CN108896758A - A kind of kit based on molten junket big coccus plate agglutination test antigen composition, preparation method and applications - Google Patents
A kind of kit based on molten junket big coccus plate agglutination test antigen composition, preparation method and applications Download PDFInfo
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- CN108896758A CN108896758A CN201810436738.0A CN201810436738A CN108896758A CN 108896758 A CN108896758 A CN 108896758A CN 201810436738 A CN201810436738 A CN 201810436738A CN 108896758 A CN108896758 A CN 108896758A
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The present invention relates to a kind of kit based on the big coccus plate agglutination test antigen composition of molten junket, preparation method and applications, include in the kit：(1) the big coccus plate agglutination test antigen of molten junket；(2) standard positive serum；(3) standard female serum；Kit of the present invention is simple to operate, can be used to detect the big pneumoniae serotype antibody of molten junket, is suitble to be widely used to promote in each base animal doctor units concerned and farm；Kit in the present invention only needs not needing any other equipment using several simple consumptive materials in test sample, can be used even if condition simpler and cruder farm；Result judgement is intuitive, and eye is seen；Operator does not need professional knowledge, it is not necessary to receive professional training, can be detected only referring to specification；Low in cost, economical and practical, user is acceptable.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to one kind is based on the big coccus plate agglutination test antigen composition of molten junket
Kit, preparation method and applications.
Background technique
The molten big coccus of junket (Macrococcus caseolyticus) belongs to megacoccus category, which includes 7 strains, molten junket
Big coccus is therein to represent strain.The bacterium can specificity slave Animal Skin and animal based food in be separated to, such as cow's milk, life
Beef, chicken, ham etc. can be also separated in China's traditional meat Guangdong style sausage；The bacterium can cause fish to fall ill, from tooth
It is separated in the fish bodies such as flounder, mackerel, largemouth bass, brings harm to aquaculture；In addition, the molten big coccus of junket can also be such that dog sends out
Disease shows the symptom of rhinitis.The bacterium also carries SCCmec original part (mecB~carrying SCCmec of methicillin-resistant
Element), performance is to penicillin medicine height drug resistance.
It is very few to the research report of the big coccus of molten junket both at home and abroad at present, only have one plant of molten big ball of junket in GenBank gene pool
The complete sequence of bacterium, considerably less, the blood of coccus big for molten junket of the molecular Biological Detection technical report of coccus big for molten junket
Clear antibody test technology is at home and abroad not reported, more without a kind of big pneumoniae serotype antibody of the molten junket of detection for realizing commercialization
Antigen.In consideration of it, being used it is necessary to develop a kind of antigen and its corresponding kit that can detect the big pneumoniae serotype antibody of molten junket
To investigate the infection state of the big coccus of molten junket.
Summary of the invention
In order to solve the above problems existing in the present technology, the present invention provides one kind based on the big coccus plate agglutination of molten junket
Test the kit and preparation method thereof of antigen composition.
It is a further object of the present invention to provide a kind of kits based on the big coccus plate agglutination test antigen composition of molten junket
Detecting the application in the molten big pneumoniae serotype antibody of junket.
It is a further object of the present invention to provide a kind of sides of molten big coccus plate agglutination test antigen detection serum antibody of junket
Method.
The technical scheme adopted by the invention is as follows：A kind of reagent based on the big coccus plate agglutination test antigen composition of molten junket
Box, the kit include：(1) the big coccus plate agglutination test antigen of molten junket；(2) standard positive serum；(3) standard female blood
Clearly.
A kind of preparation method of the kit based on the big coccus plate agglutination test antigen composition of molten junket, including walk as follows
Suddenly：
Step A, the preparation of the molten big coccus plate agglutination test antigen of junket：The big meningitidis strains of molten junket are subjected to Zengjing Granule
Afterwards, heating water bath inactivation, then centrifuging and taking precipitating are carried out, suspends to obtain with 5g/L carbolic acid physiological saline；
Step B, the preparation of standard positive serum：After taking the big coccus of molten junket to carry out Zengjing Granule, centrifuging and taking precipitating carries out water
Bath inactivation, mixes with Freund's complete adjuvant, and the immune healthy duck for being uninfected by the big coccus of molten junket separates blood from duck wing venous blood collection
Clearly, antibody titer is detected with agar gel diffusion test, potency is up to 1:32 or more be standard positive serum；
Step C, the preparation of standard female serum：The arteria carotis blood of healthy duck is taken, the room temperature mistake into big centrifuge tube is collected
Night, centrifugation take supernatant, detect through agar gel diffusion test, and the molten big pneumoniae serotype antibody of junket is negative to get arriving standard female blood
Clearly；
Step D, by the big coccus plate agglutination test antigen of the molten junket of above-mentioned preparation, standard positive serum, standard female blood
It is assembled into the kit based on the big coccus plate agglutination test antigen composition of molten junket clearly.
Further, the big meningitidis strains of molten junket that the big coccus plate agglutination test antigen of molten junket is prepared in the step A are protected
It is stored in China typical culture collection center, deposit number CCTCC No:M 2018116.
A kind of method of the big coccus plate agglutination test antigen detection serum antibody of molten junket, includes the following steps：
Step 1: taking out the big coccus plate agglutination test antigen of molten junket of 4 DEG C of preservations, restores to room temperature, sufficiently shake up；
Step 2: serum to be checked, standard positive serum, each 25ul of standard female serum is added dropwise respectively on a glass；
Step 3: being separately added into the big coccus plate agglutination test antigen 25 μ l of the molten junket sufficiently shaken up, mix well；
Step 4: be stored at room temperature 2~5 minutes, the solidifying of the big coccus plate agglutination test antigen of molten junket and corresponding serum is observed
Collect phenomenon；
Step 5: determining positive, negative findings according to whether it is aggregated.
Beneficial effects of the present invention are：
A. kit is simple to operate, is suitble to be widely used to promote in each base animal doctor units concerned and farm,
Kit in the present invention only needs the first-class simple consumptive material of glass plate, pipettor, pipettor gun in test sample, does not need to appoint
What its equipment can be used even if condition simpler and cruder farm；
B. result judgement is intuitive, and eye is seen；
C. operator does not need professional knowledge, it is not necessary to receive professional training, can be detected only referring to specification；
D. low in cost, economical and practical, user is acceptable.
Detailed description of the invention
Fig. 1 is the big coccus indirect hemagglutination antigen examination criteria positive serum of molten junket, standard female serum and saprophytic grape
Coccus, Staphylococcus sciuri, staphylococcus aureus, enterococcus faecalis, enterococcus faecium positive serum；
In Fig. 1, "+" indicates examination criteria positive serum, and "-" indicates examination criteria negative serum, " saprophytic " expression detection
The positive serum of staphylococcus saprophyticus, " squirrel " indicate the positive serum of detection Staphylococcus sciuri, " golden yellow " expression detection gold
The positive serum of staphylococcus aureus；" excrement intestines " indicate the positive serum of detection enterococcus faecalis；" dung intestines " indicate detection dung intestines ball
The positive serum of bacterium.It is equal that the molten big coccus plate agglutination test antigen of junket detects corresponding standard positive serum, standard female serum
It sets up；Detect staphylococcus saprophyticus, Staphylococcus sciuri, staphylococcus aureus, enterococcus faecalis, enterococcus faecium positive blood
Clearly, result is feminine gender；
Fig. 2 is to detect 2 times of standard positive serums being serially diluted and former times with the big coccus plate agglutination test antigen of molten junket
Standard female serum；
In Fig. 2,1:2~1:32 indicate the extension rate of serum, the standard positive serum antibody titer which detects
It is 1:32, as a result standard female serum is set up without agglutination；
Fig. 3 is that 2 times of standard positive serums being serially diluted and former times standard female serum are detected with agar gel diffusion test；
In Fig. 3,1:2~1:32 indicate the extension rate of serum, and this method detects that standard positive serum antibody titer is
1:32, standard female serum, which expands band without fine jade, to be occurred, and is as a result set up.
Specific embodiment
The present invention is described in further details below with reference to embodiment.
One, kit is formed based on the big coccus plate agglutination test antigen of molten junket：
It include 4 bottles of the big coccus plate agglutination test antigen of molten junket in kit, 2ml/ bottles, 4 DEG C save；Standard positive serum
1 bottle, 0.5ml/ bottles, -20 DEG C freeze；1 bottle of standard female serum, 0.5ml/ bottles, -20 DEG C freeze.The kit detectable about 300
Part serum, storage life are 6 months.
Molten junket big coccus MCSD (Macrococcus caseolyticus MCSD) bacterial strain for preparing antigen is source
In the field separation strains of duck, it is stored in China typical culture collection center on March 11st, 2018, deposit number is
CCTCC No:M 2018116, preservation address (the Chinese Wuhan Wuhan University), the viability of the bacterial strain are survival.
Two, a kind of preparation method of the kit based on the big coccus plate agglutination test antigen composition of molten junket：
Step A, the preparation of carbolic acid physiological saline：
5g/L (w/v) carbolic acid physiological saline to be prepared, that is, weighs 9 grams of Nacl, 5 grams of phenol (carbolic acid) add deionized water,
It is settled to 1000ml, high pressure sterilization.Specific preparation amount can adjust according to actual needs.
Step B, the molten big coccus plate agglutination test antigen preparation of junket：
1) -80 DEG C of big coccus CCTCC No of molten junket for freezing the preservation of (v/v) glycerol of addition 20% are taken out:2018116 bacterium of M
The liquid spawn of strain, streak inoculation is on tryptose soya agar (TSA) plate, 37 DEG C, 5%CO2In case culture 15~
20h。
2) single colonie on picking TSA plate is connected in pancreas peptone soybean broth (TSB), 37 DEG C, 200r/min shake
15~18h of culture is swung, then presses 1 again:100 (v/v) ratios are inoculated in fresh TSB culture medium, 37 DEG C, 200r/min concussion training
6~8h is supported, until OD600 value is 1.0~1.2；
3) 70 DEG C of water-baths inactivate 45min；
4) bacterium solution after inactivation is centrifuged 15min with 4000r/min, abandons supernatant, then with 5g/L (w/v) carbolic acid physiology salt
Water washing precipitates 2 times；
5) bacterial sediment is dyed with commercialization crystal violet solution, operating method carries out to specifications.
6) it is finally precipitated, is mixed, as with 5g/L (w/v) carbolic acid physiological saline suspension thalline of bacterium solution original volume 20%
The molten big coccus plate agglutination test antigen of junket.
Step C, the preparation of standard positive serum：
Take -80 DEG C of big coccus CCTCC No of molten junket for freezing the preservation of (v/v) glycerol of addition 20%:2018116 bacterial strain of M
Liquid spawn dips bacterium solution with oese, streak inoculation on TSA plate, be placed in 37 DEG C, culture 15 in 5% CO2 case~
20h, then picking single colonie are inoculated in 5ml TSB, then 37 DEG C, 200r/min 15~18h of culture are transferred to 1ml bacterium solution
In the fresh TSB of 100ml, continue in 37 DEG C, 200r/min 6~8h of shake culture, until the OD600 value of bacterium solution reach 1.0~
1.2；After culture, whole bacterium solutions are centrifuged 15min with 4000r/min, with the phosphate buffer of concentration 0.01M, pH7.2
Bacterial sediment is washed 3 times, then uses 10ml phosphate buffer suspension thalline, carries out count of bacteria, then 70 DEG C of water-baths inactivate
45min；Above-mentioned inactivated bacterial liquid is taken to mix in equal volume with Freund's complete adjuvant, the immune healthy duck for being uninfected by the big coccus of molten junket,
Every duck is inoculated with 5,000,000,000 bacteriums.After immune 2 weeks again booster immunization it is primary；2 weeks after secondary immunity, separated from duck wing venous blood collection
Serum detects serum antibody titer with agar gel diffusion test, and potency is up to 1:32 or more be standard positive serum；After if two exempt from
Standard is not achieved in serum antibody titer, can booster immunization again, until complying with standard.
Step D, the preparation of standard female serum：
2~2.5 kilograms of weight of healthy duck is chosen, arteria carotis bloodletting is up to dead, by blood collection into big centrifuge tube,
Lid is screwed, ambient temperature overnight, then 3000r/min is centrifuged 15min, and supernatant is taken, is detected through agar gel diffusion test, the molten big coccus of junket
Serum antibody is feminine gender, as standard female serum；
Step E, by the big coccus plate agglutination test antigen of the molten junket of above-mentioned preparation, standard positive serum, standard female blood
It is assembled into the kit based on the big coccus plate agglutination test antigen composition of molten junket clearly.
Three, the application method example of the kit based on the big coccus plate agglutination test antigen composition of molten junket：
A kind of method of the big coccus plate agglutination test antigen detection serum antibody of molten junket, includes the following steps：
1. taking out the big coccus plate agglutination test antigen of molten junket of 4 DEG C of preservations on demand, restores to room temperature, sufficiently shake up；
2. serum to be checked, standard positive serum, each 25 μ l of standard female serum is added dropwise respectively on a glass；
3. being separately added into the big coccus plate agglutination test antigen 25 μ l of the molten junket sufficiently shaken up, mix well；
4. being stored at room temperature 2~5min, the agglutination phenomenon of molten junket big coccus plate agglutination test antigen and corresponding serum is observed；
5. degree whether is aggregated and is aggregated according to it to determine positive, negative findings.
6. the criterion of agglutinating reaction intensity：
1)++++：There is big purple agglutination block, liquid is clear and transparent, i.e., 100% agglutination；
2)+++：There is apparent purple agglutination piece, liquid is nearly transparent, i.e., 75% agglutination；
3)++：There are visible purple agglutination piece, liquid slight haziness, i.e., 50% agglutination；
4)+：There are small purple granular substance, opaque, i.e., 25% agglutination；
5)-：No agglutinating particle occurs, and liquid is in uniformly muddiness, i.e., without agglutination.
7. result judgement：
100% agglutination (++++) is presented to correlating in standard positive serum；No agglutination should be presented in standard negative control serum
(-)。
Under the premise of compareing qualified, seroreaction to be checked is observed.The serum that 50% agglutination (++) or more is presented determines
For the positive, 25% agglutination (+) is presented and following person is determined as feminine gender.
Four, the specificity and sensitivity Detection of the big coccus plate agglutination test antigen of molten junket：
Specific detection：With molten junket big coccus plate agglutination test antigen detection staphylococcus saprophyticus, Staphylococcus sciuri,
Staphylococcus aureus, enterococcus faecalis, enterococcus faecium positive serum, shown in Fig. 1, result is feminine gender, show the antigen spy
It is anisotropic preferable.
Antigen sensitive detection：Standard positive serum is successively made 1 with physiological saline:2,1:4,1:8,1:16, 1:32 times
Dilution, is added dropwise each 25 μ l of above-mentioned diluted serum sample, standard positive serum, standard female serum, so respectively on a glass
After sequentially add the 25 big coccus plate agglutination test antigens of the molten junket of μ l, mix well, be stored at room temperature 2~5min.The result shows that when
Standard positive serum is diluted to 1:When 32, shown in Fig. 2, although agglutinating reaction obviously weakens, but still it can determine that as the positive；Fig. 3
It is shown, it is identical as the testing result of agar gel diffusion test.Therefore, when examination criteria positive serum, the sensibility of both methods
It is close.
Five, kit detects field sample
It is clear to 178 parts of duck bloods for picking up from scale duck, it is anti-with the molten big coccus plate agglutination test of junket of the present invention
Former and agar gel diffusion test is detected respectively.The result shows that the molten junket that the molten big coccus plate agglutination test antigen of junket detects
Big coccus positive rate is 42.1% (75/178), and the positive rate that agar gel diffusion test detects is 38.2% (68/178), two kinds
The coincidence rate of method testing result is 96.1% (171/178), and the results are shown in Table 1.
The testing result of table 1 kit of the invention and agar gel diffusion test
The above result shows that the big coccus plate agglutination test of molten junket involved in the present invention is anti-when detecting field sample
Former and matched kit has higher sensibility than agar gel diffusion test, can effectively detect the intracorporal molten big coccus blood of junket of duck
Clear antibody.In addition, this method is simple to operate, result judgement is intuitive, can be applicable in actual production, and it is big to carry out molten junket
Coccus epidemiological survey.
The present invention is not limited to above-mentioned preferred forms, anyone can show that other are various under the inspiration of the present invention
The product of form, however, make any variation in its shape or structure, it is all that there is skill identical or similar to the present application
Art scheme, is within the scope of the present invention.
Claims (5)
1. a kind of kit based on the big coccus plate agglutination test antigen composition of molten junket, it is characterised in that：The kit packet
It includes：(1) the big coccus plate agglutination test antigen of molten junket；(2) standard positive serum；(3) standard female serum.
2. a kind of preparation side for preparing the kit based on the big coccus plate agglutination test antigen composition of molten junket in claim 1
Method, which is characterized in that include the following steps：
Step A, the preparation of the molten big coccus plate agglutination test antigen of junket：After the big meningitidis strains of molten junket are carried out Zengjing Granule, into
The inactivation of row heating water bath, then centrifuging and taking precipitating, suspend to obtain with 5g/L carbolic acid physiological saline；
Step B, the preparation of standard positive serum：After taking the big coccus of molten junket to carry out Zengjing Granule, centrifuging and taking precipitating carries out water-bath and goes out
It is living, it is mixed with Freund's complete adjuvant, the immune healthy duck for being uninfected by the big coccus of molten junket separates serum from duck wing venous blood collection,
Antibody titer is detected with agar gel diffusion test, potency is up to 1:32 or more be standard positive serum；
Step C, the preparation of standard female serum：The arteria carotis blood of healthy duck is taken, is collected into big centrifuge tube, ambient temperature overnight, from
The heart takes supernatant, detects through agar gel diffusion test, and the molten big pneumoniae serotype antibody of junket is negative to get arriving standard female serum；
Step D, by the big coccus plate agglutination test antigen of the molten junket of above-mentioned preparation, standard positive serum, standard female serum group
Dress up the kit based on the big coccus plate agglutination test antigen composition of molten junket.
3. as claimed in claim 2 in preparation claim 1 based on molten junket big coccus plate agglutination test antigen composition
The preparation method of kit, it is characterised in that：The molten junket that the big coccus plate agglutination test antigen of molten junket is prepared in the step A is big
Meningitidis strains are stored in China typical culture collection center, and deposit number is CCTCC No:M 2018116.
4. a kind of kit based on the big coccus plate agglutination test antigen composition of molten junket as described in claim 1 is molten in detection
Application in the big pneumoniae serotype antibody of junket.
5. a kind of method of the big coccus plate agglutination test antigen detection serum antibody of molten junket, which is characterized in that including walking as follows
Suddenly：
Step 1: taking out the big coccus plate agglutination test antigen of molten junket of 4 DEG C of preservations, restores to room temperature, sufficiently shake up；
Step 2: serum to be checked, standard positive serum, each 25ul of standard female serum is added dropwise respectively on a glass；
Step 3: being separately added into the big 25 μ l of coccus plate agglutination test antigen of the molten junket sufficiently shaken up, mix well；
Step 4: being stored at room temperature 2~5 minutes, it is existing with the agglutination of corresponding serum to observe the big coccus plate agglutination test antigen of molten junket
As；
Step 5: determining positive, negative findings according to whether it is aggregated.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810436738.0A CN108896758A (en) | 2018-05-09 | 2018-05-09 | A kind of kit based on molten junket big coccus plate agglutination test antigen composition, preparation method and applications |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810436738.0A CN108896758A (en) | 2018-05-09 | 2018-05-09 | A kind of kit based on molten junket big coccus plate agglutination test antigen composition, preparation method and applications |
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