CN101597566B - Method for detecting drug sensitivity of mycobacterium tuberculosis by using closed micropore card - Google Patents
Method for detecting drug sensitivity of mycobacterium tuberculosis by using closed micropore card Download PDFInfo
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- CN101597566B CN101597566B CN2009100654955A CN200910065495A CN101597566B CN 101597566 B CN101597566 B CN 101597566B CN 2009100654955 A CN2009100654955 A CN 2009100654955A CN 200910065495 A CN200910065495 A CN 200910065495A CN 101597566 B CN101597566 B CN 101597566B
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Abstract
The invention discloses a method for detecting drug sensitive of Mycobacterium tuberculosis by using a closed micropore card, comprising an oblong card body with test micropores and an upper cover; the test micropores are evenly distributed on the card body in multiple rows and columns; a ring-shaped groove surrounding the test micropores is arranged on the card body; a card groove is arranged on the periphery of the card body outside the ring-shaped groove; a card plate arranged on the periphery of the upper cover is matched with the card groove; the inner surface of the upper cover is provided with sealed convex rings corresponding to the micropores. The invention also discloses a method for detecting drug sensitivity of mycobacterium tuberculosis by using the card, which can identify the clinically isolated strains of the mycobacterium tuberculosis to be detected and determine the minimum inhibitory concentration of antituberculosis drugs at the same time. Setting of growth indicator pores facilitates the drug sensitivity tests of the clinically isolated strains at different growth speeds to be completed smoothly and ensures the reliability of the results. The tightness of the micropore card ensures the safety of the operators.
Description
Technical field
The present invention relates to a kind of vitro detection compositions and methods, especially relate to a kind of method of using the closed micropore card to detect drug sensitivity of mycobacterium tuberculosis.
Background technology
Tuberculosis is a kind of ancient disease, Be Controlled once once, but along with AIDS VICTIMS's popular with immunosuppressant therapy of increasing, global in recent years tuberculosis is in rising trend.Especially China, because treatment restriction of condition and increasing of AIDS patient, tuberculosis is very severe in China's situation.And the method that the tubercule bacillus susceptibility detects in the most of hospital of current China still is solid medium absolute concentration method and scaling method, and additive method all can not get popularizing owing to the restriction or the cost too expensive of state of the art at present.Yet the solid susceptibility method time is very long, can reach two months, seriously delays patient's timely diagnosis and treatment.And on the testing method of tuberculosis susceptibility, the liquid susceptibility is because method more fast progressively is subjected to the attention of clinical detection.This kind method also is the method that WHO encourages employing, and especially in developing country, liquid susceptibility method will further be urged its development owing to cheap.
In current liquid nutrient medium antibiotics susceptibility test, the most frequently used is to adopt MTT as the bacterial growth indicator, if MTT becomes red-purple from yellow, then representing has bacterial growth in the culture, judge the resistance situation of mycobacterium tuberculosis with this.MTT has cytotoxicity, and the growth of mycobacterium tuberculosis is had certain influence.Alamar Blue is as a kind of novel developer, and by wide coverage, it does not influence the normal growth of cell as the indicator of cell growth, follow-up growing state that can observation of cell after adding.The domestic method discussion to some extent that Alamar Blue method is detected mycobacterium tuberculosis.Present method promptly is to adopt Alamar Blue as the tubercule bacillus indicator of growing, and its colour developing is more sensitiveer than mtt assay, has shortened the time of detection.
In the drug sensitive test to mycobacterium tuberculosis, because bottled culture volume is big, the microbiotic that needs is more and make tuberculosis susceptibility cost of expert testimony costliness.On microwell plate, carry out the tuberculosis drug sensitive test, though considerable research has been arranged, yet, because the test aperture on the microwell plate is not relative with the external world airtight, make that the tuberculosis drug sensitive test is difficult to break through on microwell plate, can't enter the clinical detection laboratory.Closed micropore plate at present method design is exactly in order to solve these difficult problems.
Summary of the invention
The object of the present invention is to provide a kind of low cost to carry out the closed micropore card of drug sensitive test, and use this closed micropore card to detect the method for drug sensitivity of mycobacterium tuberculosis.
For achieving the above object, the present invention can take following technical proposals:
Use closed micropore card of the present invention detects the method for drug sensitivity of mycobacterium tuberculosis, comprise the rectangular card body and the loam cake that have test microvia, the mode that described test microvia is multirow, multiple row (4 * 9,4 * 10,4 * 11 or 4 * 12) is distributed on the card body; On described card body, offer annular recesses around test microvia, card body periphery place outside described annular recesses is provided with draw-in groove, clamp and described draw-in groove that described loam cake periphery is provided with are complementary, and are provided with and the corresponding sealing bulge loop of each micropore at the internal surface of described loam cake.
For guaranteeing to form good sealing between loam cake and card body, Mycobacterium tuberculosis is not polluted the environment in culturing process, to guarantee operator's safety, be pasted with flexible sealing ring on the inwall of described draw-in groove.
The loam cake clamp of closed micropore card of the present invention snaps in the draw-in groove of card body, the sealing bulge loop of upper interior surface effectively seals pairing micropore, can guarantee that whole culturing process carries out in an airtight system, the annular recesses of offering around test microvia is used for pouring into sterilized water, prevents the excessive vaporization of moisture in the drug sensitive test process; Whole tuberculosis drug sensitive test can be carried out in being equipped with the laboratory of Biohazard Safety Equipment.
Use above-mentioned closed micropore card to detect the method for drug sensitivity of mycobacterium tuberculosis, may further comprise the steps:
The first step: be divided into three zones with arranging the test microvia that is provided with on the closed micropore card card body: susceptibility is identified district's multiple row; Bacterial type is identified district's 1 row; Growth indicator 2 row: one classifies the thalli growth telltale hole as, and one classifies substratum Quality Control hole as;
Second step: the loam cake of closed micropore card is opened, and the micropore of susceptibility being identified the district is by the row classification, and each row adds a kind of microbiotic, and with antibiotic amount substratum doubling dilution in each micropore in the delegation, antibiotic concentration reduces successively; Identify that at bacterial type multiple hole interpolation PNB identifies that substratum and TCH identify substratum in the micropore of distinguishing; In the micropore of growth indicator, add with susceptibility and identify the substratum that the district is identical;
The 3rd step: add tubercule bacillus bacteria suspension to susceptibility respectively and identify that district, bacterial type are identified in the micropore in district and in the thalli growth telltale hole of growth indicator, the substratum Quality Control does not add bacteria suspension in the hole, is blank through dilution;
The 4th step: will add sterilized water in the annular recesses of offering around all micropores on the closed micropore card, cover the loam cake of closed micropore card, after microwell plate seals with loam cake, put into 37 ℃ of incubator 3-8 days;
The 5th step: cultured microwell plate in the 4th step is taken out, first row adds Alamar Blue in the growth indicator, 37 ℃ of incubated overnight, observed in second day, if growth telltale hole color becomes pink or colourless by original blueness, it is blue that substratum Quality Control hole keeps, prove that then thalline reaches the concentration that makes the colour-changing agent variable color, identify that in susceptibility district and bacterial type evaluation district add the Alamar Blue of same amount, 37 ℃ of overnight incubation, observed the variable color situation in second day, minimum inhibition concentration is defined as the drug level of the blue micropore adjacent with pink micropore, and according to the variable color situation judgement bacterial type of identifying substratum, non-Mycobacterium tuberculosis PNB (p-nitrobenzoic acid) identifies that substratum and TCH (thiophene-2-carboxylic acid hydrazine) identify that the substratum color is pink, Mycobacterium tuberculosis var.bovis PNB (p-nitrobenzoic acid) identifies that substratum and TCH (thiophene-2-carboxylic acid hydrazine) identify that the substratum color is blueness, human-like Mycobacterium tuberculosis TCH (thiophene-2-carboxylic acid hydrazine) identifies substratum color pink, and PNB (p-nitrobenzoic acid) identifies the substratum color blue; If two hole colors all become pink, illustrate that then this experiment is invalid; If growth indicator two hole colors keep blue, then in growth second row, two holes, indicator, add Alamar Blue, determination methods is the same, and the like.
Use closed micropore card of the present invention can carry out bacterial type evaluation and antitubercular agent minimum inhibition concentration mensuration to the mycobacterium tuberculosis clinical isolates strain that will detect simultaneously according to the method described above, the setting of growth telltale hole, make the drug sensitive test of the clinical separation strain that the speed of growth is different to finish smoothly, guaranteed result's reliability.
Description of drawings
Fig. 1 is the structural representation of closed micropore card of the present invention.
Fig. 2 is that the A-A of Fig. 1 is to view.
Embodiment
As shown in Figure 1, 2, use closed micropore card of the present invention detects the method for drug sensitivity of mycobacterium tuberculosis, comprises the rectangular card body 2 and the loam cake 3 that have test microvia 1, and the mode that described test microvia 1 is 4 row, 12 row is distributed on the card body 2; Offer annular recesses 4 around test microvia 1 on described card body 2, the card body periphery place outside described annular recesses 4 is provided with draw-in groove 5, is pasted with flexiplast wear ring 7 on the inwall of described draw-in groove 5; The clamps 6 that described loam cake 3 peripheries are provided with are complementary with described draw-in groove 5, are provided with and each test microvia 1 corresponding sealing bulge loop at the internal surface of described transparent plastics loam cake 3.
Detect embodiment 1:
48 test microvia (4 row, 12 row) of arranging on the closed micropore card card body are divided into three zones: susceptibility is identified district's 9 row; Bacterial type is identified district's 1 row; Growth indicator 2 row: one classifies the thalli growth telltale hole as, and one classifies substratum Quality Control hole as; Identify that in susceptibility district's first row add respectively with Mycobacterium tuberculosis liquid nutrient medium dissolved vazadrine (8 μ g/ml), Rifampin (4 μ g/ml), Vetstrep (32 μ g/ml), Tibutol (128 μ g/ml) 200ml (Rifampin with dmso solution then add substratum again), in the micropore of back, with the mycobacterium tuberculosis liquid nutrient medium microbiotic is carried out doubling dilution successively, identifying that the Kong Zhongfu hole adds the mycobacterium tuberculosis that contains PNB and TCH and identify substratum, in each hole of growth indicator, adding and susceptibility is identified and distinguished identical but do not contain antibiotic liquid nutrient medium;
To be diluted to the concentration of 3 Maxwell turbidity with physiological saline through the mycobacterium tuberculosis of separation and purification, 80ul is added in every hole, does not add bacteria suspension in the substratum Quality Control hole of the indicator secondary series of wherein growing; The drug sensitive plate that inoculation is good seals with the transparent plastics loam cake, put 37 ℃ of incubators and cultivate taking-up in 3 days, add 5-10%Alamar Blue 25-50 μ l at the growth telltale hole, 37 ℃ of incubated overnight, observed in second day, growth telltale hole color becomes pink by original blueness, it is blue that substratum Quality Control hole keeps, the proof thalline reaches the concentration that makes the colour-changing agent variable color, identify that in susceptibility district and bacterial type evaluation district add the Alamar Blue of same amount, 37 ℃ of overnight incubation were observed the variable color situation in second day;
In the susceptibility zone, observe the color of four lines micropore: gradient hole, the first row vazadrine 2 μ g/ml bluenesss, 1 μ g/ml hole and back micropore color are pink, illustrate that minimum inhibition concentration is 2 μ g/ml; The second row Rifampin, 0.063 μ g/ml color blue, 0.031 μ g/ml color pink, then minimum inhibition concentration is 0.063 μ g/ml; The third line 0.125 color pink, 0.250 μ g/ml hole color blue, then minimum inhibition concentration is 0.250 μ g/ml; Fourth line 8 μ g/ml hole color pink, 16 μ g/ml hole color blue, then minimum inhibition concentration is 16 μ g/ml.From identifying the multiple hole result of substratum, PNB (p-nitrobenzoic acid) identifies that substratum and TCH (thiophene-2-carboxylic acid hydrazine) identify that the substratum color does not all change, and illustrates that then this clinical isolates strain is a Mycobacterium tuberculosis var.bovis.
Detect embodiment 2:
48 test microvia (4 row, 12 row) of arranging on the closed micropore card card body are divided into three zones: susceptibility is identified district's 9 row; Bacterial type is identified district's 1 row; Growth indicator 2 row: one classifies the thalli growth telltale hole as, and one classifies substratum Quality Control hole as; Identify that in susceptibility district's first row add respectively with Mycobacterium tuberculosis liquid nutrient medium dissolved vazadrine (8 μ g/ml), Rifampin (4 μ g/ml), Vetstrep (32 μ g/ml), Tibutol (128 μ g/ml) 200ml (Rifampin with dmso solution then add substratum again), in the micropore of back, with the mycobacterium tuberculosis liquid nutrient medium microbiotic is carried out doubling dilution successively, identifying that the Kong Zhongfu hole adds the mycobacterium tuberculosis that contains PNB and TCH and identify substratum, in each hole of growth indicator, adding and susceptibility is identified and distinguished identical but do not contain antibiotic liquid nutrient medium;
To be diluted to the concentration of 2 Maxwell turbidity with physiological saline through the mycobacterium tuberculosis of separation and purification, 40ul is added in every hole, does not add bacteria suspension in the substratum Quality Control hole of the indicator secondary series of wherein growing;
The drug sensitive plate that inoculation is good seals with the transparent plastics loam cake, put 37 ℃ of incubators and cultivate taking-up in 5 days, add 5-10%Alamar Blue 25-50 μ l at the growth telltale hole, 37 ℃ of incubated overnight, observed in second day, two holes are pink simultaneously, illustrate that then substratum can make the developer variable color, this invalidate the test.
Detect embodiment 3:
48 test microvia (4 row, 12 row) of arranging on the closed micropore card card body are divided into three zones: susceptibility is identified district's 9 row; Bacterial type is identified district's 1 row; Growth indicator 2 row: one classifies the thalli growth telltale hole as, and one classifies substratum Quality Control hole as; Identify that in susceptibility district's first row add respectively with Mycobacterium tuberculosis liquid nutrient medium dissolved vazadrine (8 μ g/ml), Rifampin (4 μ g/ml), Vetstrep (32 μ g/ml), Tibutol (128 μ g/ml) 200ml (Rifampin with dmso solution then add substratum again), in the micropore of back, with the mycobacterium tuberculosis liquid nutrient medium microbiotic is carried out doubling dilution successively, identifying that the Kong Zhongfu hole adds the mycobacterium tuberculosis that contains PNB and TCH and identify substratum, in each hole of growth indicator, adding and susceptibility is identified and distinguished identical but do not contain antibiotic liquid nutrient medium;
To be diluted to the concentration of 1 Maxwell turbidity with physiological saline through the mycobacterium tuberculosis of separation and purification, 20ul is added in every hole, does not add bacteria suspension in the substratum Quality Control hole of the indicator secondary series of wherein growing;
The drug sensitive plate that inoculation is good seals with the transparent plastics loam cake, put 37 ℃ of incubators and cultivate taking-up in 8 days, add 5～10%Alamar Blue 25-50 μ l at the growth telltale hole, 37 ℃ of incubated overnight were observed in second day, and two holes are blue simultaneously, illustrate that thalli growth does not also reach developer variable color concentration, need add 5～10%Alamar Blue 25-50 μ l again at growth indicating area second row, cultivate 24 hours for 37 ℃, observe separately again.
Detect embodiment 4:
48 test microvia (4 row, 12 row) of arranging on the closed micropore card card body are divided into three zones: susceptibility is identified district's 9 row; Bacterial type is identified district's 1 row; Growth indicator 2 row: one classifies the thalli growth telltale hole as, and one classifies substratum Quality Control hole as; Identify that in susceptibility district's first row add respectively with Mycobacterium tuberculosis liquid nutrient medium dissolved Moxifloxacin (6 μ g/ml), Ofloxacine USP 23 (8 μ g/ml), Gatifloxacin (2 μ g/ml), capromycin (4 μ g/ml) 200ml (water-fast microbiotic is added substratum after with dmso solution again), in the micropore of back, with the mycobacterium tuberculosis liquid nutrient medium microbiotic is carried out doubling dilution successively, identifying that the Kong Zhongfu hole adds the mycobacterium tuberculosis that contains PNB and TCH and identify substratum, in each hole of growth indicator, adding and susceptibility is identified and distinguished identical but do not contain antibiotic liquid nutrient medium;
To be diluted to the concentration of 2 Maxwell turbidity with physiological saline through the mycobacterium tuberculosis of separation and purification, 20 μ l are added in every hole, and bacteria suspension is not added in the substratum Quality Control hole of the indicator secondary series of wherein growing;
The drug sensitive plate that inoculation is good seals with the transparent plastics loam cake, put 37 ℃ of incubators and cultivate taking-up in 5 days, add Alamar Blue at the growth telltale hole, 37 ℃ of incubated overnight were observed in second day, growth telltale hole color becomes pink or colourless by original blueness, it is blue that substratum Quality Control hole keeps, and proves that thalline reaches the concentration that makes the colour-changing agent variable color, identifies that in susceptibility district and bacterial type evaluation district add the Alamar Blue of same amount, 37 ℃ of overnight incubation were observed the variable color situation in second day.Minimum inhibition concentration is that adjacent pink micropore color is a minimum inhibition concentration for blue micropore drug level, the result is 0.25 μ g/ml for the MIC of Moxifloxacin, the MIC of Ofloxacine USP 23 is 2 μ g/ml, and the MIC of Gatifloxacin is 0.5 μ g/ml, and the MIC of capromycin is 0.5 μ g/ml.
Claims (1)
1. a non-diagnostic purpose, use the closed micropore card to detect the method for drug sensitivity of mycobacterium tuberculosis, it is characterized in that: described closed micropore card comprises rectangular card body (2) and the loam cake (3) that has test microvia (1), and the mode that described test microvia (1) is multirow, multiple row is distributed on the card body (2); Upward offer annular recesses (4) at described card body (2) around test microvia (1), card body periphery place outside described annular recesses (4) is provided with draw-in groove (5), the clamp (6) that described loam cake (3) periphery is provided with is complementary with described draw-in groove (5), is provided with and the corresponding sealing bulge loop of each test microvia (1) at the internal surface of described loam cake (3); Be pasted with flexible sealing ring (7) on the inwall of described draw-in groove (5);
Concrete detection step is as follows:
The first step: be divided into three zones with arranging the test microvia that is provided with on the closed micropore card card body: susceptibility is identified district's multiple row; Bacterial type is identified district's 1 row; Growth indicator 2 row: one classifies the thalli growth telltale hole as, and one classifies substratum Quality Control hole as;
Second step: the loam cake of closed micropore card is opened, and the micropore of susceptibility being identified the district is by the row classification, and each row adds a kind of microbiotic, and with antibiotic amount substratum doubling dilution in each micropore in the delegation, antibiotic concentration reduces successively; Identify that at bacterial type multiple hole interpolation PNB identifies that substratum and TCH identify substratum in the micropore of distinguishing; In the micropore of growth indicator, add with susceptibility and identify the substratum that the district is identical;
The 3rd step: add tubercule bacillus bacteria suspension to susceptibility respectively and identify that district, bacterial type are identified in the micropore in district and in the thalli growth telltale hole of growth indicator, the substratum Quality Control does not add bacteria suspension in the hole, is blank through dilution;
The 4th step: will add sterilized water in the annular recesses of offering around all micropores on the closed micropore card, cover the loam cake of closed micropore card, after microwell plate seals with loam cake, put into 37 ℃ of incubator 3-8 days;
The 5th step: cultured microwell plate in the 4th step is taken out, first row adds Alamar Blue in the growth indicator, 37 ℃ of incubated overnight, observed in second day, if growth telltale hole color becomes pink by original blueness, it is blue that substratum Quality Control hole keeps, prove that then thalline reaches the concentration that makes the colour-changing agent variable color, identify that in susceptibility district and bacterial type evaluation district add the Alamar Blue of same amount, 37 ℃ of overnight incubation, observed the variable color situation in second day, minimum inhibition concentration is defined as the drug level of the blue micropore adjacent with pink micropore, and according to the variable color situation judgement bacterial type of identifying substratum, non-Mycobacterium tuberculosis PNB identifies that substratum and TCH identify that the substratum color is pink, Mycobacterium tuberculosis var.bovis PNB identifies that substratum and TCH identify that the substratum color is blueness, and human-like Mycobacterium tuberculosis TCH identifies substratum color pink, and PNB identifies the substratum color blue; If two hole colors all become pink, illustrate that then this experiment is invalid; If growth indicator two hole colors keep blue, then in growth second row, two holes, indicator, add Alamar Blue, determination methods is the same, and the like.
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102146430B (en) * | 2011-01-27 | 2013-06-19 | 兰州大学 | Mycobacterium tuberculosis medicament sensitive phenotype detection method and application of method |
| CN103820310B (en) * | 2014-02-15 | 2017-11-07 | 江苏中新医药有限公司 | A kind of drug sensitivity test concentration gradient detection kit and method of testing |
| CN106070089A (en) * | 2016-08-17 | 2016-11-09 | 福建农林大学 | A kind of orifice plate combinative structure |
| CN109294868B (en) * | 2018-09-28 | 2021-11-19 | 杭州莱约科技有限公司 | Sample loading device of biological reaction container |
| CN110591902A (en) * | 2019-10-12 | 2019-12-20 | 重庆金域医学检验所有限公司 | Biological indicator assisted crushing culture device and using method thereof |
| CN116150829B (en) * | 2023-02-08 | 2023-11-24 | 珠海美华医疗科技有限公司 | Design method of trace drug sensitive test card with S-shaped flow channel |
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| CN101065186A (en) * | 2004-07-08 | 2007-10-31 | 科峻实验室仪器公司 | Microplate with temporary seals |
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