CN108956247A - A kind of staphylococcus saprophyticus serum antibody slide agglutination detection kit and its application - Google Patents
A kind of staphylococcus saprophyticus serum antibody slide agglutination detection kit and its application Download PDFInfo
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- 230000004520 agglutination Effects 0.000 title claims abstract description 60
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/34—Purifying; Cleaning
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
- G01N2001/4083—Concentrating samples by other techniques involving separation of suspended solids sedimentation
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Abstract
The present invention relates to a kind of staphylococcus saprophyticus serum antibody slide agglutination detection kit and its application, the kit includes: (1) staphylococcus saprophyticus slide agglutination test antigen；(2) standard positive serum；(3) standard female serum；(4) glass plate；A kind of staphylococcus saprophyticus Serum Antibody Detection kit provided by the invention can be used to detect staphylococcus saprophyticus serum antibody, the kit is simple to operate, it is suitble to be widely popularized and use in each base animal doctor units concerned and farm, testing result is intuitive, stablizes, is reliable, is conducive to be widely used in base.
Description
Technical field
The invention belongs to the detection reagent technical fields of microorganism, and in particular to a kind of staphylococcus saprophyticus serum antibody inspection
Test agent box and its application.
Background technique
Staphylococcus saprophyticus (Staphylococcus saprophyticus) belongs to coagulase-negative staphylococci category, is
One kind being widely distributed in nature gram-positive cocci, is conditionity pathogenic bacteria, has important clinical meaning.Meanwhile it is rotten
Raw staphylococcus is also the main pathogenic fungi and Greece's sausage for being cow subclinical mastitis, boiled salted duck and Chinese the Dong nationality's tradition
Common strain in fermented meat.It is very few to the research report of staphylococcus saprophyticus both at home and abroad at present, for staphylococcus saprophyticus
Molecular Biological Detection technology relies primarily on laboratory bacterial separation, PCR molecular biology identification, for staphylococcus saprophyticus
Serum Antibody Detection technology is at home and abroad not reported, and then lacks the serology of quickly and effectively staphylococcus saprophyticus in base
Diagnostic method, this may make base cause mistaken diagnosis to staphylococcus saprophyticus infection and fail to pinpoint a disease in diagnosis, and causing to Animal husbandry production need not
The loss wanted.
Summary of the invention
In order to solve the deficiencies in the prior art and the above problem, the present invention provides a kind of staphylococcus saprophyticus serum antibodies
Slide agglutination detection kit.The kit includes: (1) staphylococcus saprophyticus slide agglutination test antigen；(2) standard male
Property serum；(3) standard female serum；(4) glass plate.
Preferably, the staphylococcus saprophyticus slide agglutination test antigen is by staphylococcus saprophyticus CCTCC M2018117
Bacterial strain is prepared.
Preferably, the staphylococcus saprophyticus slide agglutination test antigen is after staphylococcus saprophyticus is carried out Zengjing Granule
Heating water bath inactivation, then centrifuging and taking precipitating are carried out, suspends to obtain with 5g/L (w/v) carbolic acid physiological saline.
Preferably, the staphylococcus saprophyticus slide agglutination test antigen is specifically prepared as follows:
(1) the staphylococcus saprophyticus glycerol stock frozen is taken out, streak inoculation is in tryptose soya agar (TSA) plate
On, 37 DEG C, 5%CO216~20h is cultivated in case；
(2) single colonie on picking TSA plate is connected in pancreas peptone soybean broth (TSB), 37 DEG C, 200r/min shake
12~15h of culture is swung, is then inoculated in fresh TSB culture medium, 37 DEG C, 200r/min by volume again for the ratio of 1:100
6~8h of shake culture, until OD600Value 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 crystal violet solution；
(6) it is finally precipitated, is mixed, i.e., with 5g/L (w/v) carbolic acid physiological saline suspension thalline of bacterium solution original volume 20%
For staphylococcus saprophyticus slide agglutination test antigen.
Preferably, the standard positive serum is prepared as follows:
(1) -80 DEG C of staphylococcus saprophyticus glycerol stocks frozen are taken, dip bacterium solution with oese, streak inoculation is in TSA plate
On, it is placed in 37 DEG C, 5%CO215~20h is cultivated in case, then picking single colonie is inoculated in 5ml TSB, 37 DEG C, 200r/min training
15~18h is supported, then 1ml bacterium solution is transferred in the fresh TSB of 100ml, continues to cultivate 6~8h in 37 DEG C, 200r/min, until
The OD of bacterium solution600Value reaches 1.0~1.2；
(2) after cultivating, whole bacterium solutions are centrifuged 15min with 4000r/min, with the phosphoric acid of concentration 0.01M, pH7.2
Salt buffer by bacterial sediment wash 3 times, then use 10ml phosphate buffer suspension thalline, progress count of bacteria, then 70 DEG C
Water-bath inactivates 45min；
(3) above-mentioned inactivated bacterial liquid is taken to mix in equal volume with Freund's complete adjuvant, it is immune to be uninfected by staphylococcus saprophyticus
Healthy duck, every duck are inoculated with 5,000,000,000 bacteriums.Booster immunization is primary again after 2 weeks after immune.By 2 weeks after secondary immunity, from duck
Wing venous blood sampling separation serum detects antibody titer with agar gel diffusion test, and it is standard positive blood that potency, which reaches 1:32 or more,
Clearly.If two, which exempt from rear serum antibody titer, is not achieved standard, can booster immunization again, until complying with standard.
Preferably, the standard female serum is prepared as follows:
The arteria carotis blood of 2~2.5 kilograms of weight of healthy duck is collected, collects into big centrifuge tube, screws lid, room temperature mistake
Night, then 15min is centrifuged with 3000r/min, take supernatant to obtain standard female serum.
It is a further object of the present invention to provide above-mentioned staphylococcus saprophyticus Serum Antibody Detection kits to detect saprophytic Portugal
Application in grape pneumoniae serotype antibody, the application are the applications of the diagnosing and treating purpose of non-disease.
Further, the method for kit detection staphylococcus saprophyticus serum antibody includes the following steps:
Serum to be checked, standard positive serum, each 25 μ l of standard female serum is added dropwise in the first step respectively on a glass；
Second step is separately added into the plate agglutination test antigen 25 μ l sufficiently shaken up, mixes well；
Third step is stored at room temperature 2~5min, observes the agglutination phenomenon of antigen and corresponding serum；
4th step is aggregated situation according to it to determine positive, negative findings, and 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.
The invention has the benefit that
A. the antigen in the present invention is staphylococcus saprophyticus antigen, has specificity well, can truly detect corruption
Raw staphylococcus serum antibody；
B. result judgement is intuitive, and eye is seen；
C. kit is simple to operate, the present invention in kit only needed in test sample glass plate, pipettor,
The first-class simple consumptive material of pipettor gun, does not need any other instrument and equipment, can be used even if condition simple and crude farm yet,
It is suitble to be widely used to promote in each base animal doctor units concerned and farm, carries out staphylococcus saprophyticus epidemiological survey；
D. the kit is low in cost, economical and practical, and user is acceptable, and safety and stability is nontoxic, non-environmental-pollution；
E. testing result is reproducible, stablizes, reliably, can accurately detect staphylococcus saprophyticus serum antibody.
Detailed description of the invention
Fig. 1: detection of the staphylococcus saprophyticus slide agglutination test antigen to standard positive serum, standard female serum；It is right
The positive serum detection of staphylococcus, Pasteurella, streptococcus, enterococcus faecalis, enterococcus faecium；
Fig. 2: staphylococcus saprophyticus slide agglutination test antigen detects 2 times of standard positive serums being serially diluted and former times is marked
Quasi- negative serum；
Note: the extension rate of 1:2~1:32 expression serum.
Fig. 3: agar gel diffusion test detects 2 times of standard positive serums being serially diluted and former times standard female serum；
Note: the extension rate of 1:2~1:32 expression serum.
Specific embodiment
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation
Example is only a part of the invention, rather than the whole invented.Based on the embodiments of the present invention, ordinary skill people
Member's every other embodiment obtained without making creative work, shall fall within the protection scope of the present invention.
For preparing the staphylococcus saprophyticus bacterial strain (Staphylococcus saprophyticus) of antigen as field point
From strain.It was preserved in China typical culture collection center (CCTCC) on 03 11st, 2018, preservation address: Wuhan University, bacterium
Strain deposit number is CCTCC M2018117, preservation title: staphylococcus saprophyticus SSD (Staphylococcus
saprophyticus SSD)。
A kind of preparation of the staphylococcus saprophyticus serum antibody slide agglutination detection kit of embodiment 1
It include 4 bottles of staphylococcus saprophyticus slide agglutination test antigen in the kit, 1ml/ bottles, 4 DEG C save；Standard positive
1 bottle of serum, 0.5ml/ bottles, -20 DEG C freeze；1 bottle of standard female serum, 0.5ml/ bottles, -20 DEG C freeze；(the disinfection of 1 piece of glass plate
It afterwards, can Reusability).The kit can detect about 150 parts of serum, and storage life is 6 months.
1. the preparation of standard positive serum:
Take -80 DEG C of staphylococcus saprophyticus glycerol stocks frozen, dip bacterium solution with oese, streak inoculation on TSA plate,
It is placed in 37 DEG C, cultivates 15~20h in 5%CO2 case, then picking single colonie is inoculated in 5ml TSB, 37 DEG C, 200r/min culture
Then 15~18h is transferred to 1ml bacterium solution in the fresh TSB of 100ml, continue in 37 DEG C, 200r/min 6~8h of shake culture, directly
To the OD of bacterium solution600Value reaches 1.0~1.2.After culture, whole bacterium solutions are centrifuged 15min with 4000r/min, use concentration
The phosphate buffer of 0.01M, pH7.2 by bacterial sediment wash 3 times, then use 10ml phosphate buffer suspension thalline, progress
Count of bacteria, then 70 DEG C of water-baths inactivate 45min.It takes above-mentioned inactivated bacterial liquid to mix in equal volume with Freund's complete adjuvant, is immunized not
The healthy duck of staphylococcus saprophyticus was infected, every duck is inoculated with 5,000,000,000 bacteriums.After immune 2 weeks again booster immunization it is primary.It is secondary
By 2 weeks after immune, serum is separated from duck wing venous blood collection, with agar gel diffusion test detection serum antibody titer, potency is up to 1:
32 or more be standard positive serum.If two, which exempt from rear serum antibody titer, is not achieved standard, can booster immunization again, until symbol
Standardization.
2. 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 taking supernatant is standard female serum；
3. the preparation of carbolic acid physiological saline:
It is spare to prepare 5g/L (w/v) carbolic acid physiological saline, that is, weighs 9 grams of Nacl, 5 grams of phenol (carbolic acid), add from
Sub- water is settled to 1000ml, high pressure sterilization.Specific preparation amount can adjust according to actual needs.
4. prepared by staphylococcus saprophyticus slide agglutination test antigen:
1) take out the staphylococcus saprophyticus glycerol stock that freezes, streak inoculation on tryptose soya agar (TSA) plate,
37 DEG C, 5%CO215~20h is cultivated in case.
2) single colonie on picking TSA plate is connected in pancreas peptone soybean broth (TSB), 37 DEG C, 200r/min concussion
15~18h is cultivated, is then inoculated in fresh TSB culture medium, 37 DEG C, 200r/min shake by volume again for the ratio of 1:100
6~8h of culture is swung, until OD600Value 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%
Staphylococcus saprophyticus plate agglutination test antigen.
A kind of staphylococcus saprophyticus serum antibody slide agglutination detection kit prepared by 2 embodiment 1 of embodiment makes
Use method
One, the antigen of 4 DEG C of preservations is taken out on demand, is restored to room temperature, is sufficiently shaken up；
Two, serum to be checked, standard positive serum, each 25 μ l of standard female serum is added dropwise respectively on a glass；
Three, it is separately added into the plate agglutination test antigen 25 μ l sufficiently shaken up, is mixed well；
Four, it is stored at room temperature 2~5min, observes the agglutination phenomenon of antigen and corresponding serum
Five, degree whether is aggregated and is aggregated according to it to determine positive, negative findings.The judgement mark of agglutinating reaction intensity
It is quasi-:
1) ++++: there is big purple agglutination block, liquid is clear and transparent, i.e., 100% agglutination；
2) +++: there is apparent purple agglutinator, liquid is nearly transparent, i.e., 75% agglutination；
3) ++: there is visible purple agglutinator, liquid is not very transparent, i.e., 50% agglutination；
4)+: there are small purple granular substance, opaque, i.e., 25% agglutination；
5)-: liquid mixing, in uniformly muddiness, i.e., without agglutination.
Result judgement: 100% agglutination (++++) is presented to correlating in standard positive serum；Standard negative control serum should be presented
Without agglutination (-).Under the premise of compareing qualified, seroreaction to be checked is observed.The serum that 50% agglutination (++) or more is presented is sentenced
It is set to the positive, 25% agglutination (+) is presented and following person is determined as feminine gender.
It is rotten in a kind of staphylococcus saprophyticus serum antibody slide agglutination detection kit prepared by 3 embodiment 1 of embodiment
The specificity and sensitivity Detection of raw staphylococcus slide agglutination test antigen:
Specific detection: staphylococcus saprophyticus slide agglutination test antigen is to standard positive serum, standard female serum
Detection, as shown in Figure 1, result is set up；With staphylococcus saprophyticus slide agglutination test antigen detection Pasteurella, staphylococcus,
Enterococcus faecalis, enterococcus faecium, streptococcus serum show that the antigentic specificity is preferable as shown in Figure 1, result is feminine gender.
Antigen sensitive detection: standard positive serum is successively made into 1:2,1:4,1:8,1:16,1:32 times with physiological saline
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 25 μ l staphylococcus saprophyticus slide agglutination test antigens, mix well, be stored at room temperature 2~5min.As a result such as Fig. 2
It is shown, when standard positive serum is diluted to 1:32, although agglutinating reaction obviously weakens, but still can determine that as the positive, this is anti-
The standard positive serum antibody titer that original detects is 1:32, as a result standard female serum is set up without agglutination；It is tried with AGP test
2 times of standard positive serums being serially diluted of detection and former times standard female serum are tested, as a result as shown in figure 3, this method detects
Standard positive serum antibody titer is 1:32, and standard female serum, which expands band without fine jade, to be occurred, testing result and staphylococcus saprophyticus
The result that slide agglutination test antigen detects is identical, therefore both methods sensibility having the same.
It is rotten in a kind of staphylococcus saprophyticus serum antibody slide agglutination detection kit prepared by 4 embodiment 1 of embodiment
Raw staphylococcus slide agglutination test antigen and agar gel diffusion test are to the detection sensitivity of field sample to comparing the scale of picking up from
98 parts of serum for changing farm, with staphylococcus saprophyticus plate agglutination test antigen of the present invention and agar gel diffusion test point
It is not detected.The result shows that the staphylococcus saprophyticus positive rate that staphylococcus saprophyticus slide agglutination test antigen detects is
33.6% (33/98), the positive rate that agar gel diffusion test detects are 31.6% (31/98), the symbol of two methods testing result
Conjunction rate is 97.9% (96/98).The result shows that staphylococcus saprophyticus slide agglutination test antigen and mating involved in the present invention
Kit there is higher sensibility in field trials than agar gel diffusion test, can effectively detect staphylococcus saprophyticus serum
Antibody.
Claims (8)
1. a kind of staphylococcus saprophyticus serum antibody slide agglutination detection kit, which is characterized in that the kit includes:
(1) staphylococcus saprophyticus slide agglutination test antigen；(2) standard positive serum；(3) standard female serum；(4) glass plate.
2. a kind of staphylococcus saprophyticus serum antibody slide agglutination detection kit according to claim 1, feature exist
In the staphylococcus saprophyticus slide agglutination test antigen is prepared by staphylococcus saprophyticus CCTCC M2018117 bacterial strain
It arrives.
3. a kind of staphylococcus saprophyticus serum antibody slide agglutination detection kit according to claim 1, feature exist
In the staphylococcus saprophyticus slide agglutination test antigen is to carry out water-bath after staphylococcus saprophyticus to be carried out to Zengjing Granule to add
Heat inactivation, then centrifuging and taking precipitating, suspend to obtain with 5g/L (w/v) carbolic acid physiological saline.
4. a kind of staphylococcus saprophyticus serum antibody slide agglutination detection kit according to claim 3, feature exist
In what the staphylococcus saprophyticus slide agglutination test antigen was specifically prepared as follows:
(1) the staphylococcus saprophyticus glycerol stock that freezes is taken out, streak inoculation is on tryptose soya agar (TSA) plate, and 37
DEG C, 5%CO215~20h is cultivated in case；
(2) single colonie on picking TSA plate is connected in pancreas peptone soybean broth (TSB), 37 DEG C, 200r/min concussion training
12~15h is supported, is then inoculated in fresh TSB culture medium, 37 DEG C, 200r/min concussion by volume again for the ratio of 1:100
6~8h is cultivated, until OD600Value 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 physiological saline
Washing 2 times；
(5) bacterial sediment is dyed with crystal violet solution；
(6) it is finally precipitated, is mixed with 5g/L (w/v) carbolic acid physiological saline suspension thalline of bacterium solution original volume 20%, it is as rotten
Raw staphylococcus slide agglutination test antigen.
5. a kind of staphylococcus saprophyticus serum antibody slide agglutination detection kit according to claim 1, feature exist
In the standard positive serum is prepared as follows:
(1) -80 DEG C of staphylococcus saprophyticus glycerol stocks frozen are taken, dip bacterium solution with oese, streak inoculation on TSA plate,
It is placed in 37 DEG C, 5%CO216~20h is cultivated in case, then picking single colonie is inoculated in 5ml TSB, 37 DEG C, 200r/min culture
Then 12~15h is transferred to 1ml bacterium solution in the fresh TSB of 100ml, continue to cultivate 6~8h in 37 DEG C, 200r/min, until bacterium
The OD of liquid600Value reaches 1.0~1.2；
(2) after cultivating, whole bacterium solutions are centrifuged 15min with 4000r/min, it is slow with the phosphate of concentration 0.01M, pH7.2
Fliud flushing by bacterial sediment wash 3 times, then use 10ml phosphate buffer suspension thalline, progress count of bacteria, then 70 DEG C of water-baths
Inactivate 45min；
(3) above-mentioned inactivated bacterial liquid is taken to mix in equal volume with Freund's complete adjuvant, the immune health for being uninfected by staphylococcus saprophyticus
Duck, every duck are inoculated with 5,000,000,000 bacteriums, and booster immunization is primary again after being immunized 2 weeks, by 2 weeks after secondary immunity, from duck wing vein
Blood sampling separation serum detects antibody titer with agar gel diffusion test, and it is standard positive serum that potency, which reaches 1:32 or more, if two
Exempt from rear serum antibody titer and standard be not achieved, can booster immunization again, until complying with standard.
6. a kind of staphylococcus saprophyticus serum antibody slide agglutination detection kit according to claim 1, feature exist
In the standard female serum is prepared as follows:
The arteria carotis blood of 2~2.5 kilograms of weight of healthy duck is collected, collects into big centrifuge tube, screws lid, ambient temperature overnight,
15min is centrifuged with 3000r/min again, supernatant is taken to obtain standard female serum.
7. application of the kit described in claim 1~6 any one in detection staphylococcus saprophyticus serum antibody, described
Application be non-disease diagnosing and treating purpose application.
8. application of the kit according to claim 7 in detection staphylococcus saprophyticus serum antibody, which is characterized in that
The method of detection staphylococcus saprophyticus serum antibody includes the following steps:
Serum to be checked, standard positive serum, each 25ul of standard female serum is added dropwise in the first step respectively on a glass；
Second step is separately added into the agglutination test antigen 25 μ l sufficiently shaken up, mixes well；
Third step is stored at room temperature 2~5 minutes, observes the agglutination phenomenon of antigen and corresponding serum；
4th step is aggregated situation according to it to determine positive, negative findings, and the serum that 50% agglutination or more is presented is determined as sun
Property, 25% agglutination is presented and following person is determined as feminine gender.
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| CN110938670A (en) * | 2019-11-07 | 2020-03-31 | 南方医科大学南方医院 | Application of reaction of microbial liquid and serum vesicles in detection of microbial infection |
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| CN110938670A (en) * | 2019-11-07 | 2020-03-31 | 南方医科大学南方医院 | Application of reaction of microbial liquid and serum vesicles in detection of microbial infection |
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Application publication date: 20181207 |