CN106226536B - A kind of position phase polymorphism test method of salmonella H antigens - Google Patents

A kind of position phase polymorphism test method of salmonella H antigens Download PDF

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CN106226536B
CN106226536B CN201610618448.9A CN201610618448A CN106226536B CN 106226536 B CN106226536 B CN 106226536B CN 201610618448 A CN201610618448 A CN 201610618448A CN 106226536 B CN106226536 B CN 106226536B
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韩涛
郑小严
戴明
高宇
柯振华
张芳
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FUJIAN INSPECTION AND RESEARCH INSTITUTE FOR PRODUCT QUALITY
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    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract

The invention discloses a kind of position phase polymorphism test method of salmonella H antigens, it carries out the agglutination reaction of serum of O antigens to Salmonella strains to be checked, 1st phase and the 2nd phase H factors are examined according to a point public sentiment condition for O antigens, when it is not single-phase bacterium only to detect a phase and bacterial strain to be checked, it is defined as aimed strain, known phase H factor serums and aimed strain are added in sterile vegetative meat soup afterwards, bacterial sediment is collected by centrifugation after culture, the slide agglutination inspection that bacteria suspension is used to treat phase-detecting H antigens is obtained after washing bacterium.The slide agglutination speed of H antigens is fast after our tagmeme phase polymorphism, phenomenon is obvious, interference is small, reproducible, success rate is high, easy to operate, has higher feasibility and practicality.

Description

A kind of position phase polymorphism test method of salmonella H antigens
Technical field
The present invention relates to a kind of position phase polymorphism test method of salmonella H antigens.
Background technology
Salmonella is zoonosis pathogen significant on public hygienics, its correct serotype It is significant in infectious disease control.2500 kinds of Salmonella serogroup has been detected at present, existing more than 290 serotype in China Report.The Serotype Identification of salmonella is more complicated, and its serotype is together decided on by O antigens, the 1st phase and the 2nd phase H antigens etc. (Some only have a phase H factors), and the measure of H antigens is the Main Basiss of salmonella serotype.H antigens are located at flagellum On, it is in the nature unstable proteantigen, there is the factor such as stronger specificity, heating, dry, freezing or Ethanol Treatment Flagella structural is damaged, causes H antigenicities to weaken, so as to bring difficulty to appraisal.For example, there is experimenter to find in freezer Resting period it is longer sample separation salmonella strain, reach H antigenicity restore needed for passage number it is more.Daily identification work The flagellum depauperation of salmonella is frequently encountered in work and the indefinite situation of parting, in standard GB/T 4789.4-2010 In recommend simple flat board method, glass-tube method and the traditional position phase polymorphism method of 3 kinds of voltage regulator tube method to induce another phase H factors.Other Scientific worker also discloses that the modification method of some phase polymorphisms experiment, such as nutrient broth-nutrient agar method, coubling dilution Deng.Wherein simple flat board method is widely used because its is simple and convenient, but its distinct disadvantage is that success rate is low;Glass-tube method and voltage regulator tube method Material there are certain requirements, such as capillary and small voltage regulator tube, and complex operation.In addition the above method uses nutrient agar or semisolid Substrate of the soft agar as strain growth, the presence of agar suppress the mobility of culture medium, and the kinetic characteristic of this and flagellum is present Antagonism, so as to hinder the antigenic recoveries of H.In addition, salmonella can break through low concentration agar, diffuse to inside culture medium Growth, cause thalline picking difficult, therefore when carrying out the slide agglutination test of H antigens, culture medium is inevitably with thalline Into in serum drop, and can not be completely dispersed, small solid medium particle is easily obscured with serum agglutination particle, so as to The judgement of severe jamming aggegation result.In addition, the agglutinator of H antigens to be cotton-shaped, is not easy to observe, generally required in experiment picking compared with For more thalline to strengthen aggegation effect, this is conflicting with the purpose for reducing solid medium interference.In view of the foregoing, at present Position phase polymorphism test method exist interference is big, success rate is low, complex operation, it is time-consuming more the shortcomings of, can not meet increasing Inspection work demand.
The content of the invention
For disadvantages mentioned above, it is an object of the invention to provide a kind of success rate is high, reproducible, interference is low, operation letter Just the efficiently position phase polymorphism test method of salmonella H antigens, to reach above-mentioned purpose, the technical solution adopted by the present invention It is:
A kind of position phase polymorphism test method of salmonella H antigens, comprises the following steps:
A. Salmonella strains to be checked are carried out with the agglutination reaction of serum of O antigens, divides public sentiment condition successively according to O antigens The 1st phase and the 2nd phase H factors are checked, when it is not single-phase bacterium only to detect a phase and bacterial strain to be checked, is defined as aimed strain;
B. known phase H factor serums are taken into sterile vegetative meat soup(The ratio between volume of serum and nutrient broth is 1:200~ 1:800), mix, vaccination target bacterial strain, in 36 DEG C of ± 1 DEG C of culture h of 18 h ~ 24, obtain culture;
C. culture is transferred in sterile centrifugation tube, 5 ~ 10 min is centrifuged under the conditions of 5000 ~ 8000 r/min, shifting is abandoned Supernatant, retain the bacterial sediment of centrifugation bottom of the tube;
D. sterile saline is added into the centrifuge tube containing bacterial sediment(The volume of sterile saline and culture The ratio between be 1:1~1:3), with pipettor gently pressure-vaccum, until thalline suspends completely, again under the conditions of 5000 ~ 8000 r/min 5 ~ 10 min are centrifuged, supernatant is abandoned in shifting, and bacterium step is washed in completion;Washing bacterium step can be repeated 1 times or repeatedly;
E. washed to completion in the centrifuge tube of bacterium step and add sterile saline(The volume of bacterial sediment and sterile physiological salt The ratio between be 1:2~1:5), with pipettor, gently pressure-vaccum mixes, and obtains bacteria suspension;
F. 10 ~ 15 μ L are taken to treat that phase-detecting H factor serums are added dropwise on clean slide, taking 1 ~ 2 μ L bacteria suspensions to be added to serum In drop, mix and tilt shake slide, agglutination phenomenon is observed within 1 min;Simultaneously in another region sterile physiological of slide Salt solution replaces treating phase-detecting H factor serums, is used as and compares after being mixed with bacteria suspension.
Compared with prior art, the beneficial effects of the invention are as follows:Culture containing agar is replaced using the nutrient broth of liquid Substrate of the base as strain growth, eliminate the antagonism that interference and flagellum of the agar to slide agglutination are grown.Liquid The composition of culture medium is uniform, and microorganism can fully contact and utilize the nutriment in culture medium, motion of the flagellum as salmonella Organ, motion effect can be fully played in liquid medium within and is significantly induced to grow, more conducively H is antigenic extensive It is multiple.During agglutination test, higher H antigenicities become apparent from agglutination phenomenon, and without the interference of solid medium particle, aggegation knot Fruit is easier to observe and judged.
The present invention is by being collected by centrifugation bacterial sediment, it is easier to obtains more thalline to realize the mesh of enhancing aggegation effect , thalline relative distribution used is suspended in bacteria suspension during agglutination test, it is easier to is spread in serum uniform, makes aggegation Speed faster, phenomenon become apparent from, disturb it is smaller.In addition, the experimental condition and each component that specify that each step quantitative in method Dosage, make that the repeatability of phase polymorphism experiment is more preferable, has higher success rate.
Embodiment
In order that the objects, technical solutions and advantages of the present invention become apparent from, with reference to embodiments, the present invention is carried out It is further to describe in detail, it will be appreciated that described embodiment is only explaining the present invention.Every skill based on the present invention The simplification or equivalent variations that art scheme is made, belong to protection scope of the present invention.
Embodiment 1:
The blind sample examination of 2015 food security sampling observation detection Cheng Jian mechanisms of state food pharmaceuticals administration general bureau(Salmonella Examine), totally 10 parts of samples, numbering are CODE1 ~ 10, if detection salmonella need to carry out serotype to isolated strains.Through inspection Survey, totally 4 parts of samples are that salmonella is positive by CODE1, CODE4, CODE5, CODE6 in 10 parts of samples.Enter according to the following steps afterwards Row follow-up test:
A. 4 Salmonella strains to be checked are carried out with the agglutination reaction of serum of O antigens, divides public sentiment condition according to O antigens It is examined in the 1st phase and the 2nd phase H factors, qualification result CODE1(O 7:H r)、CODE4(O 4:H i)、CODE5(O 4:H f,g,s)、CODE6(O 19:H i), wherein CODE5 bacterial strain to be checked is single-phase bacterium, so determining CODE1, CODE4, CODE6 The salmonella positive strain of totally 3 samples is aimed strain.
B. known phase H factor serums are taken into sterile vegetative meat soup(The ratio between volume of serum and nutrient broth is 1:200), Mix, vaccination target bacterial strain, in 36 DEG C of ± 1 DEG C of 18 h of culture, obtain culture;
C. culture is transferred in sterile centrifugation tube, 10 min is centrifuged under the conditions of 5000 r/min, supernatant is abandoned in shifting, is protected Stay the bacterial sediment of centrifugation bottom of the tube;
D. sterile saline is added into the centrifuge tube containing bacterial sediment(The volume of sterile saline and culture The ratio between be 1:1), with pipettor gently pressure-vaccum, until thalline suspends completely, centrifuge 10 under the conditions of 5000 r/min again Supernatant is abandoned in min, shifting, and bacterium step is washed in completion;
E. washed to completion in the centrifuge tube of bacterium step and add sterile saline(The volume of bacterial sediment and sterile physiological salt The ratio between be 1:2), with pipettor, gently pressure-vaccum mixes, and obtains bacteria suspension;
F. 10 μ L are taken to treat that phase-detecting H factor serums are added dropwise on clean slide, taking 1 μ L bacteria suspensions to be added to serum drop In, mix and tilt shake slide, agglutination phenomenon is observed within 1 min;Simultaneously in another region sterile saline of slide Instead for the treatment of phase-detecting H factor serums, it is used as and compares after being mixed with bacteria suspension;H antigens position phase polymorphism result of the test is CODE1(H 7)、 CODE4(H 2)、CODE6(H z6).
The complete qualification result of serotype is as shown in table 1, while carries out multiple put down to qualification result with simple flat board method Row checking, confirmatory experiment show that the qualification result of two methods is identical, and completely the same with blind sample examination measurement result.
Embodiment 2:
This grade of blind sample examination of food security sampling observation detection Cheng Jian mechanisms of office of state food pharmaceuticals administration general bureau in 2016 Totally 10 parts of samples, numbering are CODE1 ~ 10 for work, wherein salmonella inspection, and report detects or do not detected salmonella.Through inspection Surveying CODE2, CODE7, CODE8 in 10 parts of samples, totally 3 samples are that salmonella is positive.Subsequently tried according to the following steps afterwards Test:
A. 3 Salmonella strains to be checked are carried out with the agglutination reaction of serum of O antigens, divides public sentiment condition according to O antigens It is examined in the 1st phase and the 2nd phase H factors, inspection result CODE2(O 7:H c);CODE7(O 4:H f,g,s)、CODE8(O 7:H c), wherein CODE7 bacterial strain to be checked is single-phase bacterium, so determining that the salmonella of CODE2, CODE8 totally 2 samples is positive Bacterial strain is aimed strain.
B. known phase H factor serums are taken into sterile vegetative meat soup(The ratio between volume of serum and nutrient broth is 1:500), Mix, vaccination target bacterial strain, in 36 DEG C of ± 1 DEG C of 20 h of culture, obtain culture;
C. culture is transferred in sterile centrifugation tube, 5 min is centrifuged under the conditions of 6000 r/min, supernatant is abandoned in shifting, is protected Stay the bacterial sediment of centrifugation bottom of the tube;
D. sterile saline is added into the centrifuge tube containing bacterial sediment(The volume of sterile saline and culture The ratio between be 1:2), with pipettor gently pressure-vaccum, until thalline suspends completely, 5 min are centrifuged under the conditions of 6000 r/min again, Supernatant is abandoned in shifting, and bacterium step is washed in completion;
E. washed to completion in the centrifuge tube of bacterium step and add sterile saline(The volume of bacterial sediment and sterile physiological salt The ratio between be 1:3), with pipettor, gently pressure-vaccum mixes, and obtains bacteria suspension;
F. 13 μ L are taken to treat that phase-detecting H factor serums are added dropwise on clean slide, taking 1 μ L bacteria suspensions to be added to serum drop In, mix and tilt shake slide, agglutination phenomenon is observed within 1 min;Simultaneously in another region sterile saline of slide Instead for the treatment of phase-detecting H factor serums, it is used as and compares after being mixed with bacteria suspension;H antigens position phase polymorphism result of the test is CODE2(H 5)、 CODE8(H 5).
The complete qualification result of serotype is as shown in table 2, equally serotype result is carried out with simple flat board method parallel Checking, the result show that the qualification result of two methods is completely the same.
Embodiment 3:
To one plant of salmonella typhimurium(Strain number:CGMCC1.1174)Carry out the confirmatory experiment of serotype.Will Follow-up test is carried out according to the following steps after bacterial strain activation:
A. Salmonella strains to be checked are carried out with the agglutination reaction of serum of O antigens, divides public sentiment condition successively according to O antigens The 1st phase and the 2nd phase H factors are checked, qualification result is O 4:H i, so being defined as aimed strain;
B. known phase H factor serums are taken into sterile vegetative meat soup(The ratio between volume of serum and nutrient broth is 1:800), Mix, vaccination target bacterial strain, in 36 DEG C of ± 1 DEG C of 24 h of culture, obtain culture;
C. culture is transferred in sterile centrifugation tube, 5 min is centrifuged under the conditions of 8000 r/min, supernatant is abandoned in shifting, is protected Stay the bacterial sediment of centrifugation bottom of the tube;
D. sterile saline is added into the centrifuge tube containing bacterial sediment(The volume of sterile saline and culture The ratio between be 1:3), with pipettor gently pressure-vaccum, until thalline suspends completely, 5 min are centrifuged under the conditions of 8000 r/min again, Supernatant is abandoned in shifting, and bacterium step is washed in completion;Bacterium step is washed to repeat 1 time;
E. washed to completion in the centrifuge tube of bacterium step and add sterile saline(The volume of bacterial sediment and sterile physiological salt The ratio between be 1:5), with pipettor, gently pressure-vaccum mixes, and obtains bacteria suspension;
F. 15 μ L are taken to treat that phase-detecting H factor serums are added dropwise on clean slide, taking 2 μ L bacteria suspensions to be added to serum drop In, mix and tilt shake slide, agglutination phenomenon is observed within 1 min;Simultaneously in another region sterile saline of slide Instead for the treatment of phase-detecting H factor serums, it is used as and compares after being mixed with bacteria suspension;
The slide agglutination inspection result of 2nd phase H antigens of aimed strain is H 2, meets the serum of salmonella typhimurium Type feature.
In the implementation process of above-mentioned 3 embodiments, find H when using technical scheme after the phase polymorphism of position because Sub- serum agglutination speed faster, phenomenon become apparent from, disturb small, the repeatability of position phase polymorphism experiment is more preferable, has higher success rate.Reliably Qualification result also illustrate that the position phase polymorphism test method of salmonella H antigens of the present invention has higher feasibility and practicality Property.
Those of ordinary skill in the art are familiar with it should be appreciated that the experimental condition of technical solution of the present invention is included necessarily Scope, such as:The ratio between volume of serum and nutrient broth is 1 during training objective bacterial strain:200~1:800, the culture of aimed strain Time is 18 ~ 24 h, when culture is centrifuged centrifugal rotational speed be 5000 ~ 8000 r/min, centrifugation time be 5 ~ 10 min, wash bacterium The ratio between sterile saline and the volume of culture are 1 in step:1~1:3, washing bacterium step can be repeated 1 times or repeatedly, make The ratio between volume of bacterial sediment and sterile physiological salt is 1 during standby bacteria suspension:2~1:5, it is added dropwise on clean slide and treats the phase-detecting H factors The dripping quantity of serum is 10 ~ 15 μ L during serum, and the addition of bacteria suspension is 1 ~ 2 μ L when bacteria suspension is added into serum drop. When using the experimental condition in above range, technical scheme can also reach embodiment 1, embodiment 2 or embodiment 3 Identical effect.
Above by description of listed embodiment, the basic ideas and basic principles of the present invention are elaborated.Obviously, institute The embodiment stated is only the part of the embodiment of the present invention, and non-limiting exhaustion.Based on the embodiment in the present invention, ability All other embodiment that domain those of ordinary skill is obtained on the premise of creative work is not paid, belongs to the present invention Protection domain.

Claims (1)

1. a kind of position phase polymorphism test method of the salmonella H antigens of non-disease diagnosis and therapeutic purposes, comprises the following steps:
A. Salmonella strains to be checked are carried out with the agglutination reaction of serum of O antigens, is examined according to a point public sentiment condition for O antigens 1st phase and the 2nd phase H factors, when it is not single-phase bacterium only to detect a phase and bacterial strain to be checked, it is defined as aimed strain;
B. taking known phase H factor serums, the ratio between volume of serum and nutrient broth is 1 into sterile vegetative meat soup:200~1: 800, mix, vaccination target bacterial strain, in 36 DEG C of ± 1 DEG C of culture h of 18 h ~ 24, obtain culture;
C. culture is transferred in sterile centrifugation tube, 5 ~ 10 min is centrifuged under the conditions of 5000 ~ 8000 r/min, shifting is abandoned Clearly, the bacterial sediment of centrifugation bottom of the tube is retained;
D. sterile saline is added into the centrifuge tube containing bacterial sediment, the ratio between volume of sterile saline and culture For 1:1~1:3, with pipettor gently pressure-vaccum, until thalline suspends completely, again under the conditions of 5000 ~ 8000 r/min centrifugation 5 ~ Supernatant is abandoned in 10 min, shifting, and bacterium step is washed in completion;Bacterium step is washed to be repeated 1 times or repeatedly;
E. washed to completion in the centrifuge tube of bacterium step and add sterile saline, the ratio between volume of bacterial sediment and sterile physiological salt For 1:2~1:5, with pipettor, gently pressure-vaccum mixes, and obtains bacteria suspension;
F. 10 ~ 15 μ L are taken to treat that phase-detecting H factor serums are added dropwise on clean slide, taking 1 ~ 2 μ L bacteria suspensions to be added to serum drop In, mix and tilt shake slide, agglutination phenomenon is observed within 1 min;Simultaneously in another region sterile saline of slide Instead for the treatment of phase-detecting H factor serums, it is used as and compares after being mixed with bacteria suspension.
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