis (β haemolysis)
Petite.After Gram’s staining, microscopy is observed, and it is gram-positive cocci, becomes catenation.Gu
In body culture medium, growth is many in short chain or botryoidalis, then becomes the spherical arrangement of typical chain in fluid medium.
The streptokinase test positive, bacitracin sensitization test is positive.All meeting lists every testing result person, can sentence
Disconnected bacterial strain to be checked is beta hemolytic streptococcus.
For many years, mainly rely in the detection of laboratory animal pathogenic microorganism traditional separation and Culture and
Biochemical identification method, it is universally recognized classical way on microbiology, easily draws clear and definite conclusion,
Experimental result can be verified repeatedly, it is possible at most of labs.But these methods or
Time-consumingly, or specificity is low, and needs testing staff to the colonial morphology of pathogenic microorganism, physical and chemical reaction
Matter etc. have and judge accurately, and just can draw correct conclusion, this just requires that testing staff must have in a down-to-earth manner
Microbiology theoretical basis and abundant the pathogenic microorganism examination experience.
Therefore, for the detection technique of laboratory animal pathogenic microorganism, in addition it is also necessary to improved, to improve
Detection efficiency, improves detection accuracy.
Summary of the invention
It is an object of the invention to provide a kind of Laboratory Animal Resource quality quick monitoring method.
In a first aspect of the present invention, it is provided that objective microbe in a kind of specificity identification Laboratory Animal Resource
The method of infection conditions, described method includes:
(1) with the DNA of Laboratory Animal Resource testing sample as template, with objective microbe specific primer
Carry out PCR amplification, it is thus achieved that pcr amplification product；
(2) analyzing pcr amplification product, if recording positive products, then showing that there is objective microbe infects；
Wherein, described objective microbe is staphylococcus aureus, and its PCR primer is selected from: SEQ ID
The primer of NO:1 and SEQ ID NO:2, SEQ ID NO:3 and the primer of SEQ ID NO:4, SEQ
ID NO:5 and the primer of SEQ ID NO:6, SEQ ID NO:7 and the primer of SEQ ID NO:8,
SEQ ID NO:9 and the primer of SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12's
Primer, or SEQ ID NO:13 and the primer of SEQ ID NO:14；Or
Described objective microbe is Klebsiella pneumonia, and its PCR primer is selected from: SEQ ID NO:15
With the primer of SEQ ID NO:16, or SEQ ID NO:17 and the primer of SEQ ID NO:18；Or
Described objective microbe is beta hemolytic streptococcus, and its PCR primer is selected from: SEQ ID NO:
The primer of 19 and SEQ ID NO:20, SEQ ID NO:21 and the primer of SEQ ID NO:22, SEQ
ID NO:23 and the primer of SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26 draws
Thing, SEQ ID NO:27 and the primer of SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:
The primer of 30, or SEQ ID NO:31 and the primer of SEQ ID NO:32.
In a preference, described Laboratory Animal Resource is freezing Laboratory Animal Resource.
In another preference, described Laboratory Animal Resource includes, but is not limited to: animal embryo, sperm,
Ovum.
In another preference, in step (2), by pcr amplification product is carried out electrophoresis, if electrophoresis strip
Band is positive, then show that there is objective microbe infects.
In another preference, in step (2), by pcr amplification product is carried out electrophoresis, if electrophoresis strip
Band is positive, is checked order by the DNA in positive band, by the gene sequence of sequencing result Yu objective microbe
Row carry out BLAST and compare, thus accurately determine in pcr amplification product whether there is objective microbe.
In another aspect of this invention, it is provided that a kind of PCR primer, described primer is the golden yellow Fructus Vitis viniferae of amplification
The primer of coccus, is selected from: SEQ ID NO:1 and the primer of SEQ ID NO:2, SEQ ID NO:3 and
The primer of SEQ ID NO:4, SEQ ID NO:5 and the primer of SEQ ID NO:6, SEQ ID NO:7
With the primer of SEQ ID NO:8, SEQ ID NO:9 and the primer of SEQ ID NO:10, SEQ ID NO:
The primer of 11 and SEQ ID NO:12, or SEQ ID NO:13 and the primer of SEQ ID NO:14；
Or
Described primer is the primer of amplification staphylococcus aureus, is selected from: SEQ ID NO:15 and SEQ ID
The primer of NO:16, or SEQ ID NO:17 and the primer of SEQ ID NO:18；Or
Described primer is the primer of amplification beta hemolytic streptococcus, is selected from: SEQ ID NO:19 and SEQ
The primer of ID NO:20, SEQ ID NO:21 and the primer of SEQ ID NO:22, SEQ ID NO:23
With the primer of SEQ ID NO:24, SEQ ID NO:25 and the primer of SEQ ID NO:26, SEQ ID
The primer of NO:27 and SEQ ID NO:28, SEQ ID NO:29 and the primer of SEQ ID NO:30,
Or SEQ ID NO:31 and the primer of SEQ ID NO:32.
In another aspect of this invention, it is provided that the purposes of described primer, dynamic for specificity identification experiment
Objective microbe infection conditions in goods and materials source.
In another aspect of this invention, it is provided that one target in specificity identification Laboratory Animal Resource is micro-
The test kit of biological infection situation, including:
The primer of amplification staphylococcus aureus, is selected from: SEQ ID NO:1 and SEQ ID NO:2 draws
Thing, SEQ ID NO:3 and the primer of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6
Primer, SEQ ID NO:7 and the primer of SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:
The primer of 10, SEQ ID NO:11 and the primer of SEQ ID NO:12, or SEQ ID NO:13 and
The primer of SEQ ID NO:14；And/or
The primer of amplification staphylococcus aureus, is selected from: SEQ ID NO:15's and SEQ ID NO:16
Primer, or SEQ ID NO:17 and the primer of SEQ ID NO:18；And/or
The primer of amplification beta hemolytic streptococcus, is selected from: SEQ ID NO:19 and SEQ ID NO:20
Primer, SEQ ID NO:21 and the primer of SEQ ID NO:22, SEQ ID NO:23 and SEQ ID
The primer of NO:24, SEQ ID NO:25 and the primer of SEQ ID NO:26, SEQ ID NO:27 and
The primer of SEQ ID NO:28, SEQ ID NO:29 and the primer of SEQ ID NO:30, or SEQ ID
The primer of NO:31 and SEQ ID NO:32.
In a preference, described test kit also includes: containing the inspection of objective microbe reference culture
Survey standard substance.
In another preference, described test kit also includes selected from following reagent: DNA extraction reagent,
PCR buffer, in archaeal dna polymerase, and/or explanation identification experiment Animal resources, objective microbe infects feelings
The operation instructions of the method for condition.
The other side of the present invention, due to this disclosure, is aobvious to those skilled in the art
And be clear to.
Accompanying drawing explanation
Fig. 1, utilize positive criteria bacterial strain set up PCR system and screening primer schematic flow sheet.
Fig. 2, the schematic flow sheet of method for quick for freezing resource.
Fig. 3, staphylococcus aureus primer 1 is utilized to carry out PCR amplification, the purpose band that electrophoresis obtains
Sequence B last result.
Fig. 4, staphylococcus aureus primer 2 is utilized to carry out PCR amplification, the purpose band that electrophoresis obtains
Sequence B last result.
Fig. 5, Klebsiella pneumonia primer 1 is utilized to carry out PCR amplification, the purpose band that electrophoresis obtains
Sequence B last result.
Fig. 6, Klebsiella pneumonia primer 2 is utilized to carry out PCR amplification, the purpose band that electrophoresis obtains
Sequence B last result.
Fig. 7, beta hemolytic streptococcus primer 1 is utilized to carry out PCR amplification, the purpose bar that electrophoresis obtains
The sequence B last result of band.
Fig. 8, beta hemolytic streptococcus primer 2 is utilized to carry out PCR amplification, the purpose bar that electrophoresis obtains
The sequence B last result of band.
Detailed description of the invention
The present inventor is through in-depth study, and disclosing one first can mesh in specificity identification Laboratory Animal Resource
The method of mark microorganism infection conditions, screening obtains the extraordinary PCR primer of specificity, has detection
Sensitivity and accuracy.
As used herein, described " objective microbe " including: staphylococcus aureus, kerekou pneumonia primary
One or more in Salmonella, beta hemolytic streptococcus.
The present inventor is by the screening to primer, it is thus achieved that one can specificity identification Laboratory Animal Resource, especially
Being the primer that in freezing Laboratory Animal Resource, objective microbe infects, its specificity is good, for target
The DNA sample generation specific amplification of microorganism, and be there is not spy in the DNA not having objective microbe
Specific amplification sample.
Therefore, the present invention provides a kind of primer, and described primer is the primer of amplification staphylococcus aureus,
It is selected from: SEQ ID NO:1 and the primer of SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4
Primer, SEQ ID NO:5 and the primer of SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:
The primer of 8, SEQ ID NO:9 and the primer of SEQ ID NO:10, SEQ ID NO:11 and SEQ ID
The primer of NO:12, or SEQ ID NO:13 and the primer of SEQ ID NO:14.Or, described primer
It is the primer of amplification staphylococcus aureus, is selected from: SEQ ID NO:15 and SEQ ID NO:16 draws
Thing, or SEQ ID NO:17 and the primer of SEQ ID NO:18；Or described primer is amplification beta hemolysis
The streptococcic primer of property, is selected from: SEQ ID NO:19 and the primer of SEQ ID NO:20, SEQ ID NO:
The primer of 21 and SEQ ID NO:22, SEQ ID NO:23 and the primer of SEQ ID NO:24, SEQ
ID NO:25 and the primer of SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28 draws
Thing, SEQ ID NO:29 and the primer of SEQ ID NO:30, or SEQ ID NO:31 and SEQ ID NO:
The primer of 32
These primers of the present invention can also use radiosiotope, biotin, enzyme, fluorescein or other
Chemiluminescent substance is marked.
Utilize primer and the PCR amplification system of the present invention, only need to carry out PCR reaction and/or agarose
Gel electrophoresis, and by judging the presence or absence of corresponding PCR primer, it is possible to judge accurately and rapidly to treat
Whether test sample product exist the infection of objective microbe, and required sample size is little, for the mesh of trace
Mark microorganism also can detect effectively.
Polymerase chain reaction (PCR) technology is technology well known to those skilled in the art, and its ultimate principle is
The method of external enzyme' s catalysis specific DNA fragment.The invention provides the round pcr of a kind of optimization.
The method of the DNA obtaining testing sample can take traditional phenol/chloroform/isoamyl alcohol method, or can adopt
The DNA extraction kit being purchased with some, this kind of test kit is well known to those skilled in the art.
The invention still further relates to a kind of objective microbe infection conditions in specificity identification Laboratory Animal Resource
Test kit, containing foregoing primer in described test kit.Additionally, described test kit also may be used
Containing specific binding probe occurring with a certain site in the amplified production of described primer, described
Probe portability detectable signal such as fluorescence signal, thus be applied to fluorescence quantitative PCR detection.
Additionally, described test kit also can be containing other for objective microbe sense in identification experiment Animal resources
The reagent that dye situation is useful, such as (but not limited to):
(A) various PCR reaction reagent, such as but not limited to: Taq enzyme, PCR buffer, dNTP,
Archaeal dna polymerase etc.；Or
(B) reagent needed for various extractions DNA (i.e. preparing PCR reaction template), such as but not limited to:
Phenol, chloroform, isoamyl alcohol, NaCl etc.；Or
(C) test kit of DNA is extracted.
Additionally, also can be containing objective microbe infection conditions in identification experiment Animal resources in described test kit
Operation instructions and/or S.O.P..Test kit of the present invention can realize quickly detecting, criticizing
The purpose of objective microbe infection conditions in amount test experience Animal resources, substantially reduces laboratory animal money
Source is detected the time needed for pathogenic microorganism and spent reagent.
Compared with traditional morphological analysis method, round pcr more specificity and accuracy, will not be by outward
Boundary's factor (such as temperature change, chemicals) affects and changes, and its sensitivity is high, quick, easy,
It can not only detect efficient pathogenic microorganism, and can detect death and be difficult to the cause of disease cultivated
Microorganism, correlation technique and skill requirement to testing staff are the highest.
The PCR method of the present invention only need to be by required in freezing experiment Animal resources, freezing and resuscitation process
Solution, the frozen material of aquesterilisa that preserves frozen thing liquid nitrogen directly extract the carrying of test kit with minim DNA
Take the DNA of microorganism, design corresponding primer and the laggard performing PCR of corresponding condition (polymerase chain reaction),
Thus quickly understand the microorganism situation in freezing resource.
In addition it is also possible to use LAMP method to replace above-mentioned PCR method three kinds of pathogenic microorganisms of detection,
Time needed for the method is shorter, need not use PCR instrument, and need not by electricity in experimentation
The mode of swimming detects amplified production, can be carried out the detection of amplified production by turbidimetry etc., required for it
Instrument cost lower.LAMP technology is mainly by the identifying purpose gene of four different primer specifics
On six particular sequences, then by having the BstDNA polymerase of strand-displacement activity, at 60 DEG C-65 DEG C
It is catalyzed the synthesis of new chain under constant temperature, thus experiment purpose gene efficient, quickly expands.
Below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that these embodiments are only used for
The present invention is described rather than limits the scope of the present invention.The reality of unreceipted actual conditions in the following example
Proved recipe method, generally writes according to normal condition such as J. Pehanorm Brooker etc., Molecular Cloning: A Laboratory guide, the 3rd
Version, Science Press, the condition described in 2002, or according to the condition proposed by manufacturer.
Embodiment 1, the formulation of experimental technique route and utilize positive criteria bacterial strain screening primer
1, the PCR method of positive control is set up
The objective microbe that this experiment need to detect is: staphylococcus aureus, beta hemolytic streptococcus,
Klebsiella pneumonia.
Above-mentioned three strain reference cultures are bought as sun from Chinese medicine Culture Collection (CMCC)
Property comparison, this three strains reference culture is recovered, extracts the DNA of reference culture after recovery, according to
The gene order design specific primer corresponding thereto of each reference culture, to corresponding primer screening and
PCR system optimizes, comparison after PCR primer being checked order, and verifies positive criteria bacterial strain accuracy, builds with this
Vertical PCR method.Flow process such as Fig. 1.
2, primer screening
Repeatedly study screening through substantial amounts of, the present inventor optimize be applicable to carry out staphylococcus aureus,
Klebsiella pneumonia, the PCR primer of beta hemolytic streptococcus.Specific as follows:
(1), the primer of staphylococcus aureus and PCR condition and PCR amplification system
Primer and amplification condition such as table 1 for staphylococcus aureus.
Table 1
PCR amplification system is as follows:
(2), the primer of Klebsiella pneumonia and PCR condition
Primer and amplification condition such as table 2 for Klebsiella pneumonia.
Table 2
PCR amplification system is as follows:
(3), the primer of beta hemolytic streptococcus and PCR condition
Primer and amplification condition such as table 3 for beta hemolytic streptococcus.
Table 3
PCR amplification system is as follows:
Embodiment 2, positive criteria bacterial strain PCR result product sequencing result BLAST
The positive criteria bacterial strain PCR primer obtained is carried out electrophoresis, order-checking.The sequencing result of acquisition is entered
Row BLAST.Parameter is as follows:
Score is comparison score, and two sequence similarities are the highest；
E Value value is the least, the most credible；
Identities is concordance, it is simply that the ratio that two sequence holdings are consistent.
1, staphylococcus aureus primer 1
Utilize staphylococcus aureus primer 1, it is thus achieved that purpose band 122bp.Blast result is as shown in Figure 3.
2, staphylococcus aureus primer 2
Utilize staphylococcus aureus primer 2, it is thus achieved that purpose band 132bp.Blast result is as shown in Figure 4.
3, Klebsiella pneumonia primer 1
Utilize Klebsiella pneumonia primer 1, it is thus achieved that purpose band 368bp.Blast result is as shown in Figure 5.
4, Klebsiella pneumonia primer 2
Utilize Klebsiella pneumonia primer 2, it is thus achieved that purpose band 423bp.Blast result is as shown in Figure 6.
5, beta hemolytic streptococcus primer 3
Utilize beta hemolytic streptococcus primer 3, it is thus achieved that purpose band 346bp.Blast result such as Fig. 7
Shown in.
6, beta hemolytic streptococcus primer 5
Utilize beta hemolytic streptococcus primer 5, it is thus achieved that purpose band 423bp.Blast result such as Fig. 8
Shown in.
Illustrated by above-mentioned experimental result, staphylococcus aureus, Klebsiella pneumonia and hemolytic
Streptococcus uses PCR method detection to be feasible, and detection accuracy is high.
Embodiment 3, the foundation of method for quick for freezing resource
By solution required in freezing resource (embryo and sperm etc.), freezing and resuscitation process, containing preserving
The aquesterilisa of frozen thing liquid nitrogen extracts test kit (purchased from QIAGEN) with minim DNA respectively and extracts DNA.
With previous embodiment 1 provide for staphylococcus aureus, Klebsiella pneumonia, B-mode molten
Courageous and upright streptococcic PCR primer and corresponding PCR condition and PCR reaction system are to above-mentioned sample DNA
Carry out PCR reaction, if obtaining positive band, with the presence of corresponding bacterial strain in the most provable freezing resource, with
Time can reclaim PCR primer and carry out DNA sequencing and further confirm that.
Operating process is as shown in Figure 2.
The all documents mentioned in the present invention are incorporated as reference the most in this application, just as each literary composition
Offer and be individually recited as with reference to like that.In addition, it is to be understood that reading the above-mentioned teachings of the present invention
Afterwards, the present invention can be made various changes or modifications by those skilled in the art, and these equivalent form of values are same
Fall within the application appended claims limited range.
Claims (10)
1. a method for objective microbe infection conditions, its feature in specificity identification Laboratory Animal Resource
Being, described method includes:
(1) with the DNA of Laboratory Animal Resource testing sample as template, with objective microbe specific primer
Carry out PCR amplification, it is thus achieved that pcr amplification product；
(2) analyzing pcr amplification product, if recording positive products, then showing that there is objective microbe infects；
Wherein, described objective microbe is staphylococcus aureus, and its PCR primer is selected from: SEQ ID
The primer of NO:1 and SEQ ID NO:2, SEQ ID NO:3 and the primer of SEQ ID NO:4, SEQ
ID NO:5 and the primer of SEQ ID NO:6, SEQ ID NO:7 and the primer of SEQ ID NO:8,
SEQ ID NO:9 and the primer of SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12's
Primer, or SEQ ID NO:13 and the primer of SEQ ID NO:14；Or
Described objective microbe is Klebsiella pneumonia, and its PCR primer is selected from: SEQ ID NO:15
With the primer of SEQ ID NO:16, or SEQ ID NO:17 and the primer of SEQ ID NO:18；Or
Described objective microbe is beta hemolytic streptococcus, and its PCR primer is selected from: SEQ ID NO:
The primer of 19 and SEQ ID NO:20, SEQ ID NO:21 and the primer of SEQ ID NO:22, SEQ
ID NO:23 and the primer of SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26 draws
Thing, SEQ ID NO:27 and the primer of SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:
The primer of 30, or SEQ ID NO:31 and the primer of SEQ ID NO:32.
2. the method for claim 1, it is characterised in that described Laboratory Animal Resource is freezing
Laboratory Animal Resource.
3. the method for claim 1, it is characterised in that described Laboratory Animal Resource includes:
Animal embryo, sperm, ovum.
4. the method for claim 1, it is characterised in that in step (2), by expanding PCR
Product carries out electrophoresis, if electrophoretic band is positive, then shows that there is objective microbe infects.
5. the method for claim 1, it is characterised in that in step (2), by expanding PCR
Product carries out electrophoresis, if electrophoretic band is positive, is checked order by the DNA in positive band, order-checking is tied
Fruit carries out BLAST with the gene order of objective microbe and compares, thus accurately determines pcr amplification product
In whether there is objective microbe.
6. a PCR primer, it is characterised in that described primer is the primer of amplification staphylococcus aureus,
It is selected from: SEQ ID NO:1 and the primer of SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4
Primer, SEQ ID NO:5 and the primer of SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:
The primer of 8, SEQ ID NO:9 and the primer of SEQ ID NO:10, SEQ ID NO:11 and SEQ ID
The primer of NO:12, or SEQ ID NO:13 and the primer of SEQ ID NO:14；Or
Described primer is the primer of amplification staphylococcus aureus, is selected from: SEQ ID NO:15 and SEQ ID
The primer of NO:16, or SEQ ID NO:17 and the primer of SEQ ID NO:18；Or
Described primer is the primer of amplification beta hemolytic streptococcus, is selected from: SEQ ID NO:19 and SEQ
The primer of ID NO:20, SEQ ID NO:21 and the primer of SEQ ID NO:22, SEQ ID NO:23
With the primer of SEQ ID NO:24, SEQ ID NO:25 and the primer of SEQ ID NO:26, SEQ ID
The primer of NO:27 and SEQ ID NO:28, SEQ ID NO:29 and the primer of SEQ ID NO:30,
Or SEQ ID NO:31 and the primer of SEQ ID NO:32.
7. the purposes of the primer described in claim 6, target in specificity identification Laboratory Animal Resource
Microorganism infection conditions.
8. a test kit for objective microbe infection conditions in specificity identification Laboratory Animal Resource,
It is characterized in that, including:
The primer of amplification staphylococcus aureus, is selected from: SEQ ID NO:1 and SEQ ID NO:2 draws
Thing, SEQ ID NO:3 and the primer of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6
Primer, SEQ ID NO:7 and the primer of SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:
The primer of 10, SEQ ID NO:11 and the primer of SEQ ID NO:12, or SEQ ID NO:13 and
The primer of SEQ ID NO:14；And/or
The primer of amplification staphylococcus aureus, is selected from: SEQ ID NO:15's and SEQ ID NO:16
Primer, or SEQ ID NO:17 and the primer of SEQ ID NO:18；And/or
The primer of amplification beta hemolytic streptococcus, is selected from: SEQ ID NO:19 and SEQ ID NO:20
Primer, SEQ ID NO:21 and the primer of SEQ ID NO:22, SEQ ID NO:23 and SEQ ID
The primer of NO:24, SEQ ID NO:25 and the primer of SEQ ID NO:26, SEQ ID NO:27 and
The primer of SEQ ID NO:28, SEQ ID NO:29 and the primer of SEQ ID NO:30, or SEQ ID
The primer of NO:31 and SEQ ID NO:32.
9. test kit as claimed in claim 8, it is characterised in that also include in described test kit: contain
There are the examination criteria product of objective microbe reference culture.
10. test kit as claimed in claim 8, it is characterised in that also include in described test kit being selected from
Following reagent: DNA extraction reagent, PCR buffer, archaeal dna polymerase, and/or explanation are identified real
Test the operation instructions of the method for objective microbe infection conditions in Animal resources.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106909631A (en) * | 2017-01-23 | 2017-06-30 | 中国农业大学 | Experimental animal individual traceability method and device |
| CN107513582A (en) * | 2017-10-23 | 2017-12-26 | 蔡慧娜 | Authentication chip, kit and the authentication method of positive blood culture |
| CN107904321A (en) * | 2017-12-21 | 2018-04-13 | 天津宝坻紫荆科技有限公司 | Staphylococcus aureus in food PCR detection primers, probe and detection method |
| CN107904287A (en) * | 2017-12-28 | 2018-04-13 | 吉林出入境检验检疫局检验检疫技术中心 | Staphylococcus aureus and bacillus cereus quick determination method |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101407835A (en) * | 2007-10-12 | 2009-04-15 | 统一企业(中国)投资有限公司 | Genetic marker and method for detecting rhamnose bacterium lacticum |
| CN101570782A (en) * | 2009-03-20 | 2009-11-04 | 杨春华 | Detection kit and detection method for 8 species of pathogenic bacteria in dairy products |
| CN103421906A (en) * | 2013-08-23 | 2013-12-04 | 中国检验检疫科学研究院 | Composition and method for detecting drug resistance of staphylococcus aureus |
| CN104419703A (en) * | 2013-09-11 | 2015-03-18 | 中国科学院上海生命科学研究院 | Method for quickly detecting common pathogenic bacteria with high flux |
-
2015
- 2015-05-28 CN CN201510281845.7A patent/CN106282322A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101407835A (en) * | 2007-10-12 | 2009-04-15 | 统一企业(中国)投资有限公司 | Genetic marker and method for detecting rhamnose bacterium lacticum |
| CN101570782A (en) * | 2009-03-20 | 2009-11-04 | 杨春华 | Detection kit and detection method for 8 species of pathogenic bacteria in dairy products |
| CN103421906A (en) * | 2013-08-23 | 2013-12-04 | 中国检验检疫科学研究院 | Composition and method for detecting drug resistance of staphylococcus aureus |
| CN104419703A (en) * | 2013-09-11 | 2015-03-18 | 中国科学院上海生命科学研究院 | Method for quickly detecting common pathogenic bacteria with high flux |
Non-Patent Citations (4)
| Title |
|---|
| 徐平等: "系统低温生物学技术在实验小鼠资源保存中的建立与应用", 《中国比较医学杂志》 * |
| 李伟: "SPF级大鼠、小鼠五种病原菌检测方法的建立和应用", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
| 林霖等: "荧光PCR快速检测A、C、G群溶血性链球菌", 《食品工业科技》 * |
| 苏明会: "不同季节牦牛精液生产中的细菌污染", 《畜牧兽医杂志》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106909631A (en) * | 2017-01-23 | 2017-06-30 | 中国农业大学 | Experimental animal individual traceability method and device |
| CN106909631B (en) * | 2017-01-23 | 2019-10-08 | 中国农业大学 | Experimental animal individual traceability method and device |
| CN107513582A (en) * | 2017-10-23 | 2017-12-26 | 蔡慧娜 | Authentication chip, kit and the authentication method of positive blood culture |
| CN107904321A (en) * | 2017-12-21 | 2018-04-13 | 天津宝坻紫荆科技有限公司 | Staphylococcus aureus in food PCR detection primers, probe and detection method |
| CN107904287A (en) * | 2017-12-28 | 2018-04-13 | 吉林出入境检验检疫局检验检疫技术中心 | Staphylococcus aureus and bacillus cereus quick determination method |
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