CN105759034A - Mycoplasma pneumoniae detection kit - Google Patents
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- CN105759034A CN105759034A CN201610194372.1A CN201610194372A CN105759034A CN 105759034 A CN105759034 A CN 105759034A CN 201610194372 A CN201610194372 A CN 201610194372A CN 105759034 A CN105759034 A CN 105759034A
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
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- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/30—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Mycoplasmatales, e.g. Pleuropneumonia-like organisms [PPLO]
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Abstract
The invention discloses a kit and detection method for mycoplasma pneumoniae IgM on the basis of a fluorescence immunoassay chromatographic technique.The method comprises the steps that fluorescein serves as a marking material, and a mouse antihuman IgM mu chain monoclonal antibody is marked.A mycoplasma pneumoniae specific IgM antibody in serum is combined with a fluorescein marked antibody to form a reaction compound which is caught by a mycoplasma pneumoniae antigen, the mycoplasma pneumoniae IgM catch yield is in positive correlation with signal strength of the fluorescent antibody, and the mycoplasma pneumoniae IgM in the serum can be detected out by means of an immunofluorescence detector reading.The method has the advantages of being short in time, good in specificity, good in accuracy and stability and the like, and the coincidence rate of the detection result with that of a European Union mycoplasma pneumoniae IgM detection kit is high.A new method for rapidly and accurately detecting the mycoplasma pneumoniae IgM in clinic is provided, and a good market prospect is achieved.
Description
Technical field
The present invention relates to the detection method of a kind of mycoplasma pneumoniae, be specifically related to the detection method of a kind of mycoplasma pneumoniae based on fluorescence immune chromatography technology, further relate to adopt the detection kit of the method.
Background technology
Mycoplasma pneumoniae (Mycoplasmapneumoniae, MP) be a kind of can without the prokaryote bred voluntarily in life culture medium, not easy dyeing, it does not have cell wall.The research display MP respiratory tract infection caused is had to account for 20-40% (Principi, N., etal:Clininfectiousdiseases, 2001 of community acquired pneumonia；32:1281-1289.).According to statistics, about annual about 100,000 adults are because of the infection hospitalization of MP.The clinical symptoms of mycoplasma pneumoniae is inconspicuous, has also the general respiratory symptoms such as simply headache, pharyngalgia, heating, cough, and due to the acellular wall of MP, therefore its treatment causing infection and other antibacterial and viral infection are different on Therapeutic Method.Therefore quickly, Accurate Diagnosis MP infects critically important for treating.
At present, the main method of detection MP is several aspects such as MP separation and Culture, PCR detection, cold agglutination test, serum specific antibody detection, is respectively arranged with pluses and minuses.Pathogen isolation cultivation is considered as the goldstandard of medical diagnosis on disease, but MP fostering requirement condition is significantly high, the time longer (about 6 weeks) needed, therefore for the treatment of clinical patients without practicality, so seldom by cultivating, laboratory diagnoses whether patient is MP infection.The current PCR method detection P1 albumen of MP, 16SrRNA and other protein gene have had a large amount of report (Waveren, G., etal:JClinmicrobiology, 1999；37:14-17.), but PCR method complicated operation, it is desirable to have relevant instrument, laboratory technician has to pass through special training, so being difficult to promote in some grass-roots units.Cold agglutination test is nonspecific reaction, except can being positive when MP infects, also seen in hepatopathy, hemolytic anemia, infectious monocytosis etc., if and first time blood specimen collection later, or the resistance of patient is more weak, testing result is likely to as negative (Sanchez-Vargas, F.M., etal:Clinmicrobiologyandinfection, 2008；14: 105-117.).
Most widely used Serologic detection is ELISA, it is necessary to minimal amount of serum can be carried out this test.But enzyme linked immunosorbent assay (ELISA) test kit complex operation, the drawback wasted time and energy hinders its application in real-time test.The Serology test that can be applied to real-time test is immune chromatography method, the detection equipment that the method is simple to operate, quick, convenient, result is prone to judgement, need not be special, is widely used in each field of Clinical Laboratory, food safety supervision and environmental monitoring.On market, major part immunochromatography product adopts gold colloidal as detection label, (Kosack, C.S., etal:Jvirologicalmethods, 2014 based on quantitative and semi-quantitative；204:6-10.).But when Application Optics instrument carries out half-quantitative detection, colloidal gold strip is typically only capable to the optical signalling on 10-20 μm of nitrocellulose filter surface of detection, have lost the signal of nearly 80-90%.
The detection of MP serum antibody has IgM and IgG two kinds, IgM just can be detected (Talkington, D.F., etal:Clinicalanddiagnosticlaboratoryimmunology, 2004 after infecting 7～10 days；11:862-867.).Owing to the incubation period of MP infection is 2～3 weeks, individually detection IgG is very easy to false negative and causes wrong diagnosis and escape, and IgM is typically in infecting one week after and occurs, within 3～4 weeks, peak, when patient occurs that symptom is gone to a doctor, IgM antibody has reached higher level, therefore the detection of IgM antibody contributes to the early diagnosis of MP.
At present, the research adopting fluorescence immune chromatography method detection mycoplasma pneumoniae IgM antibody both at home and abroad have not been reported.Select fluorescein to substitute gold colloidal as marker material, set up fluorescence immune chromatography method and will be provided with colloidal gold immunochromatographimethod advantage simple, quick, can have again higher sensitivity, be conducive to quick, Accurate Diagnosis that MP infects.
Summary of the invention
The invention also discloses a kind of Mycoplasma pneumonia detection kit based on fluorescence immune chromatography technology, this Mycoplasma pneumonia detection kit includes the sample pad, fluorescence pad, nitrocellulose filter and the adsorptive pads that attach successively on polrvinyl chloride (PVC) base plate；Wherein fluorescence pad is the fiberglass packing of IgM antibody and the fluorescein-labelled goat anti-rabbit igg antibody being coated with fluorescein-labelled mouse-anti people；Nitrocellulose filter detection line is coated with mycoplasma pneumoniae antigen, and nature controlling line is coated with rabbit igg antibody.The fluorescein of labelling is near-infrared fluorescent element, it is preferable that fluorescein Alexa647.The IgM antibody of mouse-anti people is IgM μ chain monoclonal antibody.Nitrocellulose filter adopts SatouriusCN140.Mycoplasma pneumoniae antigen is attached most importance to and is organized P1 antigen and native antigen, and wherein mycoplasma pneumoniae restructuring P1 antigen is purchased from Shenzhen Fei Peng Biological Co., Ltd., and native antigen is purchased from Microbix company.P1 albumen is one of the main immunogenic albumen and attachment proteins of mycoplasma pneumoniae, has become major target (Li, W., etal:Scientificreports, 2015 of mycoplasma pneumoniae Clinical detection at present；5:15539.).The mycoplasma pneumoniae restructuring P1 antigen that the present invention selects is the C-terminal fragment of P1 albumen, has higher immunogenicity (Chourasia, B.K., etal:BMCmicrobiology, 2014；14:108.).Native antigen is mycoplasma pneumoniae reference culture FH, adopts ether inactivation, it is possible to catch most of mycoplasma pneumoniae specific IgM as far as possible.The specificity of comprehensive P1 Detection of antigen mycoplasma pneumoniae of recombinating, and the sensitivity of native antigen detection mycoplasma pneumoniae, it is possible to improve the performance of detectable of the present invention.
The invention also discloses fluorescein Alexa647 purposes in preparing fluorescence immune chromatography detectable, the particularly purposes in preparing mycoplasma pneumoniae detectable.The present invention selects fluorescein Alexa647 as novel fluorescence marker material, and it is a kind of bright stable far red light dyestuff, and human eye is invisible but is able to be received by most imaging system.In the method for the detection mycoplasma pneumoniae of the present invention, AlexaThe NHS ester of 647 can be combined with the first amino of antibody and not damage the biological activity of antibody, compared to other similar fluorescence, has higher fluorescence intensity, better light resistance, it is possible to be substantially reduced the interference of background fluorescence.Current Alexa647 are not also applied to mycoplasma pneumoniae immunochromatographiassay assay reagent, compared to gold colloidal, Alexa647 can improve the sensitivity of reagent as marker material；Compared to the microsphere marker material such as quantum dot and time resolution, Alexa647 accuracies that ensure that reagent, CV value is lower.
The invention also discloses a kind of method detecting mycoplasma pneumoniae, the method is based on fluorescence immune chromatography technology, adopts detection kit disclosed by the invention, the mycoplasma pneumoniae IgM antibody in detection serum sample.
In the detection serum sample of the present invention, the method for mycoplasma pneumoniae comprises the following steps: use fluorescein as marker material, the IgM μ chain monoclonal antibody of labelling mouse anti human.When serum mixes with buffer, and it is added drop-wise on immunochromatography film, mycoplasma pneumoniae specific IgM antibodies in serum is combined formation reaction complex with fluorescein labelled antibody, reaction complex moves forward along nitrocellulose filter along with chromatography effect, detected coated mycoplasma pneumoniae restructuring P1 antigen and native antigen on line by nitrocellulose filter to catch, the signal intensity of the mycoplasma pneumoniae IgM quantity of the catch in serum and fluorescent antibody is proportionate, by Immunofluorescence test instrument reading, can detect that the mycoplasma pneumoniae IgM in serum.
Wherein fluorescein is near-infrared fluorescent element, it is preferable that fluorescein Alexa647.The IgM antibody of mouse-anti people is IgM μ chain monoclonal antibody, and mycoplasma pneumoniae antigen is attached most importance to and organized P1 antigen and native antigen, and restructuring P1 antigen is the C-terminal fragment of P1 albumen, and native antigen is mycoplasma pneumoniae reference culture FH.Nitrocellulose filter adopts SatouriusCN140, and sample diluting liquid surfactant adds 2%Tween-20, relatively other surfactant, and chromatographic effect is faster.
Test kit to the present invention, determines Cutoff value by drawing ROC curve, the performance of reagent is investigated.Result shows, (1) is 10min based on the optimum reacting time of the mycoplasma pneumoniae IgM quick detection reagent of fluorescence immune chromatography technology, it is possible to quickly obtain testing result；(2) there is good specificity, with other several frequently seen respiratory pathogen no cross reaction；(3) having good accuracy, within-run and between-run analysis coefficient is respectively less than 15%；(4) there is good stability, it is possible to preserve 2 years in room temperature；(5) higher with European Union's mycoplasma pneumoniae IgM detection kit testing result coincidence rate.So the Clinical detection that is established as of this method provides a kind of means quick and precisely detecting mycoplasma pneumoniae IgM.
The test kit quickly detecting mycoplasma pneumoniae based on fluorescence immune chromatography technology of the present invention, combines the advantage quickly and easily of the sensitive and accurate of fluorescence and chromatography method.The fluorescein selected is the light of a kind of 651nm of absorption wavelength and can inspire the fluorescent dye of 672nm wavelength, and the fluorescence of long wavelength can effectively avoid the interference of other background substance fluorescence.Stable covalentlying bind on antibody by being connected with the R-NH2 group of antibody, this is the basis that this reagent can stably preserve.It addition, fluorescein AlexaThe use of 647 effectively reduce batch between batch in difference, this is that the reliability of testing result provides guarantee.The detection method of the present invention, fast and convenient, it is only necessary to 10min is obtained with accurate result, it is not necessary to testing staff to be giveed training and just can directly operate, be very easy to Clinical detection.
The traget antibody present invention selects the Anti-human IgMμ chain monoclonal antibody of high special, the single-minded IgM caught in serum, is not combined with IgG or other kinds of immunoglobulin, therefore has the IgM antibody specificity of height.(ForghaniB, etal:JClinmicrobial, 1984；19:606-609.) be coated while native antigen and recombinant antigen, being effectively reduced the situation of missing inspection flase drop, especially the use of recombiant protein, improves detected signal value, its specificity is very high, reduces the probability of the respiratory pathogen cross reaction common with other.The combination of two kinds of antigens uses, and is better than the result of use of antigen alone.
Different nitrocellulose filters is compared by the present invention, by comparing the film of two kinds of different pore sizes of SatouriusCN95 and SatouriusCN140, the impact of fluorescence immune chromatography result is selected.It is shown that it is bigger to compare CN95, CN140 yin and yang attribute difference.The present invention has also carried out the selection optimization Test of surfactant in sample diluting liquid.PBS (pH7.4) adds 2%NP-40,2%Tween-20,2%Tween-80 and 2%Triton-X100, the impact on the present invention of the relatively more different surfactants.Find that the Tween-20 of 2% runs line the best in chromatography process, chromatographic effect can be reached faster.
MP is widely present in all over the world, it is possible to cause endemic, early diagnosis to be conducive to shortening the course of disease of mycoplasma pneumoniae patient.The present invention overcomes enzyme linked immunosorbent assay (ELISA) test kit complex operation, the drawback wasted time and energy and colloidal gold method cannot quantify, sensitivity is relatively low, and the defect failed to pinpoint a disease in diagnosis being likely to occur, and compares sensitivity with European Union ELISA kit suitable.The detection sensitivity of the inventive method is higher than gold-immunochromatographyreagent reagent for assay, and can realize quantitatively.
For clinic provide a kind of not only quickly but also can accurately detect the new method of mycoplasma pneumoniae IgM, infect for early diagnosis MP and offer help, there is good market prospect.
Accompanying drawing explanation
The optimization figure of Fig. 1 film.Wherein N1N2N3N4 represents negative serum respectively, and P1P2P3P4 represents positive serum respectively.
The selection figure of surfactant in Fig. 2 sample diluting liquid.
Fig. 3 detects the determination figure of time.Observe and survey the situation of change that a negative and positive serum T peak/C peak area ratio increases over time.
Fig. 4 ROC curve determines Cutoff value.Detect 148 parts of positives and 61 parts of negative serums, analyze Cutoff value by ROC curve in SPSS16.0.
Fig. 5 stability test figure.
Detailed description of the invention
The preparation of embodiment Test paper
One, material:
Fluorescein Alexa647:Invitrogen company
P1 recombinant antigen: Shenzhen Fei Peng Biological Co., Ltd.
MP native antigen: Microbix company
The IgM μ chain monoclonal antibody of mouse anti human: Shenzhen Fei Peng Biological Co., Ltd.
Rabbit igg antibody and goat anti-rabbit igg antibody: Sigma company
Sucrose, PVP and Tween-20 etc.: Chemical Reagent Co., Ltd., Sinopharm Group
Mycoplasma pneumoniae IgM gold-immunochromatographyreagent reagent for assay box: Weifang Kanghua Biotech. Co., Ltd.
European Union's mycoplasma pneumoniae IgMELISA detection kit: EUROIMMUNUS company
Influenza A virus, Influenza B virus, respiratory syncytial virus, adenovirus, Chlamydia pneumoniae, the hot rickettsia of Q, parainfluenza virus, the respiratory pathogen positive serums such as legionella pneumophilia, MP positive serum and MP negative serum: Shenyang Disease Control and Prevention Center, hospital of PLA 304 and hospital of PLA 307.
Buffer solution is as follows:
Sample pad treatment fluid (50mmol/LNa2B4O7.10H2O, 1%PVP (wt/vol), 0.2%Casein-Na (wt/vol), 0.5%Tween-20 (vol/vol), 0.1%NaN3(wt/vol))；
Sample diluting liquid (10mol/LPBS, 2%Tween-20 (v/v) (pH7.4))；
Fluorescent antibody complex preserves liquid: (50mmol/LTris-HCl, 1%BSA (wt/vol), 0.5%Tween-20 (vol/vol), 10%Sucrose (wt/vol) and 0.05%Proclin-300 (vol/vol) (pH8.0)).
Two, method:
(1) preparation of fluorescein and antibody complex
Take 1mg antibody, be dissolved in the PBS buffer solution of quantitative 0.01MpH according to final concentration of 2mg/ml dilution ratio, mixing, stand 10min.Then in reaction system, add the NaHCO of 1M50 μ l3Solution, reaction mixing, stand 15min.According to corresponding proportion, add the fluorescent dye of 10 μ L.Reaction mixing, at room temperature stands 2h.Then move into 4 DEG C of refrigerator overnight.Again complex good for labelling is crossed post through gel SephadexG25, remove and be not associated with the fluorescein of antibody and be not associated with the antibody of fluorescein, to reduce non-specific responding.Finally complex good for post excessively is carried out that preservation is concentrated by ultrafiltration stand-by.
Adopting fluorescent antibody complex to preserve liquid and dilute fluorescein-labelled Mus anti-IgMμchainantibody and goat anti-rabbit antibody, 8 μ l/cm are sprayed on fiberglass packing.Use PBS to be diluted to 0.75mg/ml, 0.175mg/ml P1 recombinant antigen and MP native antigen respectively and be coated on detection line position, use PBS to be diluted to 1.5mg/ml rabbit igg antibody and be coated on nature controlling line position.
(2) preparation of chromatograph test strip
1. the assembling of chromatograph test strip
Chromatography paper slip mainly includes five parts, nitrocellulose filter (NC film), absorbent paper, sample pad, fluorescence pad, PVC base plate.NC film T line is coated MP native antigen and P1 recombinant antigen, and C line is coated with the rabbit igg antibody of suitable concn, dries 3h in 37 ° of baking ovens.Sample pad is soaked through sample pad treatment fluid, dries 12h in 37 ° of baking ovens.Fluorescence pad is sprayed with the Mus anti-IgMμchainantibody of mark fluorescent element and the goat anti-rabbit igg antibody of mark fluorescent element, in 37 ° of oven for drying 1h.Use automatic cutting machine to be cut into 3.6mm/ strip after sample pad, fluorescence pad, NC film, absorbent paper having been assembled in order, load detection card stand-by.
2. operating procedure
Taking 1 μ l serum uses sample diluting liquid according to after 1: 100 dilution, drips in sample well, identifies T line and the crest of C line position fluorescence signal when 10min respectively by fluorescence detector, and instrument calculates PeakArea automatically, and draws T peak/C peak area ratio..
(3) optimization of tomographic system
1. the optimization of film
The relatively film of two kinds of different pore sizes of SatouriusCN95 and the SatouriusCN140 impact on fluorescence immune chromatography result.Detection serum is 4 parts of MP negative serums and 4 parts of MP positive serums, utilizes instrument to detect T peak/C peak area ratio, it is determined that optimal NC film.
2. the selection of surfactant in sample diluting liquid
10mol/LPBS buffer (pH7.4) adds 2%NP-40,2%Tween-20,2%Tween-80 and 2%Triton-X100, the impact on the present invention of the relatively more different surfactants.
3. the determination of chromatography time
1 part of MP negative serum and 1 part of MP positive serum are respectively adopted after diluent is diluted, drip the well in test strips and carry out chromatography, start timing from application of sample, detect T peak/C peak area ratio every 1min.
(4) detection actual sample determines Cutoff value
Adopt test kit of the present invention that 34 parts of MP positive serums and 166 parts of MP negative serums are detected, and determine its Cutoff value by ROC curve in SPSS16.0.
(5) detection systematic function is investigated
1. specificity is investigated
Adopt test kit of the present invention detection influenza A virus, Influenza B virus, respiratory syncytial virus, adenovirus, Chlamydia pneumoniae, the hot rickettsia of Q, parainfluenza virus, each three parts of the positive serum of several pathogenic organisms of respiratory tract of legionella pneumophilia, every part is repeated for three times, investigates specificity situation.
2. sensitivity is investigated
Adopt test kit of the present invention and colloidal gold immunochromatographimethod method, 11 parts of MP positive serums and 7 parts of MP negative serums are detected, compares the sensitivity of two kinds of methods.
3. precision is investigated
The reagent strip of two batches of preparations detects same a feminine gender positive serum respectively, and every batch is detected 10 times, calculates batch interior coefficient of variation (CV) between criticizing respectively, investigates accuracy.
4. study on the stability
Ready reagent strip is placed heating 0 day in 60 DEG C of electric drying oven with forced convections, and 2 days, 4 days, 6 days, 8 days, the strong sun of detection, weak sun, each three times of negative serum, investigated acceleration for stabilization implementations respectively.
(6) investigate with the concordance of European Union's mycoplasma pneumoniae IgMELISA detection kit
Use the present invention and European Union's test kit 47 parts of MP positive serums of detection and 339 parts of negative serums respectively, use SPSS16.0 software four fold table check analysis to draw this method and European Union's test kit comparison of coherence result, and calculate positive coincidence rate, negative match-rate and Kappa coefficient etc..European Union's mycoplasma pneumoniae IgM detection kit is the Mycoplasma pneumonia detection kit commonly used on the market, close with Cleaning Principle of the present invention, all for MPIgM antibody test.
Three, result:
(1) optimization of tomographic system
1. the optimization of film
Relatively two kinds of model NC film testing results of SatouriusCN95 and CN140, the i.e. difference of T peak/C peak area ratio.Bigger relative to CN95, CN140 yin and yang attribute difference, therefore the present invention selects CN140 film as the suitableeest NC film (see accompanying drawing 1).
2. the selection of surfactant in sample diluting liquid
Adopting different diluents that 1 part of MP positive serum is detected, it is best that the Tween-20 of 2% runs line in chromatography process, can reach chromatographic effect faster, so we select Tween-20 as the optimum surfactant (see accompanying drawing 2) of the present invention.
3. the determination of chromatography time
We detect MP yin and yang attribute serum T peak/C peak area ratio situation over time.Start T peak/C peak area ratio from 5min to tend towards stability, that is chromatography uses instrument to detect after starting 5min, result zero difference, but consider the accuracy of detection and the adequacy etc. of reaction, the present invention selects 10min as optimum detection time (see accompanying drawing 3).
(2) detection actual sample determines Cutoff value
Adopting the present invention that 34 parts of MP positive serums and 166 parts of MP negative serums are detected, by SPSS16.0ROC tracing analysis, area under curve is 0.986 (p < 0.001).Trade-off curve keeps left most a bit (sensitivity add specificity maximum a bit) determine that Cutoff is 0.3830, be negative lower than 0.3830, higher than 0.3830 for the positive (see accompanying drawing 4).
(3) detection systematic function is investigated
1. specificity
As shown in table 1, the present invention is used to detect swine flu, second stream, respiratory syncytial virus, adenovirus, parainfluenza virus, Chlamydia pneumoniae, the hot rickettsia of Q, each 3 parts of positive serums of legionella pneumophilia.The detected value of these several pathogen positive serums is all below Cutoff value, it was shown that the method specificity that the present invention detects mycoplasma pneumoniae is higher, does not have cross reaction with these several respiratory tract infection class pathogen.
Several respiratory pathogen Virus monitory result of table 1
2. sensitivity
As shown in table 2, to detect the result of 15 parts of serum completely the same with the result of European Union's mycoplasma pneumoniae IgMELISA detection kit for the present invention.And adopt colloidal gold kit to detect, and 7 parts of MP negative serum results are consistent with above two detection method, but 8 parts of MP positive serums detect 5 parts of negative findingses, it can be seen that, the sensitivity of gold-immunochromatographyreagent reagent for assay box is lower than the inventive method.
The comparison of table 2 present invention and gold-immunochromatographyreagent reagent for assay box testing result
In addition, being diluted by 3 parts of MP positive serums, testing result is as shown in table 3.For with a positive serum, gold-immunochromatographyreagent reagent for assay box can only detect the MP specific IgM antibodies of 1: 10 dilution, and fluorescence immune chromatography method can detect the MP specific IgM antibodies of 1: 1000 dilution.By the comparison of above two aspects, Alexa of the present invention647 are compared to gold colloidal is more suitable for label and is applied to tomographic system.
Table 3 present invention compares with gold-immunochromatographyreagent reagent for assay box sensitivity
3. precision
As shown in table 4, the present invention detect MP strong positive serum, weak positive serum and negative serum batch between CV respectively 3.28%, 6.04% and 6%, the CV respectively 3.45%, 4.89% and 10.14% in crowd, can control below 15%.Show that the present invention has good repeatability, this systematic error greatly reduced during detection, make result more reliable.
Batch interior difference between 4 batches, table
4. stability
Test kit of the present invention difference high temperature under 60 DEG C of conditions accelerates 0 day, 2 days, 4 days, 6 days, after 8 days, and Virus monitory result (see accompanying drawing 5).0 day, 2 days, 4 days, within 6 days, testing result deviation was both less than 10%, and change is little, and when the 8th day, positive and weak sun detected value declined 24%, 14% respectively compared to the 6th day by force, declined by a big margin, and negative detected value raised 19% compared to the 6th day, and entire change is obvious.60 DEG C are accelerated within 6 days, to be approximately equal to 25 DEG C of room temperature and place 2 years, and therefore test kit of the present invention is minimum can stably preserve about 2 years.
(4) investigate with the concordance of European Union's mycoplasma pneumoniae IgM detection kit
As shown in table 5, the comparison of the present invention and European Union's test kit, positive coincidence rate is 93.62%, negative match-rate is 96.76%, total coincidence rate is 96.37%, four fold table inspection in SPSS16.0 software is used to draw the concordance better (Kappa coefficient is 0.842, p < 0.001) of two test kits.
The concordance of table 5 and European Union's test kit is investigated
Claims (10)
1. the Mycoplasma pneumonia detection kit based on fluorescence immune chromatography technology, including the sample pad attached successively on polrvinyl chloride base plate, fluorescence pad, nitrocellulose filter and adsorptive pads, wherein fluorescence pad is the fiberglass packing of IgM antibody and the fluorescein-labelled goat anti-rabbit igg antibody being coated with fluorescein-labelled mouse-anti people；Having detection line and nature controlling line on nitrocellulose filter, detection line is coated with mycoplasma pneumoniae antigen, and nature controlling line is coated with rabbit igg antibody.
2. detection kit according to claim 1, wherein fluorescein is near-infrared fluorescent element.
3. detection kit according to claim 2, wherein fluorescein is fluorescein
4. detection kit according to claim 1, wherein the IgM antibody of mouse-anti people is IgM μ chain monoclonal antibody.
5. detection kit according to claim 1, wherein nitrocellulose filter adopts SatouriusCN140.
6. detection kit according to claim 1, wherein mycoplasma pneumoniae antigen is attached most importance to group P1 antigen and native antigen.
7. fluoresceinPurposes in preparing fluorescence immune chromatography detectable.
8. purposes according to claim 7, wherein detectable is mycoplasma pneumoniae antibody detectable.
9. a fluorescence immune chromatography detection method for mycoplasma pneumoniae antibody, comprises the following steps: use fluorescein as marker material, the IgM antibody of labelling mouse-anti people；After mycoplasma pneumoniae IgM antibody in serum mixes with sample diluting liquid, be combined formation reaction complex with fluorescein labelled antibody；Along with chromatography effect moves forward along nitrocellulose filter, detected coated mycoplasma pneumoniae antigen on line by nitrocellulose filter and catch；By Immunofluorescence test instrument, detect the mycoplasma pneumoniae IgM antibody in serum.
10. detection method according to claim 9, wherein fluorescein is fluoresceinThe IgM antibody of mouse-anti people is IgM μ chain monoclonal antibody, and sample diluting liquid surfactant adds 2%Tween-20, and nitrocellulose filter adopts SatouriusCN140.
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106198966A (en) * | 2016-09-06 | 2016-12-07 | 中国农业大学 | A kind of near-infrared fluorescent immune chromatography reagent kit detecting aflatoxin and application thereof |
| CN108950053A (en) * | 2018-08-25 | 2018-12-07 | 潍坊德诺泰克生物科技有限公司 | For detecting the primed probe group and its application of Blastomyces dermatitidis |
| CN109022429A (en) * | 2018-07-27 | 2018-12-18 | 山东德诺生物科技有限公司 | For detecting the primed probe group and its application of rs671 |
| CN110133255A (en) * | 2018-02-02 | 2019-08-16 | 中国人民解放军军事科学院军事医学研究院 | A kind of quickly SERS- immunochromatography detection method with highly sensitive detection mycoplasma pneumoniae infection |
| CN111122861A (en) * | 2020-01-20 | 2020-05-08 | 浙江大学 | Kit for detecting specific IgE of serum mycoplasma pneumoniae |
| CN111172242A (en) * | 2019-12-19 | 2020-05-19 | 武汉中帜生物科技股份有限公司 | Kit for joint detection of influenza A virus and influenza B virus based on double amplification technology and application thereof |
| CN111856003A (en) * | 2020-02-04 | 2020-10-30 | 潍坊市康华生物技术有限公司 | Novel coronavirus (2019-nCoV) IgM and IgG antibody detection test strip, kit and method |
| CN113624975A (en) * | 2021-08-20 | 2021-11-09 | 浙江嘉孚生物科技有限公司 | Time-resolved immunofluorescence chromatography reagent strip and application thereof in qualitative detection of mycoplasma and chlamydia for non-diagnosis purpose |
| CN113817032A (en) * | 2021-09-28 | 2021-12-21 | 山东硕景生物科技有限公司 | Natural antigen P1 for detecting anti-mycoplasma pneumoniae antibody and preparation method and application thereof |
| CN114397451A (en) * | 2022-01-25 | 2022-04-26 | 山东康华生物医疗科技股份有限公司 | Chlamydia pneumoniae antigen detection plate and detection box |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106198966A (en) * | 2016-09-06 | 2016-12-07 | 中国农业大学 | A kind of near-infrared fluorescent immune chromatography reagent kit detecting aflatoxin and application thereof |
| CN110133255A (en) * | 2018-02-02 | 2019-08-16 | 中国人民解放军军事科学院军事医学研究院 | A kind of quickly SERS- immunochromatography detection method with highly sensitive detection mycoplasma pneumoniae infection |
| CN109022429A (en) * | 2018-07-27 | 2018-12-18 | 山东德诺生物科技有限公司 | For detecting the primed probe group and its application of rs671 |
| CN108950053A (en) * | 2018-08-25 | 2018-12-07 | 潍坊德诺泰克生物科技有限公司 | For detecting the primed probe group and its application of Blastomyces dermatitidis |
| CN111172242A (en) * | 2019-12-19 | 2020-05-19 | 武汉中帜生物科技股份有限公司 | Kit for joint detection of influenza A virus and influenza B virus based on double amplification technology and application thereof |
| CN111172242B (en) * | 2019-12-19 | 2023-06-02 | 武汉中帜生物科技股份有限公司 | Kit for combined detection of influenza A and B virus based on double amplification technology and application thereof |
| CN111122861A (en) * | 2020-01-20 | 2020-05-08 | 浙江大学 | Kit for detecting specific IgE of serum mycoplasma pneumoniae |
| CN111856003A (en) * | 2020-02-04 | 2020-10-30 | 潍坊市康华生物技术有限公司 | Novel coronavirus (2019-nCoV) IgM and IgG antibody detection test strip, kit and method |
| CN113624975A (en) * | 2021-08-20 | 2021-11-09 | 浙江嘉孚生物科技有限公司 | Time-resolved immunofluorescence chromatography reagent strip and application thereof in qualitative detection of mycoplasma and chlamydia for non-diagnosis purpose |
| CN113817032A (en) * | 2021-09-28 | 2021-12-21 | 山东硕景生物科技有限公司 | Natural antigen P1 for detecting anti-mycoplasma pneumoniae antibody and preparation method and application thereof |
| CN114397451A (en) * | 2022-01-25 | 2022-04-26 | 山东康华生物医疗科技股份有限公司 | Chlamydia pneumoniae antigen detection plate and detection box |
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