CN101748187B - Quick detection reagent for drug sensitivity of mycoplasma pneumoniae and preparation method - Google Patents
Quick detection reagent for drug sensitivity of mycoplasma pneumoniae and preparation method Download PDFInfo
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Abstract
The invention discloses a quick detection reagent for the drug sensitivity of mycoplasma pneumoniae and a preparation method. The quick detection reagent comprises two parts of a pneumonia type mycoplasma culture medium and a drug sensitive plate. The pneumonia type mycoplasma culture medium comprises fresh bovine serum, fresh yeast juice, glucose, phenol red and bacteriostatic agent. By controlling the pH value and the concentration of the glucose and the phenol red in a culture solution, the reaction speed of the reagent is enhanced. The drug sensitive plate is a 24-hole culture plate and is marked with numbers of 1-12, each number is marked with a hole A and a hole B, and the total number of the holes is 24. The numbers of 1-11 are respectively coated with 11 medicines, and the hole A and the hole B of each number are respectively coated with the high-concentration liquor and low-concentration liquor of the same medicine. The invention has stable performance, visual judgment standard and quick reaction and can report a result within 24 hours. The invention can quickly detect mycoplasma pneumoniae, can detect that the infected mycoplasma pneumoniae is sensitive to which medicine, and has a direct guide function to the pharmacy treatment. The invention provides a guarantee for the quick diagnosis and the effective treatment of the infection disease of mycoplasma pneumoniae.
Description
Technical field
The present invention relates to external diagnosis reagent and preparation method, particularly the susceptibility detection reagent and the preparation method that detect of respiratory tract that causes because of mycoplasma and pulmonary disorder.
Background technology
Mycoplasma pneumoniae is the common disease substance of respiratory tract and pneumonia infection disease, and recall rate accounts for respiratory tract infection crowd's 20%～40%.Mycoplasma pneumoniae infection can cause upper respiratory tract infection and mycoplasma pneumonia, its administrated method with viral, bacterial infection is all different, the mycoplasma pneumoniae that Different Individual infects is also different to the susceptibility of medicine, the mycoplasma pneumoniae diagnosis adds the detection reagent of susceptibility, can guarantee mycoplasma pneumoniae disease early diagnosis and science medication, make the patient obtain timely and effective treatment.More to mycoplasma laboratory diagnosis technology both at home and abroad at present, as specific antibody detection technique, metabolic inhibition test, Southern Blot, PCR method etc.The antibody test technology is prone to cross reaction.Metabolic inhibition test need add specific antibody and suppress the mycoplasma growth, and technology is complicated, and clinical application is few.Southern Blot experimental technique requires high, is difficult for generally carrying out, and false positive and false negative appear in PCR method easily, by the health ministry Clinical Laboratory of ordering to forbid being used for.And the culture method specific culture medium that to be the life characteristic according to mycoplasma adopt detects the method for mycoplasma.This method can not only be diagnosed mycoplasma infection specifically, can also with the drug sensitive plate combination, the mycoplasma that is infected is carried out the susceptibility analysis.The mycoplasma culture medium of each hospital's use has single Ureaplasma urealyticum substratum, single mycoplasma hominis substratum and combined mycoplasma substratum at present.And the existing people of single mycoplasma culture medium has declared Chinese patent (application number: 9611701.0).The combined mycoplasma substratum also has Chinese patent (application number: 00113758.1).Above mycoplasma culture medium all is applied to the diagnosis of the mycoplasma infection of urethra reproductive tract, and is used for the existing commodity appearance of mycoplasma pneumoniae detection reagent of respiratory tract and lung's mycoplasma infection, but does not see the patent documentation report.The drug sensitivity of mycoplasma pneumoniae detection reagent is not seen dependent merchandise and patent documentation report so far.
Summary of the invention
The object of the present invention is to provide a kind of quick detection reagent for drug sensitivity of mycoplasma pneumoniae and preparation method, detect the mycoplasma pneumoniae that is infected when this reagent can detect mycoplasma pneumoniae specifically which kind of is used always the susceptibility sense, be mainly used in the diagnosis of the respiratory tract of the pediatrics department and Pneumology Department and lung's mycoplasma infection and instruct the clinical application treatment, for the diagnosis early of mycoplasma pneumoniae infection disease and effectively treatment give security.
For reaching above purpose, the present invention takes following technical scheme to be achieved:
A kind of quick detection reagent for drug sensitivity of mycoplasma pneumoniae comprises mycoplasma pneumoniae substratum and drug sensitive plate, it is characterized in that, described mycoplasma pneumoniae substratum comprises following component by mass percentage:
Basic medium 70～90%;
Calf serum 10～30%;
Yeast powder 0.5～2.5%;
Glucose 0.1～0.5%;
The penicillin that also comprises phenol red, the 10～200,000 units/100ml that adds 0.08～0.2ppm in the above-mentioned mycoplasma pneumoniae substratum;
Wherein basic medium comprises following component by mass percentage:
Mother liquor 96～99%
Sodium-chlor 0.3～0.7%
Potassium primary phosphate 0.1～0.3%
Peptone 0.3～3%
Wherein mother liquor is that beef leach liquor and pig stomach or lung digestive fluid are by 4: 6～6: 4 mixing solutions of mass ratio;
Described drug sensitive plate is one 24 well culture plate, indicate 1～No. 12, indicate A, B two holes for every number, wherein 1～No. 11 hole is coated with 11 kinds of conventional treatment mycoplasma medicines respectively, and these medicines filter out from following 7 big class medicines: beta-lactam class, Macrolide, aminoglycosides, tetracyclines, lincomycin class, Lu's mycin class, quinolones; Wrap respectively by the soup with a kind of low, high two kinds of different concns of medicine in the A of each number, B two holes, the high density soup is the dose of once being grown up and is dissolved in 1～5ml damping fluid, the lower concentration soup is 15～25 times of high density soup redilution, and the 12nd hole is drug coated not, gives over to contrast.
A kind of preparation method of above-mentioned quick detection reagent for drug sensitivity of mycoplasma pneumoniae comprises the steps:
One, the preparation of mycoplasma pneumoniae substratum
(1) extracts fresh beef leach liquor, pig stomach or lung digestive fluid respectively, in 4: 6～6: 4 ratio of mass ratio, with the mixed substratum mother liquor that gets of beef leach liquor and pig stomach or lung digestive fluid;
(2) above substratum mother liquor and following material are mixed by mass percentage:
Mother liquor 96～99%
Sodium-chlor 0.3～0.7%
Potassium primary phosphate 0.1～0.3%
Peptone 0.3～3%
After treating that sodium-chlor, potassium primary phosphate and peptone dissolve fully, boil filtration, get filtrate, promptly make basic culture solution;
(3) basic medium and following material are mixed by mass percentage,
Basic medium 70～90%
Calf serum 10～30%
Yeast powder 0.5～2.5%
Glucose 0.1～0.5%
Add: phenol red 0.08～0.2pp m, penicillin 10～200,000 units/100ml, regulating the pH value is 6.0～8.0, promptly makes the mycoplasma pneumoniae substratum.
Two, the preparation of drug sensitive plate
(1) with hole of mark, per two holes of 24 well culture plates number, to 12, every number two holes are designated as A, B hole respectively number from 1 mark in the hole, through slightly wash, fine purifiation, 40 ℃ of constant temperature dry standby;
(2) from following 7 big class medicines: filter out 11 kinds of conventional treatment mycoplasma medicines beta-lactam class, Macrolide, aminoglycosides, tetracyclines, lincomycin class, Lu's mycin class, the quinolones;
(3) every kind of medicine is dissolved in 1～5ml damping fluid by the each dose of adult, treat that medicine dissolves fully after, centrifugal extraction supernatant is made into two kinds of concentration again: the high density soup is the dose 1～5ml that once is grown up, the lower concentration soup is 15～25 times of high density soup redilution,
(4) draw low, the high density soup of each medicine respectively with sample injector, divide to be added in drug sensitive plate 1-11 number A, B two holes separately, leave standstill 30 minutes after, sucking-off discards.
In the such scheme, described 1～No. 11 hole is wrapped by 11 kinds of medicines and A, B two holes concentration low, the high density soup as shown in the table respectively:
Medicine name lower concentration soup (mg/ml) high density soup (mg/ml)
Doxycycline Hyclate 0.6～1.0 9.0～12.0
Tsiklomitsin 1.6～2.0 16.0～30.0
Clarithromycin 0.4～0.8 6.25～14.0
Josamycin 0.3～0.6 4.0～8.0
Minocycline hydrochloride 0.3～0.6 4.0～8.0
Azythromycin 1.4～1.8 14.0～28.0
Sparfloxacin 0.8～1.2 9.0～17.0
Erythromycin 1.4～1.8 16.0～28.0
Lomefloxacin hydrochloride 1.0～3.0 17.0～35.0
Ofloxacin 1.0～3.0 17.0～35.0
Levofloxacin hydrochloride 0.8～1.4 12.0～20.0.
Advantage of the present invention is stable performance, and judging criterion is directly perceived, and high specificity does not need specific installation, can report the result in 24 hours.This reagent carries out clinical trial in two tame hospitals, observes 200 many cases cases, obtains better result, and the coincidence rate of reaction result reaches 100% between criticizing in synchronous blind method is criticized, and the reagent stable performance is described.The result instructs clinical application by drug sensitive reaction, produce effects and efficient reaching more than 98%.Illustrate that this reagent has direct directive function to clinical application, for the early diagnosis of mycoplasma pneumoniae infection type disease and effectively treatment assurance is provided.
Embodiment
The present invention is described in further detail below in conjunction with specific embodiment.
A kind of quick detection reagent for drug sensitivity of mycoplasma pneumoniae comprises mycoplasma pneumoniae substratum and drug sensitive plate, and the mycoplasma pneumoniae substratum comprises following component: basic medium, calf serum, yeast powder, glucose.Add the phenol red and penicillin of trace.Specific embodiment is referring to table 1.
Table 1 mycoplasma pneumoniae substratum is formed mass percent %
In the table 1, basic medium comprises following component: mother liquor, sodium-chlor, potassium primary phosphate, peptone.Specific embodiment (code name is base 1～base 4) is referring to table 2.
Table 2 basic medium is formed mass percent %
Mother liquor in the table 2 (code name is female 1～female 4) is beef leach liquor and pig stomach or lung digestive fluid press mass ratio 4: 6～6: 4 mixing solutions.Specifically as shown in table 3.
Table 3 mother liquor is formed mass percent %
| Female 1 | Female 2 | Female 3 | Female 4 | |
| The beef leach liquor | 40 | 50 | 60 | 50 |
| Pig peptic digestion liquid | 60 | 50 | ||
| Lung digestive fluid | 50 | 40 |
The drug sensitive plate of quick detection reagent for drug sensitivity of mycoplasma pneumoniae of the present invention is 24 well culture plates, indicates 1～No. 12, indicates A, B two holes, totally 24 holes every number.Wherein 1～No. 11 hole is coated with 11 kinds of medicines respectively, and wrap respectively by the different concns amount of liquid medicine with a kind of medicine in the A of each number, B two holes.No. 12 A, B two holes are drug coated not, gives over to contrast.The medicine of quilt that wrap in each hole filters out from following 7 big class medicines, and promptly how beta-lactam class, Macrolide, aminoglycosides, tetracyclines, lincomycin class, Lu's mycin class, quinolones screen according to market circulation situation at that time and decide.Drug sensitive plate specific embodiment of the present invention is as shown in table 4.
Table 4
A kind of quick detection reagent for drug sensitivity of mycoplasma pneumoniae preparation method is divided into mycoplasma pneumoniae medium preparation and drug sensitive plate and prepares two portions:
One, pneumonia type mycoplasma culture medium preparation
1. the preparation of substratum mother liquor.The extraction of fresh beef leach liquor and pig stomach or lung digestive fluid:
(1) extraction of beef leach liquor: after getting the fresh beef rubbing, add 1: 2～1: 3 distilled water, soaked 10～25 hours, boiled its filtrate of leaching.
(2) pig stomach or lung digestive fluid extract: get fresh pig stomach or lung and rub, add 1: 2～1: 3 distilled water, add 0.1～0.6% stomach en-again, digested 10～25 hours, boil filtration, get its filtrate, adjust pH is 6～7.
(3) by table 3 prescription beef leach liquor and pig stomach or lung digestive fluid are mixed and made into the mother liquor of code name for female 1～mother 4.
2. the preparation of basic medium.Use above substratum mother liquor respectively, form the preparation code name by table 2 and be base 1～basic 4 basic mediums: each basic medium all is that solid chemical material sodium-chlor, potassium primary phosphate and peptone are added in the mother liquor, after the dissolving, boil filtration fully, get filtrate promptly.
3. the preparation of mycoplasma pneumoniae inspection substratum.Mix by table 1 composition with above four kinds of basic mediums respectively, the mycoplasma pneumoniae of promptly making embodiment 1～4 detects uses substratum.Wherein regulating and control the pH value that mycoplasma pneumoniae detects with substratum is 6.0～8.0.
Two, the preparation of drug sensitive plate
＜one〉drug sensitive plate is handled
1. from there being qualification unit to buy 24 well culture plates.
2. with hole of mark, per two holes of 24 well culture plates number, to 12, every number two holes are designated as A, B hole respectively number from 1 mark in the hole, slightly wash then, fine purifiation, 40 ℃ of constant temperature dry, and be standby.
＜two〉soup preparation
1. from there being qualification unit to buy 11 kinds of conventional treatment mycoplasma medicines of the accurate word code of medicine.
2. buffer preparation: get sodium-chlor 3.5～5.5g, phosphatization potassium dihydrogen 1～5g is dissolved in the 1000ml distilled water, and adjust pH is 6～8.
3. every kind of medicine is dissolved in 1～5ml damping fluid by the each dose of adult (concentration range of each medicine B hole high density soup shown in the table 4), treat that medicine dissolves fully after, centrifugal extraction supernatant is made into two kinds of concentration liquids again.The concentration of high density soup is the dose of once being grown up, and the concentration of lower concentration soup is 15～25 times of high density soup redilution.Two kinds of concentration of medicament categories and every kind of medicine are as shown in table 4.
＜three〉drug sensitive plate bag quilt
Draw the different concns soup of various medicines respectively with sample injector, be added to leave standstill 30 minutes in drug sensitive plate 1-11 number the A, B hole after, sucking-off discards.Each title of each hole institute drug coated of drug sensitive plate and concentration are as shown in table 4.
After the pneumonia type mycoplasma culture medium packing that above preparation is finished and the drug sensitive plate combination the drug sensitivity of mycoplasma pneumoniae detection reagent.It is a kind ofly mycoplasma pneumoniae infection to be diagnosed the reagent of analyzing with susceptibility with the liquid nutrient medium cultural method.This reagent helps the mycoplasma pneumoniae growth.And contain promising mycoplasma pneumoniae and decompose the glucose that is utilized as the energy, produce H+ during breakdown of glucose, make the substratum souring, and the phenol red indicator that makes substratum is from red yellowing thereby be detected.After substratum is inoculated sample, be added to each hole of drug sensitive plate respectively and cultivate, if control wells (No. 12 hole) is positive, illustrate that sample is the mycoplasma pneumoniae positive, can analyze this moment to susceptibility.If certain medicine lower concentration, high density all can suppress mycoplasma pneumoniae growth, the reaction that all is negative of the A of corresponding drug sensitive plate, B two hole substratum illustrates that mycoplasma pneumoniae is extremely sensitive to this medicine.If certain medicine high density can suppress mycoplasma pneumoniae growth, but low dense can not, the A hole corresponding with this medicine B hole reaction that is negative that is positive illustrates that mycoplasma pneumoniae is to this medicine medium sensitivity on the drug sensitive plate.If certain medicine high density, lower concentration all can not suppress the mycoplasma pneumoniae growth, A, B two Kong Jun corresponding with this medicine on the drug sensitive plate are positive, and illustrate that mycoplasma pneumoniae produces resistance to this medicine.
Claims (2)
1. a quick detection reagent for drug sensitivity of mycoplasma pneumoniae comprises mycoplasma pneumoniae substratum and drug sensitive plate, it is characterized in that, described mycoplasma pneumoniae substratum comprises following component by mass percentage:
Basic medium 70～90%;
Calf serum 10～30%;
Yeast powder 0.5～2.5%;
Glucose 0.1～0.5%;
The penicillin that also comprises phenol red, the 10～200,000 units/100ml that adds 0.08～0.2ppm in the above-mentioned mycoplasma pneumoniae substratum;
More than the mass percent sum of each component be 100%;
In the above-mentioned mycoplasma pneumoniae substratum, basic medium comprises following component by mass percentage:
Mother liquor 96～99%
Sodium-chlor 0.3～0.7%
Potassium primary phosphate 0.1～0.3%
Peptone 0.3～3%
More than the mass percent sum of each component be 100%;
Wherein mother liquor is that beef leach liquor and pig stomach or lung digestive fluid are by 4: 6～6: 4 blended mixing solutions of mass ratio;
Described drug sensitive plate is one 24 well culture plate, indicate 1～No. 12, indicate A, B two holes for every number, wherein 1～No. 11 hole is coated with 11 kinds of conventional treatment mycoplasma medicines respectively, and these medicines filter out from following 7 big class medicines: beta-lactam class, Macrolide, aminoglycosides, tetracyclines, lincomycin class, Lu's mycin class, quinolones; Wrap respectively by the soup with a kind of low, high two kinds of different concns of medicine in the A of each number, B two holes, the high density soup is the dose of once being grown up and is dissolved in 1～5ml damping fluid, the lower concentration soup is 15～25 times of high density soup redilution, and the 12nd hole is drug coated not, gives over to contrast; Wherein said 1～No. 11 hole is wrapped by A, the B two holes concentration low, the high density soup of following 11 kinds of medicines and correspondence as shown in the table respectively:
Medicine name lower concentration soup (mg/ml) high density soup (mg/ml)
Doxycycline Hyclate 0.6～1.0 9.0～12.0
Tsiklomitsin 1.6～2.0 16.0～30.0
Clarithromycin 0.4～0.8 6.25～14.0
Josamycin 0.3～0.6 4.0～8.0
Minocycline hydrochloride 0.3～0.6 4.0～8.0
Azythromycin 1.4～1.8 14.0～28.0
Sparfloxacin 0.8～1.2 9.0～17.0
Erythromycin 1.4～1.8 16.0～28.0
Lomefloxacin hydrochloride 1.0～3.0 17.0～35.0
Ofloxacin 1.0～3.0 17.0～35.0
Levofloxacin hydrochloride 0.8～1.4 12.0～20.0.
2. the preparation method of the described quick detection reagent for drug sensitivity of mycoplasma pneumoniae of claim 1 comprises the steps:
One, the preparation of mycoplasma pneumoniae substratum
(1) extracts fresh beef leach liquor, pig stomach or lung digestive fluid respectively, in 4: 6～6: 4 ratio of mass ratio, with the mixed substratum mother liquor that gets of beef leach liquor and pig stomach or lung digestive fluid;
(2) above substratum mother liquor and following material are mixed by mass percentage:
Mother liquor 96～99%
Sodium-chlor 0.3～0.7%
Potassium primary phosphate 0.1～0.3%
Peptone 0.3～3%
More than the mass percent sum of each component be 100%;
After treating that sodium-chlor, potassium primary phosphate and peptone dissolve fully, boil filtration, get filtrate, promptly make basic culture solution;
(3) basic medium and following material are mixed by mass percentage,
Basic medium 70～90%
Calf serum 10～30%
Yeast powder 0.5～2.5%
Glucose 0.1～0.5%
Add: phenol red 0.08～0.2ppm, penicillin 10～200,000 units/100ml, regulating the pH value is 6.0～8.0, promptly makes the mycoplasma pneumoniae substratum;
More than the mass percent sum of each component be 100%;
Two, the preparation of drug sensitive plate
(1) with hole of mark, per two holes of 24 well culture plates number, to 12, every number two holes are designated as A, B hole respectively number from 1 mark in the hole, through slightly wash, fine purifiation, 40 ℃ of constant temperature dry standby;
(2) from following 7 big class medicines: filter out 11 kinds of conventional treatment mycoplasma medicines beta-lactam class, Macrolide, aminoglycosides, tetracyclines, lincomycin class, Lu's mycin class, the quinolones;
(3) every kind of medicine is dissolved in 1～5ml damping fluid by the each dose of adult, treat that medicine dissolves fully after, centrifugal extraction supernatant is made into two kinds of concentration again: the high density soup is the dose 1～5ml that once is grown up, the lower concentration soup is 15～25 times of high density soup redilution;
(4) draw low, the high density soup of each medicine respectively with sample injector, divide to be added in drug sensitive plate 1-11 number A, B two holes separately, leave standstill 30 minutes after, sucking-off discards;
Wherein said 1～No. 11 hole is wrapped by A, the B two holes concentration low, the high density soup of following 11 kinds of medicines and correspondence as shown in the table respectively:
Medicine name lower concentration soup (mg/ml) high density soup (mg/ml)
Doxycycline Hyclate 0.6～1.0 9.0～12.0
Tsiklomitsin 1.6～2.0 16.0～30.0
Clarithromycin 0.4～0.8 6.25～14.0
Josamycin 0.3～0.6 4.0～8.0
Minocycline hydrochloride 0.3～0.6 4.0～8.0
Azythromycin 1.4～1.8 14.0～28.0
Sparfloxacin 0.8～1.2 9.0～17.0
Erythromycin 1.4～1.8 16.0～28.0
Lomefloxacin hydrochloride 1.0～3.0 17.0～35.0
Ofloxacin 1.0～3.0 17.0～35.0
Levofloxacin hydrochloride 0.8～1.4 12.0～20.0.
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| CN102888441A (en) * | 2011-07-20 | 2013-01-23 | 农高惠 | Kit for fast culture, identification and drug sensitivity test of myeoplasmapneumoniae (Mp) and detection method therefor |
| CN103436468B (en) * | 2013-08-11 | 2015-10-14 | 杭州致远医学检验所有限公司 | Quick growth additive of a kind of helicobacter pylori and preparation method thereof |
| CN103667154B (en) * | 2013-12-20 | 2015-11-18 | 广西壮族自治区兽医研究所 | mycoplasma culture medium and preparation method thereof |
| CN103849673B (en) * | 2014-03-27 | 2015-03-25 | 武汉菁华时间科技有限公司 | MP (mycoplasma pneumoniae) culture and identification reagent and preparation method thereof |
| CN108179172B (en) * | 2017-12-27 | 2020-05-22 | 山东省农业科学院奶牛研究中心 | Mycoplasma bovis drug sensitivity rapid detection kit and preparation method thereof |
| CN108060109A (en) * | 2018-02-11 | 2018-05-22 | 中国医学科学院医学生物学研究所 | People's mycoplasma pneumoniae isolation medium and cultural method |
| CN113151395A (en) * | 2021-04-16 | 2021-07-23 | 河南省医疗器械检验所 | Mycoplasma pneumoniae drug sensitivity rapid detection kit and preparation method thereof |
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| CN1560611A (en) * | 2004-03-08 | 2005-01-05 | 黄威权 | Mycoplasma pneumonipae detecting reagent |
| CN101555517A (en) * | 2009-05-15 | 2009-10-14 | 钱叶林 | Mycoplasma pneumoniae rapid identification and cultivation susceptibility test kit and test method |
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| CN1560611A (en) * | 2004-03-08 | 2005-01-05 | 黄威权 | Mycoplasma pneumonipae detecting reagent |
| CN101555517A (en) * | 2009-05-15 | 2009-10-14 | 钱叶林 | Mycoplasma pneumoniae rapid identification and cultivation susceptibility test kit and test method |
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Owner name: HEILONGJIANG HUILI ZHONGKANG BIOLOGICAL TECHNOLOGY Free format text: FORMER OWNER: JIANG HONGBO Effective date: 20150521 |
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Effective date of registration: 20150521 Address after: Gloky street Xiangfang District 150049 Heilongjiang city of Harbin Province, No. 2 Patentee after: Heilongjiang Huili Biotechnology Co.,Ltd. Address before: 2005 room 1, Fangyuan Lily Garden Square, Haizhuqu District Industrial Road, Guangdong, Guangzhou, 510280 Patentee before: Jiang Hongbo |
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