CN103014120B - Trace mycobacterium susceptibility test method - Google Patents
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Abstract
The invention provides a trace mycobacterium detection method and a susceptibility test method. According to the invention, the test is carried out by using a solid medium, a liquid medium and an indicator indicating mycobacterium growth. Only a small amount of strain is needed, and susceptibility tests of a plurality of drugs and mycobacterium identification can be completed in a shorter period of time. No instrument such as microscope is needed, and a test result can be observed by naked eye.
Description
Technical field
The present invention relates to a kind of Mycobacterium drug sensitive detection method, particularly relate to a kind of reduce growth indicator to mycobacterium toxicity, less bacterium amount sample can be realized carry out micro-Mycobacterium drug sensitive detection and the method for further identification of mycobacterium and mycobacterium tuberculosis.
Background technology
Mycobacterium drug sensitive testing method main at present comprises phenotype drug sensitive test and molecule drug sensitive test.Its Middle molecule susceptibility is along with molecular biological development, the research of mycobacterium resistance mechanism progressively deeply, and the direct-detection of relevant drug resistant gene and become study hotspot, establish the molecular medicine sensitization test of many detection mycobacterium mutator genes at present, without the need to cultivating, can the various sample of direct-detection, biological safety is good, detect fast, as PCR-restriction fragment length polymorphism analysis, DNA sequencing, PCR single-strand conformation polymorphism analysis, linear probe technology, DNA chip technology etc., but molecule drug sensitive test complex operation, and sensitivity and specificity also not satisfactory.
Phenotype drug sensitive test is mainly Phages Lysis test, microscopic examination Susceptibility detection technique, oxidation-reduction indicator method.
1) Phages Lysis test (Phageamplified biologically assay, PhaB): mycobacterium tuberculosis phagocytosis physical efficiency infects the mycobacterium tuberculosis of living, do not enter and infect endobacillary phage by the disinfectant deactivation added subsequently, enter endobacillary phage to breed in a large number in thalline, cellular lysate the most at last, agar plate occurs transparent plaque.The substratum adding antitubercular agent is interior because medicine is to the suppression of tubercule bacillus, and phage can not infect dead thalline, causes plaque not formed, so show as sensitivity; Otherwise what have Plaques assay is then Resistant strain.The method just can obtain result in 48 hours, but because antitubercular agent acts on mycobacterium tuberculosis mechanism difference, some is sterilization class medicine, and some is antibacterial class medicine,, cause the method to detect the susceptibility of the drug sensitivity tests of different antitubercular agent and specificity differs greatly.
2) microscopic examination Susceptibility detection technique (microscopic observation drug susceptibility, MODS): MODS technology is a kind of liquid cultural method of one tubercule bacillus morphology being carried out to microscopic examination and Resistance detection.In liquid medium within, tubercule bacillus can secrete Trehalose 6,6-dimycolate, and whether make its expression characteristics cord structures, this structure is observed by inverted microscope, has determined whether growth of bacillus tubercle, and responsive to medicine.In the people such as Hu Zhongyi paper disclosed in the academic conference of national resistant tuberculosis in 2009, have detected 66 strain tubercule bacillus to 4 kind of one line antitubercular agent: the resistance situation of Streptomycin sulphate, vazadrine, Rifampin and Tibutol by MODS method, its susceptibility is respectively: 97.0%, 90.9%, 95.5% and 86.4%, illustrates that MODS method has good application prospect in the detection of Multiple drug-resistan Tubercle bacillus.But the method must depend on inverted microscope at present, can not naked eyes observations very intuitively.
3) oxidation-reduction indicator method (redox-indicator methods): have in the liquid nutrient medium of tubercule bacillus in inoculation and add various different concns antitubercular agent and oxidation-reduction indicator, after hatching certain hour, color change whether is there is to have judged whether this strain growth according to indicator, as indicator generation reduction reaction, then prompting has tubercule bacillus to grow, and this bacterial strain is described under this drug level to this kind of Drug-resistant.This method does not need specific apparatus, with low cost.But current various indicator all has certain toxicity to mycobacterium, need consumption or the acquisition result that cultivates one's ability of needs longer time of the bacterial strain sample of higher concentration or minimizing indicator, therefore oxidation-reduction indicator method is mainly used in the susceptibility detection of mycobacterium clinical separation strain at present, and the Susceptibility Testing that cannot be used for mycobacterium in the little clinical samples of bacteria containing amount detects.
Summary of the invention
The invention provides a kind of micro-mycobacterium detection method and susceptibility detection method, solid-liquid two-phase substratum is adopted to use together with mycobacterium growth indicator, be surprised to find that, in this culture system, described strain growth indicator does not almost have toxicity to mycobacterium, significantly to shorten detection time with existing oxidation-reduction indicator method susceptibility, and bacterial strain consumption is less, the Susceptibility Testing that can be used for clinical samples mycobacterium detects.
The present invention's first object is to provide a kind of Trace mycobacterium susceptibility test method, and step comprises:
Step 1, prepares substratum, and described substratum is made up of solid medium and liquid nutrient medium, the below of solid medium liquid medium within; Containing growth of bacillus tubercle indicator in liquid nutrient medium; And by anti-mycobacterium medicine ordinance in substratum;
Step 2, joins test strains sample in step 1 substratum;
Step 3, cultivates the substratum adding test strains sample 7 ~ 35 days, as experimental group under 37 DEG C of conditions; Observe liquid nutrient medium whether variable color, or observe in solid culture primary surface or liquid nutrient medium and whether occur being with coloured particles.
Wherein, described substratum is the substratum of available cultivation mycobacterium, comprises solid medium, liquid nutrient medium and strain growth indicator.
Solid medium, as Michaelis 7H10 agarose media, Russell medium etc., also can be other solid medium of this area, or any mixture of above-mentioned solid medium.Victoria Green WPB can also be contained in described solid medium.
Liquid nutrient medium, as the Michaelis 7H9 liquid nutrient medium containing 10%OADCA or 10%ADC, also can be other liquid nutrient medium of this area (as logical substratum of reviving), or any mixture of aforesaid liquid substratum.
Microbiotic can also be contained for suppressing the sex pheromone of common quick growth, as streptococcus aureus, suis, Pseudomonas aeruginosa, Pseudomonas aeruginosa, candida albicans etc. in described liquid nutrient medium.
In above-mentioned method, described solid medium and liquid nutrient medium volume ratio are preferably 1:1 ~ 1:5.
In above-mentioned method, described growth indicator can be XTT(3,3'-[1-(phenylamino acyl group)-3,4 tetrazoles]-two (4-methoxyl group-6-nitro) benzene sulfonic acid sodium salt), resazurin (between azo tetrahydroxy phenol) etc., now observe liquid nutrient medium whether variable color; If variable color, then test strains sample is to the not clear sense of medicine, if nondiscoloration or variable color not obvious, then test strains sample is to medicaments insensitive.
It is TTC(2 that the present invention preferably grows indicator, 3,5-triphenyltetrazolium chloride) and/or MTT(thiazole bromide blue tetrazolium), now observe in liquid nutrient medium or whether solid culture primary surface occurs being with coloured particles, if there is band coloured particles, then test strains sample is to the not clear sense of medicine, if there is not band coloured particles or band coloured particles seldom, then test strains sample is to medicaments insensitive.The concentration of described growth indicator in described liquid nutrient medium is preferably 1 μ g/ml ~ 100 μ g/ml.
In the method that the present invention is above-mentioned, preferably test strains liquid nutrient medium is cultured to 10
4cFU/ml concentration, and add test strains suspension 0.1ml in every 1ml liquid nutrient medium.
In above-mentioned method, described mycobacterium is preferably mycobacterium tuberculosis, and available antitubercular agent is as Rimactazid, Streptomycin sulphate, Tibutol, Ofloxacine USP 23, Moxifloxacin, amikacin, capromycin etc.; Antitubercular agent can join in liquid nutrient medium, also can join in solid medium.
In the method that the present invention is above-mentioned, add antitubercular agent when detecting, also can be provided with the control group not adding anti-mycobacterial infections medicine, control group add that bacterium amount is experimental group 1% ~ 10%, be preferably 10%, other condition is identical.Be cultured to the variable color of control group liquid nutrient medium or occur band coloured particles, can judge: decision method is as follows: in be measured group of pastille, solid culture primary surface occurs that band coloured particles is more than control group, then represent that test strains is insensitive to described medicine, be then judged to be the resistance of bacterial strain to this medicine; To be measured group of cell culture primary surface does not occur band coloured particles or fewer than control group representing that test strains is to described medicaments insensitive, be then judged to be that bacterial strain is to this medicine not resistance or sensitivity.
The present invention's second object is to provide a kind of micro-mycobacterium authentication method, and step comprises:
Step 1, prepares substratum, and described substratum is made up of solid medium and liquid nutrient medium, the below of solid medium liquid medium within; Containing mycobacterium growth indicator in liquid nutrient medium; And mycobacterium indentifying substance is configured in substratum; Described mycobacterium growth indicator is preferably M. tuberculosis growth indicator, and described mycobacterium indentifying substance is preferably mycobacterium tuberculosis will determine reagent;
Step 2, joins test strains sample in step 1 substratum;
Step 3, cultivates the substratum adding test strains sample 7 ~ 35 days, as experimental group under 37 DEG C of conditions; Observe liquid nutrient medium whether variable color, or observe in solid culture primary surface or liquid nutrient medium and whether occur being with coloured particles, and recording strip coloured particles number.
Wherein, described substratum is the substratum of available cultivation mycobacterium, comprises solid medium, liquid nutrient medium and strain growth indicator.
Solid medium, as Michaelis 7H10 agarose media, Russell medium etc., also can be other solid medium of this area, or any mixture of above-mentioned solid medium.Victoria Green WPB can also be contained in described solid medium.
Liquid nutrient medium Michaelis 7H9 liquid nutrient medium also can be other liquid nutrient medium of this area (as logical substratum of reviving), or any mixture of aforesaid liquid substratum.The present invention is preferably as the Michaelis 7H9 liquid nutrient medium containing 10%OADCA or 10%ADC.
Microbiotic can also be contained for suppressing the sex pheromone of common quick growth, as streptococcus aureus, suis, Pseudomonas aeruginosa, Pseudomonas aeruginosa, candida albicans etc. in described liquid nutrient medium.
In above-mentioned method, described solid medium and liquid nutrient medium volume ratio are preferably 1:1 ~ 1:5.
In above-mentioned method, described mycobacterium tuberculosis identification reagent is as p-nitrobenzoic acid, thiophene-2-carboxylic acid hydrazine etc.Core mycobacterium identification reagent can join in liquid nutrient medium, also can join in solid medium.
In above-mentioned method; described growth indicator can be XTT(3; 3'-[1-(phenylamino acyl group)-3,4 tetrazoles]-two (4-methoxyl group-6-nitro) benzene sulfonic acid sodium salt), resazurin (between azo tetrahydroxy phenol) etc., now observe liquid nutrient medium whether variable color.
It is TTC(2,3,5-triphenyltetrazolium chloride that the present invention preferably grows indicator) and/or MTT(thiazole bromide blue tetrazolium), now observe in liquid nutrient medium or whether solid culture primary surface occurs being with coloured particles.The concentration of described growth indicator in described liquid nutrient medium is preferably 1 μ g/ml ~ 100 μ g/ml.
In the method that the present invention is above-mentioned, preferably test strains liquid nutrient medium is cultured to 10
4cFU/ml concentration, and add test strains suspension 0.1ml in every 1ml liquid nutrient medium.
A kind of preferred implementation of the above-mentioned micro-mycobacterium detection method of the present invention or susceptibility detection method, wherein, cultivate described in step 3 and carry out in micro porous plate, the opening part of each culture hole is to there being convex lens, described convex lens can be the openings being arranged on culture hole, porous plate has supporting lid, and the position that lid corresponds to culture hole is provided with convex lens.
In the micro-mycobacterium detection method that the present invention is above-mentioned or susceptibility detection method, test strains sample can be sputum specimen, hydrothorax, cerebrospinal fluid, blood etc. containing or the doubtful humoral specimen containing mycobacterium, also can be the mycobacterium strain be separated.
The method that trace detection mycobacterium provided by the invention and susceptibility detect, two-phase substratum and mycobacterium growth indicator is adopted to carry out the detection of mycobacterium, unexpectedly, indicator does not almost have toxicity to test strains, thus only needs a small amount of bacterial strain sample can complete the qualification of drug susceptibility detection and bacterial classification.Even 10
-5the bacterial strain sample of mg/ml concentration, detection time only needs about 10 days, and conventional oxidation reduction indicator method needs more than 1 month detection time, and detection time significantly shortens.Simultaneously because indicator does not almost have toxicity to test strains, colour developing can also be made more obvious by the consumption strengthening indicator, it is more accurate to observe.
Because tubercule bacillus indicator can carry out the incubation growth situation of real bacterium to be measured intuitively by the change of color, therefore, susceptibility detection method of the present invention is without the need to other instruments, get final product the colour developing particle that direct visual perception produces to bacterial growth, thus judge the drug susceptibility of bacterial strain and carry out strain identification.
To sum up, the invention provides a kind of fast, the mycobacterium detection method of simple, directly perceived and economy and Mycobacterium drug sensitive detection method, the method can be used for detecting the drug susceptibility of mycobacterium, the research and development of the qualification of mycobacterium and anti-mycobacterium medicine and screening.
Embodiment
The invention provides the susceptibility detection method of a kind of micro-mycobacterium, by solid medium, liquid nutrient medium and mycobacterium growth indicator with the use of, be surprised to find that, strain growth indicator does not almost have toxicity to mycobacterium in the case, only needs a small amount of bacterial strain sample to carry out susceptibility test experience.Susceptibility detection method of the present invention, without the need to the miscellaneous equipments such as microscope and instrument, can observe susceptibility detected result intuitively by the change of indicator color.
Below by specific embodiment, be described in detail Trace mycobacterium susceptibility test method of the present invention and describe, consciousness better understands the scope of the invention, but following embodiment does not limit the scope of the invention.
embodiment 1
Get sterile micro porous plate, in culture hole, add 1ml Russell medium, then solidify and obtain solid medium.
After solid medium solidifies, add 1ml liquid nutrient medium in culture hole, in the present embodiment, liquid nutrient medium is the Michaelis 7H9 liquid nutrient medium containing 10%OADC nutritional additive.
Experimental group of the present invention: add M. tuberculosis growth indicator TTC respectively in liquid nutrient medium in different culture hole, in TTC liquid medium within, concentration is 10 μ g/ml.
Comparative group 1: do not use solid medium, and do not add any indicator in liquid nutrient medium, other conditions are identical with experimental group.
M. tuberculosis strains liquid nutrient medium is cultured to 10
4cFU/ml, adds 0.1ml mycobacterium tuberculosis suspension in culture hole.
Cover cover plate, micro porous plate is placed in 37 DEG C of incubators and cultivates, whether experimental group visual inspection there is red granules (i.e. M. tuberculosis growth), and comparative group 1 inverted microscope observes whether occur characteristic cord structures (i.e. M. tuberculosis growth).Detected result finds, two groups of Result times are roughly the same, illustrate in detection method that growing indicator does not almost have toxicity to mycobacterium tuberculosis.
Comparative group 2: do not use solid medium, the Michaelis 7H9 liquid nutrient medium 2ml of 10%OADC nutritional additive, other conditions are identical with experimental group, whether visual inspection occurs that red granules (i.e. M. tuberculosis growth) found that, more than experimental group 10 ~ 20 days of the time of red granules appearred in comparative group 2.
embodiment 2
Get sterile micro porous plate, in culture hole, add 1ml Russell medium, then solidify and obtain solid medium.
After solid medium solidifies, add 2ml liquid nutrient medium in culture hole, in the present embodiment, liquid nutrient medium is the Michaelis 7H9 liquid nutrient medium containing 10%OADC nutritional additive.
The medicine that the Killing Mycobacterium Tuberculosis such as Rimactazid, Streptomycin sulphate, Tibutol, Ofloxacine USP 23, Moxifloxacin, amikacin, capromycin and kantlex infect is added in culture hole, in liquid nutrient medium, add M. tuberculosis growth indicator MTT, in MTT liquid medium within, concentration is 20 μ g/ml simultaneously.
Mill bacterium is cultured to 10 than the clinical separation strain liquid nutrient medium after turbid
4cFU/ml, adds 0.1ml mycobacterium tuberculosis suspension in culture hole.
Cover cover plate, micro porous plate is placed in 37 DEG C of incubators and cultivates, observations after 20 days, during observation, direct naked eyes are observed through the cover plate of micro porous plate, observation is that direct naked eyes are observed through the cover plate of micro porous plate, brown granulated is there is in culture hole, illustrate and represent that in this culture hole, test strains is insensitive to this medicine, it not mycobacterium tuberculosis, brown granulated is not there is in culture hole, illustrate and represent that in this culture hole, test strains, to this medicaments insensitive, is mycobacterium tuberculosis.
embodiment 3
Get sterile micro porous plate, in culture hole, add 1ml autoclaving Michaelis 7H10 agarose solid medium.
Then add 2ml liquid nutrient medium in culture hole, in the present embodiment, liquid nutrient medium is the Michaelis 7H9 liquid nutrient medium containing 10%OADC nutritional additive.
P-nitrobenzoic acid (PNB) and thiophene-2-carboxylic acid hydrazine (TCH) is added respectively in different culture hole, in liquid nutrient medium, add 50 μ g M. tuberculosis growth indicator TTC simultaneously.
Mill bacterium is cultured to 10 than the clinical separation strain liquid nutrient medium after turbid
4cFU/ml, adds 0.1ml mycobacterium tuberculosis suspension in culture hole.
Cover cover plate, be placed in by micro porous plate in 37 DEG C of incubators and cultivate, observations after 10 days, during observation, direct naked eyes are observed through the cover plate of micro porous plate, occur red granules in PNB culture hole, illustrate that test strains is not mycobacterium tuberculosis; All not there is red granules in PNB and TCH culture hole, illustrate that test strains is bacillus tuberculosis typus humanus; If do not occur red granules in PNB culture hole, and have red granules in TCH culture hole, then illustrate that test strains is mycobacterium tuberculosis var bovis.
embodiment 4
Get sterile micro porous plate, in culture hole, add 1ml autoclaving Michaelis 7H10 agarose solid medium.Also add in solid medium and have Victoria Green WPB.
After solid medium solidifies, add 1ml liquid nutrient medium in each culture hole, in the present embodiment, liquid nutrient medium is the Michaelis 7H9 liquid nutrient medium containing 10%OADC nutritional additive.
Also add mixing microbiotic in liquid nutrient medium, suppress the growth comprising the bacteriums such as streptococcus aureus, suis, Pseudomonas aeruginosa, Pseudomonas aeruginosa, candida albicans.
In different culture hole, add the medicine that the Killing Mycobacterium Tuberculosis such as Rimactazid, Streptomycin sulphate, Tibutol, Ofloxacine USP 23, Moxifloxacin, amikacin, capromycin and kantlex infect respectively, in liquid nutrient medium, add 50 μ g M. tuberculosis growth indicator TTC simultaneously.
Mill bacterium is cultured to 10 than the clinical separation strain (testing sample) after turbid with liquid nutrient medium
4cFU/ml, adds 0.1ml mycobacterium tuberculosis suspension, as experimental group in culture hole.
Arrange control group, not with antitubercular agent in control group, and testing sample add-on is 0.01ml simultaneously.
Cover cover plate, be placed in by micro porous plate in 37 DEG C of incubators and cultivate, whether the green of observing solid medium for 2 days afterwards deepens, and as fruit green obviously deepens, indicates that other mycobacterium tuberculosis pollute, needs to re-start detection.
After 7 days, red granules is there is in control group, observation experiment group result, during observation, direct naked eyes are observed through the cover plate of micro porous plate, occur red granules in culture hole, illustrate and represent that in this culture hole, test strains is insensitive to this medicine, it not mycobacterium tuberculosis, not there is red granules in culture hole, illustrate and represent that in this culture hole, test strains, to this medicaments insensitive, is mycobacterium tuberculosis.
embodiment 5
Get aseptic 24 hole micro porous plates, add 1ml Russell medium in each culture hole, then solidify and obtain solid medium, also adding in solid medium has Victoria Green WPB.And add the medicine that the Killing Mycobacterium Tuberculosis such as Rimactazid, Streptomycin sulphate, Tibutol, Ofloxacine USP 23, Moxifloxacin, amikacin, capromycin and kantlex infect in different culture hole in solid medium respectively.
After solid medium solidifies, add 1ml liquid nutrient medium in each culture hole, in the present embodiment, liquid nutrient medium is the Michaelis 7H9 liquid nutrient medium containing 10%OADC nutritional additive.
Also add mixing microbiotic in liquid nutrient medium, suppress the growth comprising the bacteriums such as streptococcus aureus, suis, Pseudomonas aeruginosa, Pseudomonas aeruginosa, candida albicans.
100 μ g M. tuberculosis growth indicator TTC are added in liquid nutrient medium.
The sputum specimen precipitation phosphate buffered saline buffer (or physiological saline) containing mycobacterium tuberculosis through liquefaction processing washs 2 times, and liquid nutrient medium is resuspended, adds 0.1ml sputum specimen precipitation suspension liquid in each culture hole.
Mill bacterium is cultured to 10 than the clinical separation strain liquid nutrient medium after turbid
4cFU/ml, adds 0.1ml mycobacterium tuberculosis suspension in culture hole.
Cover cover plate, micro porous plate is placed in 37 DEG C of incubators and cultivates, observations after 7 days, during observation, direct naked eyes are observed through the cover plate of micro porous plate, occur red granules in culture hole, illustrate and represent that in this culture hole, test strains is insensitive to this medicine, it not mycobacterium tuberculosis, not there is red granules in culture hole, illustrate and represent that in this culture hole, test strains, to this medicaments insensitive, is mycobacterium tuberculosis.
embodiment 6
Get sterile micro porous plate, add 1ml Russell medium in culture hole, then solidify and obtain solid medium, also adding in solid medium has Victoria Green WPB.
After solid medium solidifies, add 5ml liquid nutrient medium in each culture hole, in the present embodiment, liquid nutrient medium is the Michaelis 7H9 liquid nutrient medium containing 10%OADC nutritional additive.
Also add mixing microbiotic in liquid nutrient medium, suppress the growth comprising the bacteriums such as streptococcus aureus, suis, Pseudomonas aeruginosa, Pseudomonas aeruginosa, candida albicans.
The medicine that the possible Killing Mycobacterium Tuberculosis adding new research and development in culture hole infects, adds 200 μ g M. tuberculosis growth indicator TTC simultaneously in liquid nutrient medium.
Mycobacterium tuberculosis liquid nutrient medium is cultured to 10
4cFU/ml, adds 0.1ml mycobacterium tuberculosis suspension in culture hole.
Cover cover plate, micro porous plate is placed in 37 DEG C of incubators and cultivates, observations after 20 days, during observation, direct naked eyes are observed through the cover plate of micro porous plate, red granules is there is in culture hole, illustrate that mycobacterium tuberculosis is insensitive to medicine to be measured in this culture hole, this medicine can not be used as resisting tuberculosis infection medicine; If not there is red granules in culture hole, illustrate that mycobacterium tuberculosis is to medicaments insensitive to be measured in this culture hole, this medicine can be used as resisting tuberculosis infection medicine.
Meanwhile, in above-described embodiment, cover plate is transparent cover plate, and cover plate is put position and can also be provided with convex lens corresponding to each culture hole, and by the red granules occurred in convex lens amplification culture hole, thus more convenient carrying out is observed.
embodiment 7
Victoria Green WPB adds in Michaelis 9H10 agarose solid medium, and autoclaving, the solid medium got after 1ml sterilizing adds 24 orifice plates.
Then the Michaelis 7H9 liquid nutrient medium that 1 contains 10OADC nutritional additive is added, respectively to adding Rimactazid, Streptomycin sulphate, Tibutol, Ofloxacine USP 23, Moxifloxacin, amikacin, capromycin, kantlex (above is medicine group), p-nitrobenzoic acid (PNB) and thiophene-2-carboxylic acid hydrazine (TCH) in different hole; In liquid nutrient medium, add growth indicator TTC, concentration is 50 μ g/ml simultaneously.
The sputum specimen precipitation phosphate buffered saline buffer of liquefaction processing washs 2 times, resuspended with liquid nutrient medium, adds 0.1ml sample precipitation suspension liquid in each hole; 0.01ml sample precipitation suspension liquid is added, as a control group in the hole not adding medicine and identification reagent.
Cover cover plate, sealing.37 DEG C of cultivations, by visual results on cover plate after 7 days:
1) if control group and medicine group all occur red granules, then test strains Antituberculous Drugs insensitive (resistance) is represented;
2) if red granules appears in control group, and there is not red granules in medicine group, then represent test strains Antituberculous Drugs sensitivity (not resistance);
3) if there is red granules in PNB hole, then represent that test strains is non-binding mycobacterium;
4) if equal redfree particle in PNB hole and TCH hole, then represent that test strains is bacillus tuberculosis typus humanus;
5) if PNB hole redfree particle, and in TCH hole, there is red granules, then represent that test strains is mycobacterium tuberculosis var bovis.
Although foregoing is only introduced for mycobacterium tuberculosis, with passing through foregoing description, those skilled in the art are it is understood that the present invention also may be used for the susceptibility of other mycobacteriums and preliminary strain identification; And prior art can be adopted to implement according to the indicator used and identification reagent by those skilled in the art according to the method that substratum color and red granules carry out judging.
Be described in detail specific embodiments of the invention above, but it is just as example, the present invention is not restricted to specific embodiment described above.To those skilled in the art, any equivalent modifications that the present invention is carried out and substituting also all among category of the present invention.Therefore, equalization conversion done without departing from the spirit and scope of the invention and amendment, all should contain within the scope of the invention.
Claims (5)
1. the micro-mycobacterium of non-diagnostic object is identified or a susceptibility detection method, and it is characterized in that, step comprises:
Step 1, prepares substratum, and described substratum is made up of solid medium and liquid nutrient medium, the below of solid medium liquid medium within; Containing growth of bacillus tubercle indicator TTC or MTT in liquid nutrient medium; And anti-mycobacterium medicine or mycobacterium indentifying substance are configured in substratum;
Step 2, joins test strains sample in step 1 substratum;
Step 3, cultivates the substratum adding test strains sample 7 ~ 35 days under 37 DEG C of conditions; Observe and whether occur being with coloured particles, or liquid nutrient medium whether variable color;
Wherein, described solid medium and described liquid nutrient medium volume ratio are 1:1 ~ 1:5.
2. method according to claim 1, is characterized in that, the concentration of described growth indicator in described liquid nutrient medium is 1 μ g/ml ~ 100 μ g/ml.
3. method according to claim 1, is characterized in that, described liquid nutrient medium is the Michaelis 7H9 liquid nutrient medium containing 10%OADC substratum and/or 10%ADC substratum.
4. method according to claim 1, is characterized in that, described solid medium is one or both the mixture in Michaelis 7H10 substratum, Russell medium.
5. the method according to claim 3 or 4, is characterized in that, described solid medium is Russell medium, and described liquid nutrient medium is the Michaelis 7H9 liquid nutrient medium containing 10%OADC nutritional additive.
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| CN101168766A (en) * | 2007-11-13 | 2008-04-30 | 上海市肺科医院 | Diphase Roe's authentication or drug sensitive culture medium |
| CN102146430A (en) * | 2011-01-27 | 2011-08-10 | 兰州大学 | Mycobacterium tuberculosis medicament sensitive phenotype detection method and application of method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101168766A (en) * | 2007-11-13 | 2008-04-30 | 上海市肺科医院 | Diphase Roe's authentication or drug sensitive culture medium |
| CN102146430A (en) * | 2011-01-27 | 2011-08-10 | 兰州大学 | Mycobacterium tuberculosis medicament sensitive phenotype detection method and application of method |
Non-Patent Citations (1)
| Title |
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| "MODS技术快速检测结核分枝杆菌左氧氟沙星耐药性研究";付佑辉等;《临床肺科杂志》;20090228;第14卷(第2期);全文 * |
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