CN105462900A - Use method of reagent for isolation and culture of clostridium difficile in faeces - Google Patents
Use method of reagent for isolation and culture of clostridium difficile in faeces Download PDFInfo
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- CN105462900A CN105462900A CN201511021924.0A CN201511021924A CN105462900A CN 105462900 A CN105462900 A CN 105462900A CN 201511021924 A CN201511021924 A CN 201511021924A CN 105462900 A CN105462900 A CN 105462900A
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Abstract
The invention relates to a reagent for isolation and culture of clostridium difficile in faeces and a use method. The reagent is an alcoholic solution containing sulfur sodium cholate and is applied to the fields of isolation, culture and identification of the clostridium difficile and the like. The use method particularly comprises the following steps that the reagent for isolation and culture of the clostridium difficile in the faeces is added to the faeces, mixing is conducted to be uniform, and a mixed system is obtained; standing of the mixed system is conducted; centrifugation is conducted on the mixed system subjected to standing to remove suspended particles; supernatant is taken and inoculated to a selective medium of the clostridium difficile, and culture is conducted under an anaerobic condition; yellow bacterial colonies which are rough in surface, irregular in edge and capable of producing a special odor are selected for gram staining; the bacterial colonies which are positive after gram staining and have obvious match-head-shaped spores are determined as positive colonies, and the clostridium difficile is obtained; otherwise, the bacterial colonies are determined as negative colonies and are not the clostridium difficile. The reagent for isolation and culture of the clostridium difficile in the faeces and the use method have the advantages of being easy to operate, rapid, high in positive rate and the like.
Description
Technical field
The invention belongs to clostridium difficile isolation cultivation method field in ight soil, be specifically related to the reagent for clostridium difficile separation and Culture in ight soil and using method.
Background technology
Clostridium difficile (Clostridiumdifficile, CD) clostridium difficile or Clestridium difficile is had another name called, being a kind of Gram-positive strictly anaerobic bacillus with brood cell's structure, is that the nosocomial infection of generally acknowledging in global range and antibiosis disposition are suffered from diarrhoea of paramount importance pathogenic micro-organism.It can cause antibiotic-associated diarrhea, colitis, lethality pseudomembranous enteritis etc.2013, clostridium difficile was classified as microbiotic by national sanitary portion and is correlated with first of the large pathogenic bacteria of resistant organism three, and threaten degree is appointed as " the most urgent ".Be published in recent posts " burden of U.S.'s C. difficile infection " display of " New England Journal of Medicine " in February, 2015, at the annual infected patient of the U.S. more than 500,000 people, death toll is more than 2.9 ten thousand, average case fatality rate is 5.5-6.9%, severe mortality ratio is more than 60%, and the medical expense be used for the treatment of every year is more than 10,000,000,000 U.S. dollars.In China, abuse of antibiotics and serious, according to the latest news C. difficile infection account for and suffer from diarrhoea institute in patient numbers more than 25%, 20-30% in antibiotic associated diarrhea; In tumor radiotherapy patient, infected patient is more than 30%.Easily there is antibiotics resistance, repeatedly recurrence, infectivity, high lethality in C. difficile infection; In the world, clostridium difficile becomes notorious " the super bedbug " of hospital infection and antibiotics resistance field already.
Microbial culture is clinical disease pathogenic microorganism diagnosis " gold standard ", is important means and the prerequisite of pathogenic micro-organism research.China includes clostridium difficile research in " 12 " national transmissible disease major scientific and technological project, and due to culture of isolated condition stringent, correlative study report is still less.In clinical practice, the more difficult cultivation of clostridium difficile, traces it to its cause and mainly comprises 2 points: (1) clostridium difficile culture condition is comparatively harsh, needs strictly anaerobic condition; (2) there is a large amount of miscellaneous bacteria in ight soil, affect clostridium difficile and cultivate; This reason is that current clostridium difficile cultivates undesirable main cause.
Summary of the invention
The invention provides a kind of reagent for clostridium difficile separation and Culture in ight soil, the application of this reagent in clostridium difficile separation and Culture qualification etc. and the concrete using method of this reagent, by mentioned reagent and using method, miscellaneous bacteria in ight soil can be suppressed, and improve the positive cultivation rate of clostridium difficile, ensure the accurate of qualification result.
The technical scheme that the present invention solves the problems of the technologies described above is as follows:
The invention provides a kind of using method for the reagent of clostridium difficile separation and Culture in ight soil, described reagent is the spirituous solution of sulphur Sodium cholic acid, in use, comprises the following steps:
1) in ight soil, add the spirituous solution of sulphur Sodium cholic acid, mix, obtain mixed system;
2) mixed system is left standstill;
3) by step 2) leave standstill after mixed system centrifugal, remove suspended particle;
4) get supernatant, be inoculated into clostridium difficile Selective agar medium, cultivate under anaerobic condition;
5) select surface irregularity, edge uneven and produce the yellow color colonies of special odor, carry out gramstaining; Gramstaining is positive and has the bacterium colony of obvious match head gemma to be judged to be the positive, is clostridium difficile; Otherwise being judged to be feminine gender, is not namely clostridium difficile.
The beneficial effect of employing such scheme is:
The method step is arranged rationally, utilizes suitable reagent and culture condition to suppress miscellaneous bacteria in ight soil, and improves the positive cultivation rate of clostridium difficile, ensures the accurate of qualification result.
Method of the present invention have simple to operate, required time is short, the accuracy rate of qualification result is high, cultivate the advantages such as simple and easy, positive rate is high, pollution is few, have broad application prospects, be particularly useful for hospital, R&D institution's clostridium difficile separation and Culture is applied.
Further, step 1) in, the volume percent that alcohol accounts for mixed system is 75%, and the mass percent that sulphur Sodium cholic acid accounts for mixed system is 0.1%.
Adopt the beneficial effect of above-mentioned further scheme to be: mass percent be 0.1% sulfuric acid Sodium cholic acid promote that clostridium difficile gemma generates, volume percent be 75% alcohol can kill miscellaneous bacteria in fecal sample, and clostridium difficile gemma not to be affected; Simultaneously mixing quality per-cent be 0.1% sulfuric acid Sodium cholic acid and volume percent be 75% alcohol can strengthen clostridium difficile gemma and generate, and shorten the treatment time.
The too high or too low miscellaneous bacteria being unfavorable for killing in fecal sample of ethanol concn; And sulphur Sodium cholic acid excessive concentration or be too lowly unfavorable for that the sporulation of clostridium difficile and gemma are protected.
Further, step 2) in, the time left standstill is 1 hour.
The beneficial effect of above-mentioned further scheme is adopted to be: fully to ensure that clostridium difficile gemma generates and the miscellaneous bacteria killed in ight soil, and do not sacrifice the working efficiency of staff.
Further, step 4) in, described clostridium difficile Selective agar medium is CCFA Selective agar medium.
The beneficial effect of above-mentioned further scheme is adopted to be: to be the conventional Selective agar medium of clostridium difficile, to be conducive to the growth of clostridium difficile, to avoid the hypertrophy of other miscellaneous bacterias simultaneously.
Further, step 4) in, under anaerobic condition, culture condition is: 24h-48h cultivated by 30-37 DEG C of anaerobic culture box or anaerobism bag.
The beneficial effect of above-mentioned further scheme is adopted to be: to be the conventional Selective agar medium of clostridium difficile, to be conducive to the growth of clostridium difficile.
Further, step 5) after, then carry out following steps: be judged to be that positive bacterial strain is identified by pcr amplification conservative gene by selecting.
The beneficial effect of above-mentioned further scheme is adopted to be: method of the present invention, after preliminary evaluation, can be identified with PCR and combine, the accuracy of further qualification result.
In the process of research, contriver finds unexpectedly, sulphur Sodium cholic acid together with alcohol with the use of, may be used for clostridium difficile separation and Culture in ight soil, miscellaneous bacteria in ight soil can be suppressed, and improve the positive cultivation rate of clostridium difficile, ensure the accurate of the qualification result of clostridium difficile, and there is simple, quick, the easy and high accuracy for examination of preparation.In the process of concrete operations, mixed by the spirituous solution of ight soil and sulphur Sodium cholic acid, the volume percent that alcohol accounts for total system is 50%-80%, and the mass percent that sulphur Sodium cholic acid accounts for total system is 0.05%-0.5%.
Accompanying drawing explanation
Fig. 1 is according to embodiments of the invention 1, the result that bacterium is cultivated from 25 routine clostridium difficile patient ight soil, and positive control is that in embodiment 2, PCR method detects ight soil clostridium difficile result, and method 1 is for the invention provides method, and method 1 is for the invention provides method; Method 2 is simple ethanol postincubation method; Method 3 is traditional method.
Embodiment
Be described principle of the present invention and feature below in conjunction with accompanying drawing, example, only for explaining the present invention, is not intended to limit scope of the present invention.
Embodiment 1 clostridium difficile separation and ientification
1, experiment material
Sulphur Sodium cholic acid is purchased from Sheng Tianhengchuan bio tech ltd, Hubei, clostridium difficile basic medium, cefoxitin solution, 50% yolk saline suspension, D-Cycloserine solution are all purchased from Zhaoyuan topology bio-engineering corporation, anaerobic culture box (U.S. Ke Lusen), just genome DNA extracting reagent kit DP328-02 is purchased from TIANGEN Biotech (Beijing) Co., Ltd., primer, probe are all purchased from Invirogen company, and 10 × PCRbuffer, HSTaq, dNTP are purchased from Takara company.
2, fecal sample is collected
Diarrhea patient fecal sample during collecting hospital's in November, 2014 in March, 2015 (comprise watery stool, rare just, jelly sample just etc.) 25 examples, the selected trouble age is more than 18 years old, has more than one week microbiotic, immunosuppressor or chemotherapeutics and uses; Gather patient stool sample 1-2mL, if processed not in time, Saving specimen in the glycerin bouillon EP pipe of 10% ,-20 DEG C of preservations.
3, solution, substratum preparation
(1) 1% sulphur Sodium cholic acid preparation
Take 1g sulphur Sodium cholic acid, be dissolved in 100mL distilled water, after fully dissolving, sealed packaging stores.
(2) clostridium difficile CCFA Selective agar medium preparation
According to substratum preparation specification sheets, take 11g clostridium difficile basic medium, add 200mL distilled water, 121 DEG C of autoclaving 30min; Be cooled to about 55 DEG C, add 1 cefoxitin solution, 2 50% yolk saline suspension and 1 D-Cycloserine solution, fully after mixing, be down flat plate.
4, sample process
Adopt two kinds of methods to cultivate fecal samples respectively, a kind ofly take method for the present invention, the second is ethanol postincubation method, and the third is traditional method.The inventive method is: fecal sample is inoculated in final concentration be 0.1% sulphur Sodium cholic acid, in 75% spirituous solution, aerobic process is after 1 hour, and the centrifugal 5min of 13000rmp, gets precipitation and be inoculated in freshly prepared CCFA substratum, 35 DEG C of Anaerobic culturel 48h.Ethanol postincubation method: have reported in literature at present, adopt the ethanol postincubation fecal sample of 50% to improve clostridium difficile positive rate, concrete grammar is as follows; Fecal sample is mixed with raw spirit 1: 1, processes after 1 hour, the centrifugal 5min of 13000rmp, get precipitation and be inoculated in freshly prepared CCFA substratum, 35 DEG C of Anaerobic culturel 48h.Traditional method is: directly fecal sample is inoculated in CCFA substratum, 35 DEG C of Anaerobic culturel 48h.
Each group of process is as follows:
| Grouping | 1 group | 2 groups | 3 groups |
| Ight soil | 100uL | 100uL | 100uL |
| Raw spirit | 800uL | 100uL | 0 |
| 1% sulphur Sodium cholic acid | 100uL | 0 | 0 |
| Ethanol concn | 75％ | 50％ | 0％ |
| Sulphur Sodium cholic acid concentration | 0.1％ | 0％ | 0％ |
5, result judges
Select surface irregularity, edge uneven and produce the yellow color colonies of special odor, carry out gramstaining; Gramstaining is positive, and has the suspicious bacterium colony primary dcreening operation of obvious match head gemma to be judged to be the positive.
Primary dcreening operation is judged to be that positive bacterium colony can be identified further by pcr amplification conservative gene, and the conservative gene detected in the present invention is triosephosphate isomerase (triosephosphateisomerase, TPI) gene.
Cultivation results shows, and the method preliminary screening positive findings that the present invention adopts is 6 strain clostridium difficiles, and simple ethanol postincubation preliminary screening positive findings is 4 strain clostridium difficiles, and traditional method only detects 2 strain clostridium difficiles.
Embodiment 2PCR amplification clostridium difficile bacterium colony
1, extracting genome DNA
25 parts of fecal samples and embodiment 1 positive sample, extract bacterium colony genome see ight soil genome DNA extracting reagent kit specification sheets.
2, application of sample system
| Component | Volume |
| Reaction mixture | 35μL |
| Taq enzyme | 1μL |
| Gene DNA | 4μL |
| ddH 2O | Complement to 40 μ L |
Wherein, reaction mixture comprises 8 μ L10 × PCR damping fluids, dNTPs0.8mmol, each 0.8 μm of ol of upstream and downstream primer, probe 0.6 μm of ol.
Primer in system and probe are respectively upstream and downstream primer and the probe of TPI, as follows:
TPI upstream primer: 5 '-CTAGCTAAACTAGCTCCACCTAC-3 ', SEQIDNO.1,
TPI downstream primer: 5 '-TGCAACTGCTGAAGATGCTAATG-3 ', SEQIDNO.2,
TPI probe: 5 '-FAM/AGCTCCATCTATATCACTTTGACCCA/BHQ1, SEQIDNO.3;
Wherein, FAM refers to fluorescence radiation group, and Chinese is 6-Fluoresceincarboxylic acid;
BHQ1 refers to quenching group, is one of the Black hole quencher of BiosearchTechnologies company.
3, pcr amplification program
Quantitative fluorescent PCR reaction conditions is as shown above: the first stage: 94 DEG C of 5min; Subordinate phase: 94 DEG C of 20s, 58 DEG C of 30s, 10 circulations; Phase III: 94 DEG C of 20s, 58 DEG C of 30s, 40 circulations, collect FAM signal.Reach the cycle number Ct needed for threshold value of setting using FAM as judging criterion, and present S type amplification curve for positive, otherwise be negative.
4, detected result
Be accredited as positive clone through CCFA Selective agar medium primary dcreening operation, PCR detected result is the positive.Bacterium culture identification is positive fecal sample, and extracting directly faeces DNA adopts PCR method to detect and is the positive; In addition, errorless through repeatedly verifying, extract faeces DNA and adopt PCR method to detect 1 extra strain clostridium difficile.As shown in Figure 1, the overall verification and measurement ratio of clostridium difficile 85.7% (6/7) of the method for the present invention's employing is significantly higher than simple ethanol postincubation method 57.1% (4/7) and traditional method recall rate 28.6% (2/7).
Shown in table, method 1 is for the invention provides method; Method 2 is simple ethanol postincubation method; Method 3 is traditional method; PCR method is detected as method described in embodiment 2."+" represents positive, and "-" represents negative, and the positive sample that method 1-3 detects is that bacterium cultivates that to detect with PCR are all positives.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (9)
1. for a using method for the reagent of clostridium difficile separation and Culture in ight soil, it is characterized in that, described reagent is the spirituous solution of sulphur Sodium cholic acid, in use, comprises the following steps:
1) in ight soil, add the spirituous solution of sulphur Sodium cholic acid, mix, obtain mixed system;
2) mixed system is left standstill;
3) by step 2) leave standstill after mixed system centrifugal, remove suspended particle;
4) get supernatant, be inoculated into clostridium difficile Selective agar medium, cultivate under anaerobic condition;
5) select surface irregularity, edge uneven and produce the yellow color colonies of special odor, carry out gramstaining; Gramstaining is positive and has the bacterium colony of obvious match head gemma to be judged to be the positive, is clostridium difficile; Otherwise being judged to be feminine gender, is not namely clostridium difficile.
2. a kind of using method for the reagent of clostridium difficile separation and Culture in ight soil according to claim 1, it is characterized in that, step 1) in, the volume percent that alcohol accounts for mixed system is 75%, and the mass percent that sulphur Sodium cholic acid accounts for mixed system is 0.1%.
3. a kind of using method for the reagent of clostridium difficile separation and Culture in ight soil according to claim 1, is characterized in that, step 2) in, the time left standstill is 1 hour.
4. a kind of using method for the reagent of clostridium difficile separation and Culture in ight soil according to claim 1, is characterized in that, step 4) in, described clostridium difficile Selective agar medium is CCFA Selective agar medium.
5. a kind of using method for the reagent of clostridium difficile separation and Culture in ight soil according to claim 1, is characterized in that, step 4) in, under anaerobic condition, culture condition is: 24h-48h cultivated by 30-37 DEG C of anaerobic culture box or anaerobism bag.
6. a kind of using method for the reagent of clostridium difficile separation and Culture in ight soil according to any one of claim 1 to 5, it is characterized in that, step 5) after, then carry out following steps: be judged to be that positive bacterial strain is identified by pcr amplification conservative gene by selecting.
7. the application of composite reagent in ight soil in clostridium difficile separation and Culture, is characterized in that, described composite reagent comprises sulphur Sodium cholic acid and alcohol.
8. the application of a kind of composite reagent in ight soil in clostridium difficile separation and Culture according to claim 7, is characterized in that, in described reagent and the mixed total system of ight soil, the volume percent that alcohol accounts for total system is 50%-80%.
9. the application of a kind of composite reagent in ight soil in clostridium difficile separation and Culture according to claim 7, is characterized in that, in described reagent and the mixed total system of ight soil, the mass percent that sulphur Sodium cholic acid accounts for total system is 0.05%-0.5%.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105087445A (en) * | 2015-09-07 | 2015-11-25 | 杭州市农业科学研究院 | Method for isolating and purifying photosynthetic bacteria through AnaeroPack |
| CN105838774A (en) * | 2016-05-31 | 2016-08-10 | 浙江省疾病预防控制中心 | Clostridium difficile chromogenic medium and application thereof |
| CN114480562A (en) * | 2022-01-27 | 2022-05-13 | 西南医科大学附属医院 | PFOR enzyme activity guide-based FMT donor screening method and application thereof |
| CN114540238A (en) * | 2022-03-10 | 2022-05-27 | 江南大学 | Culture medium for separating clostridium difficile from intestinal microorganisms and preparation method thereof |
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| US20110287474A1 (en) * | 2010-05-18 | 2011-11-24 | Paul Riska | Cultures and protocols for diagnosis of toxigenic clostridium difficile |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105087445A (en) * | 2015-09-07 | 2015-11-25 | 杭州市农业科学研究院 | Method for isolating and purifying photosynthetic bacteria through AnaeroPack |
| CN105838774A (en) * | 2016-05-31 | 2016-08-10 | 浙江省疾病预防控制中心 | Clostridium difficile chromogenic medium and application thereof |
| CN114480562A (en) * | 2022-01-27 | 2022-05-13 | 西南医科大学附属医院 | PFOR enzyme activity guide-based FMT donor screening method and application thereof |
| CN114480562B (en) * | 2022-01-27 | 2023-09-19 | 西南医科大学附属医院 | FMT donor screening method based on PFOR enzyme activity guidance and application thereof |
| CN114540238A (en) * | 2022-03-10 | 2022-05-27 | 江南大学 | Culture medium for separating clostridium difficile from intestinal microorganisms and preparation method thereof |
| CN114540238B (en) * | 2022-03-10 | 2023-08-08 | 江南大学 | Culture medium for separating clostridium difficile in intestinal microorganisms and preparation method thereof |
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Inventor after: Zhang Lianglu Inventor after: Zhou Lu Inventor after: Dan Yanlin Inventor before: Zhang Lianglu Inventor before: Yin Lei Inventor before: Hong Haoyan |
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Application publication date: 20160406 |