CN104502608A - Group A rotavirus chromatography test paper strip based on low-noise excitation fluorescent label - Google Patents
Group A rotavirus chromatography test paper strip based on low-noise excitation fluorescent label Download PDFInfo
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Abstract
The invention belongs to the field of food detection, and particularly relates to a group A rotavirus chromatography test paper strip based on low-noise excitation fluorescent label as well as a preparation method and application of the chromatography test paper strip. According to the invention, quantum dots are adopted as the label, the immunochromatography technology is adopted, so that the chromatography test paper strip targeting to group A rotavirus is prepared, during detection, a special portable low-noise excitation fluorescent scanner is adopted for scanning a quality control line and a detection line, and qualitative and quantitative determination of a sample is realized through a fluorescence intensity detection value of the detection line. The test paper strip is used for immediate detection of group A rotavirus in food and a pathological sample, and has the advantages of rapidness, high sensitivity, qualitative, accurate and quantitative detection, and portability.
Description
Technical field
The invention belongs to food (medical science) detection field, relate in particular to a kind of A group rotavirus immuno-chromatographic test paper strip based on low noise excitation formula fluorochrome label and preparation method thereof and application.
Background technology
Rotavirus is one of main pathogens causing infantile diarrhea, its main infection intestinal epithelial cell, thus causes cellular damage, causes diarrhoea.Rotavirus is popular in autumn and winter in summer every year, and route of infection is fecal oral route, and clinical manifestation is acute gastroenteritis, sick in osmotic diarrhea.It is extensively present in occurring in nature, and the safety of the rotavirus existed in food to the mankind has danger, and this bacterium still can growth and breeding at low temperature, that chilled food threatens one of the main pathogenic fungi of human health, therefore, in food hygiene Micro biological Tests, must be paid attention to.At present, rotavirus is as an important indicator of pathogenic microbes detect, in public health, food hygiene, animal and veterinary and inspection and quarantining for import/export, all there is important meaning, also the quick of rotavirus and high-precision quantitative detection are had higher requirement simultaneously.For seeking fast, accurately, high sensitivity, easy to operate and can be quantitative detection method, countries in the world scholar has carried out large quantity research, from the method for quick developed into based in the conventional way based on immunology, molecular biology, and constantly make further progress in practice.
Immunochromatography technique is a Novel immune detection technique effectively combining the remarkable separating power of chromatography and immune response high degree of specificity, is currently used widely in fields such as disease quick diagnosis, environmental pollutant analysis and the calibratings of the pathogenic organisms factor.Its principle is a certain zone specific antigen or antibody being fixed on nitrocellulose filter, after sample is immersed in nitrocellulose filter one end of this drying, due to capillarity, sample will move forward along this film, when migrating to the specific region of crosslinked labeling antibody, in sample, namely corresponding antigen form antigen-labelled antibody compound with this antibody generation specific binding, make this region show certain signal through the colour developing of label, luminescence or electrochemical reaction, thus realize specific immunity diagnosis.Conventional label comprises the type probe that adds lustre to (comprising collaurum, colloidal-carbon, painted microballoon etc.) and fluorescence molecule (as organic fluorescent dye, quantum dot, lanthanide complexes, upper converting phosphor particle etc.).
But above-mentioned label but to there is sensitivity limited and cannot realize the defects such as accurate quantification detection, and then limit the widespread use of immunochromatography technique.As common utilizing the enzymatic reaction of enzyme labeling catalysis chromogenic substrate and develop the color in prior art, can be used for the direct-detection of antigen or antibody, the method is applied to the detection of rotavirus in food, greatly false positive rate is reduced by monoclonal antibody, but the sensitivity of the method comparatively molecular biology method is low, and have to pass through increasing bacterium screening pure culture step, be therefore difficult to obtain testing result at short notice.
For existing colloidal gold immunochromatographimethod technology, developed the color by the aggreation of the nano particles such as collaurum, thus realize result judgement.Although this kind of method has the feature such as convenient, quick, accurate and pollution-free and is widely used in medical science and detects and clinical diagnosis, in pathogen detection, its susceptibility, specificity have the unrivaled advantage of other immunological methods, and without the need to special instruments and equipment, require low to the specialty of testing staff, have good basic unit's application and development prospect.But there is the less stable of label in this method, color is single, and sensitivity is lower, the defects such as the background interference by matrix is large, and can only qualitative detection, can not accomplish that accurate quantification detects, above-mentioned marking sensitivity is limited and cannot realize accurate quantification, limits the application of immunochromatography technique.
In addition, existing immunochromatography technique is mainly used in the detection to protide always, and for the detection of microorganism, then finding is very few, is more difficult to the quantitative detection realizing microorganism simultaneously, and for detecting for pathogenic bacteria fast, its application is subject to great restriction.
Summary of the invention
For this reason, technical matters to be solved by this invention is the problem being difficult to Quantitative detection rotavirus in prior art, and then provide a kind of based on low noise excitation formula fluorescently-labeled A group rotavirus immuno-chromatographic test paper strip, and further disclose its application.
For solving the problems of the technologies described above, one provided by the invention, based on low noise excitation formula fluorescently-labeled A group rotavirus immuno-chromatographic test paper strip, comprising:
Base plate and along the sample pad, pad, antibody carrier film and the adsorptive pads that the length direction of described base plate stick to successively on described base plate, described sample pad, pad, between antibody carrier film and adsorptive pads, successively with and only contact with adjacent regions and partly overlap; Described antibody carrier film adheres to the middle part of described base plate, is provided with detection line separately and nature controlling line, and described detection line is near described pad, and described nature controlling line is near described adsorptive pads;
Described pad is coated with low noise excitation formula fluorochrome label can with the monoclonal antibody of A group rotavirus specific binding;
Described detection line by can with the polyclonal antibody coating formation of described rotavirus specific binding; Described nature controlling line is formed by with described rotavirus and the described antibody coating that all can have nothing to do with the polyclonal antibody of rotavirus specific binding.
Described based on low noise excitation formula fluorescently-labeled A group rotavirus immuno-chromatographic test paper strip, described pad sprays described can be mouse-anti A group rotavirus capsid protein monoclonal antibody with the monoclonal antibody of A group rotavirus specific binding.
Described based on low noise excitation formula fluorescently-labeled A group rotavirus immuno-chromatographic test paper strip, what form described detection line can be goat-anti A group rotavirus polyclonal antibody with the polyclonal antibody of A group rotavirus specific binding.
Described based on low noise excitation formula fluorescently-labeled A group rotavirus immuno-chromatographic test paper strip, the antibody forming described nature controlling line is sheep anti-mouse igg polyclonal antibody.
Described based on low noise excitation formula fluorescently-labeled A group rotavirus immuno-chromatographic test paper strip, described low noise excitation formula fluorescent dye is emission wavelength is 650-1000nm.
Described based on the fluorescently-labeled rotavirus immuno-chromatographic test paper strip of low noise excitation formula, described low noise excitation formula fluorescent dye is DyLight800.
Described based on the fluorescently-labeled rotavirus immuno-chromatographic test paper strip of low noise excitation formula, described nature controlling line and described detection line be arranged in parallel, described detection line and described nature controlling line spacing 0.5cm.
The invention provides and a kind ofly prepare the described method based on the fluorescently-labeled rotavirus immuno-chromatographic test paper strip of low noise excitation formula, comprise the steps:
A) get dilute 10 times with PBS damping fluid described low noise excitation formula fluorescent dye DyLight800 (being provided by Thermo Fisher company of the U.S.) with described mouse-anti A group rotavirus capsid protein monoclonal antibody (purchased from Pierce antibody company) in mass ratio for 1:4 mixes, under room temperature condition, lucifuge reacts 2 hours, subsequently the marked product of gained is put into the bag filter containing PBS damping fluid, dialyse 4 hours for 4 DEG C, in the antibody-solutions marked product that described mark is good, add final concentration is 1.5%BSA, 0.15%Tween20 and sodium azide 0.1 ‰, 4 DEG C of preservations, for subsequent use,
B) spray on described pad dilute the above-mentioned steps A after 4000 times with chromatography buffer) in described marked product, then room temperature is air-dry, for subsequent use;
C) the PBS damping fluid containing 5%BSA, 0.1%Tween 20 is used to be sprayed in described sample pad, after room temperature is air-dry, for subsequent use;
D) get respectively described rotavirus polyclonal antibody and described with rotavirus and the antibody pen machine that all can have nothing to do with the polyclonal antibody of rotavirus specific binding on described antibody carrier film, draw described detection line and described nature controlling line, room temperature is air-dry, for subsequent use;
E) described sample pad, described pad, described antibody carrier film and described adsorptive pads that above-mentioned steps obtains are adhered to successively along on the length direction of described base plate, then cut into test strips, to obtain final product.
The invention provides a kind of by above-mentioned preparation method obtain based on the purposes of low noise excitation formula fluorescently-labeled rotavirus immuno-chromatographic test paper strip in qualitative and quantitative detection rotavirus field.
The invention provides and a kind ofly utilize the described method detecting rotavirus based on the fluorescently-labeled rotavirus immuno-chromatographic test paper strip of low noise excitation formula, comprise the steps:
A) with PBS (pH7.2) damping fluid dilution process testing sample, getting supernatant as treating sample, separately getting described PBS damping fluid as negative control, carrying out immunochromatography by described test strips respectively;
B) detection line described in fluorescent scanning and described nature controlling line, measures the fluorescence intensity in described detection line and described nature controlling line region respectively, if fluorescence emission peak appears in described nature controlling line region, then shows that this test strips is effective, otherwise then invalid; If described detection line region occurs fluorescence emission peak simultaneously, be positive findings, otherwise be then negative.
The method of described detection A group rotavirus, the step also comprising preparation standard curve and quantitatively detect: wherein:
The step of described preparation standard curve comprises: get the serial standards that A group rotavirus capsid protein antigen (Beijing Hua Da protein company) is mixed with 1-40 times of gradient concentration; And above-mentioned concentration series standard items are carried out immunochromatography by described test strips respectively, detection line described in fluorescent scanning and described nature controlling line, measure the fluorescence intensity in described detection line and described nature controlling line region respectively, make the typical curve of relation between antigen concentration and fluorescence intensity ratio, and fit equation;
The step of described quantitative detection comprises: get testing sample and carry out immunochromatography detection with described test strips, utilizes above-mentioned typical curve and working strategy to try to achieve described rotavirus concentration.
Technique scheme of the present invention has the following advantages compared to existing technology:
(1) of the present invention based on low noise excitation formula fluorescently-labeled A group rotavirus immuno-chromatographic test paper strip, the low noise excitation formula fluorescence probe that its emission spectrum is positioned at low noise excitation formula region (wavelength is 650-1100nm) has higher signal to noise ratio (S/N ratio), and ensured its high detection sensitivity thus, simultaneously because biological substrate is seldom at low noise excitation formula spectral region autofluorescence, the analysis based on this type of probe mark is detected and disturbs from background fluorescence; Because the biquadratic of scattered light intensity and wavelength is inversely proportional to, the low noise excitation formula fluorescence probe that utilizing emitted light is positioned at long-wavelength region is little by its interference.Compared with conventional colloidal gold immuno-chromatography test paper strip, the immunochromatography system based on low noise excitation formula fluorochrome label improves about 10 times to the sensitivity of target virus; Minimum detectability as rotavirus colloidal gold immuno-chromatography test paper strip is 5 μ g/mL, and is 0.5 μ g/mL based on the minimum detectability of the rotavirus immuno-chromatographic test paper strip of low noise excitation formula fluorescent dye in the present invention;
(2) method of low noise excitation formula fluorescence A group rotavirus immuno-chromatographic test paper strip qualitative detection rotavirus of the present invention has portable advantage, be applicable to scene, immediately, fast detect, immuno-chromatographic test paper strip involved by this method and low noise excitation formula Fluorescence Scanner all belong to portable movable fixture, and whole testing process can complete in 10min, be applicable to scene, immediately, fast detect, then in the detection speed of instrument, portability and on-the-spot applicability, present clear superiority relative to quantitative real-time PCR equimolecular biological method;
(3) method that low noise excitation formula fluorescence colyliform virus immunity chromatograph test strip of the present invention quantitatively detects rotavirus quantitatively can detect described rotavirus, traditional immunochromatographic method relies on the buildup effect of colloid gold particle and adds lustre to sentence read result, though there is certain quantitative relation with the concentration of target bacterium in sample in its shade, but cannot accurate quantitative analysis be carried out, and near infrared fluorescent dye is marked on specific antibody by the present invention, the fluorescence intensity of detection line directly represent the amount of the target bacterium combined with capture molecules, in addition nature controlling line not with sample change fluorescence intensity as internal reference, by the fluorescence intensity on Scanning Detction line and nature controlling line, ratio calculated, and compare with typical curve can draw sample hit virus concentration.
Accompanying drawing explanation
In order to make content of the present invention be more likely to be clearly understood, below according to a particular embodiment of the invention and by reference to the accompanying drawings, the present invention is further detailed explanation, wherein
Fig. 1 is the schematic diagram of the low noise excitation formula fluorescence A group rotavirus immuno-chromatographic test paper strip described in embodiment 1;
Fig. 2 is the A group rotavirus low noise excitation formula fluoroscopic examination figure described in embodiment 2;
Fig. 3 is the typical curve of the rotavirus described in embodiment 3.
In figure, Reference numeral is expressed as: 1-sample pad, 2-pad, 3-nitrocellulose filter, 4-detection line, 5-nature controlling line, 6-adsorptive pads, 7-base plate.
Embodiment
Embodiment 1
Described in the present embodiment based on low noise excitation formula fluorescently-labeled A group rotavirus immuno-chromatographic test paper strip as shown in Figure 1, it comprises, base plate 7 and along the sample pad 1, pad 2, antibody carrier film 3 and the adsorptive pads 6 that the length direction of described base plate 7 stick to successively on described base plate, and described sample pad 1, pad 2, between antibody carrier film 3 and adsorptive pads 6, successively with and only contact with adjacent regions and partly overlap; Described antibody carrier film 3 adheres to the middle part of described base plate 7, be provided with the nature controlling line 5 of detection line 4 separately, described detection line 4 is near described pad 2, and described nature controlling line 5 is near described adsorptive pads 6.Described nature controlling line 4 be arranged in parallel with described detection line 5, described detection line and described nature controlling line spacing 0.5cm.
Described pad 2 is coated with low noise excitation formula fluorochrome label can with the monoclonal antibody mouse-anti A group rotavirus capsid protein monoclonal antibody of rotavirus specific binding; Described detection line 4 by can with the polyclonal antibody of described rotavirus specific binding _ goat-anti A group rotavirus polyclonal antibody coating formation; Described nature controlling line 5 by with described rotavirus and the described sheep anti-mouse igg polyclonal antibody coating formation that all can have nothing to do with the polyclonal antibody of rotavirus specific binding.
Described low noise excitation formula fluorescent dye is emission wavelength is 777nm, and wavelength of transmitted light is that the low noise excitation formula fluorescent dye DyLight800 of the NHS activation of 790nm is as fluorescence molecule.
The above-mentioned preparation method based on the fluorescently-labeled rotavirus immuno-chromatographic test paper strip of low noise excitation formula, comprises the steps:
1) the fluorescently-labeled rotavirus monoclonal antibody of low noise excitation formula
Get dilute 10 times with PBS damping fluid described low noise excitation formula fluorescent dye DyLight800 (being provided by Thermo Fisher company of the U.S.) with described mouse-anti A group rotavirus capsid protein monoclonal antibody (purchased from Pierce antibody company) in mass ratio for 1:4 mixes, under room temperature condition, lucifuge reacts 2 hours, subsequently the marked product of gained is put into the bag filter containing PBS damping fluid, dialyse 4 hours for 4 DEG C, in the antibody-solutions marked product that described mark is good, add final concentration is 1.5%BSA, 0.15%Tween20 and sodium azide 0.1 ‰, 4 DEG C of preservations, for subsequent use,
2) pad is made
Choosing glass fibre element film is described pad 2, the antibody of the DyLight800 mark of spraying process (1) on described pad 2, and then room temperature is air-dry, for subsequent use;
3) sample pad is made
Choosing cellulose membrane is described sample pad 1, uses the PBS damping fluid containing 5%BSA, 0.1%Tween 20 to be sprayed in described sample pad 1, after room temperature is air-dry, for subsequent use;
4) antibody carrier film
Choosing nitrocellulose filter is described antibody carrier film 3, get described A group rotavirus polyclonal antibody and described sheep anti-mouse igg (Beijing Hua Da protein company) polyclonal antibody pen machine stroke described detection line 4 and described nature controlling line 5 on described antibody carrier film 3 respectively, room temperature is air-dry, for subsequent use;
5) immuno-chromatographic test paper strip is assembled
By the described sample pad 1 that above-mentioned steps obtains, described pad 2, described antibody carrier film 3 and described adsorptive pads 6 adhere to successively along on the length direction of described base plate 7, be specially and first lay antibody carrier film 3 in the middle of base plate 7, then adsorptive pads 6 is spread in one end closed on mutually with nature controlling line 5 of antibody carrier film 3, adsorptive pads 6 and antibody carrier film 3 are partly overlapped, then described pad 2 is spread in one end closed on mutually with described detection line 4 of antibody carrier film 3, make described antibody carrier film 3 overlapping with the one end portion of described pad 2, subsequently in the other end described sample pad 1 of partly overlapping laying again of described pad 2, finally cut into 0.5cm wide, obtain test strips.
Embodiment 2
Present embodiments provide and a kind ofly utilize the above-mentioned method of carrying out qualitative detection rotavirus based on low noise excitation formula fluorescently-labeled A group rotavirus immuno-chromatographic test paper strip, comprise the steps:
A) get strawberry sample 25g, with the PBS damping fluid dilute sample of 225mL containing 5%BSA, 0.1%Tween 20, get supernatant as sample, draw 1ml supernatant stand-by.Drawing the pretreated sample of 50 μ L is added drop-wise in sample pad 1, to be absorbed dry after drip 50 μ L chromatographic solutions again, separately get described damping fluid as negative control, carry out immunochromatography by described test strips;
B) after room temperature places 15min, the fluorescence intensity in described detection line and described nature controlling line region is measured respectively with portable low noise excitation formula Fluorescence Scanner (Beijing Bo Run Fu get development in science and technology company limited), if there is fluorescence emission peak in described nature controlling line region, then show that this test strips is effective, on the contrary then invalid; If described detection line region occurs fluorescence emission peak simultaneously, be positive findings, otherwise be then negative, figure 2 shows the qualitative detection result to above-mentioned sample.
C) salmonella, staphylococcus aureus, Shigella, colon bacillus, Listeria monocytogenes, vibrio parahaemolytious are modulated into 0.6 × 10 respectively
4cFU/mL bacterium liquid, carries out brokenly bacterium process according to the method for foregoing descriptions, draws 100 μ L and is added in sample pad through the sample drop of ultrasonic process, to be absorbed dry after drip 50 μ L chromatographic solutions again.After room temperature places 15min, with Portable near infrared Fluorescence Scanner reading.Except rotavirus sample, all there is not emission peak in detection line position in all the other bacteriums detected, the test strips of each bacterium all occurs emission peak at nature controlling line place.Show that this method is good to the detection specificity of rotavirus, with other four Pseudomonas no cross reactions.
Embodiment 3
Present embodiments provide and a kind ofly utilize the described method quantitatively detecting rotavirus based on the fluorescently-labeled rotavirus immuno-chromatographic test paper strip of low noise excitation formula.
(a) preparation standard curve: the A group rotavirus capsid protein (Beijing Hua Da protein company) getting 100 μ g/ml, with containing 5%BSA, 0.1%Tween 20, described albumen is carried out multiple serial dilution by PBS (pH7.2) dilution, makes the sample of 0.5,1,2.5,5,10,20 μ g/ml.
B () is drawn the above sample drop of 50 μ L and is added in sample pad 1,50 μ L chromatographic solutions are dripped again after absorption of sample, room temperature leaves standstill 10 minutes, above-mentioned test strips is put into portable low noise excitation formula Fluorescence Scanner (Beijing Bo Run Fu get development in science and technology company limited) reading.The A group rotavirus standard items of above-mentioned serial dilution detect respectively, and scanning obtains the fluorescent value of detection line and nature controlling line, occur that surveyed area fluorescence peak and Quality Control region fluorescence peak side show that reaction is normal simultaneously.Each dilutability sample horizontal survey twice, averages as measured value, testing result in table 1, with this value to corresponding sample concentration production standard working curve, as shown in Figure 3, and the typical curve that matching must be suitable for.
The corresponding testing result of table 1 variable concentrations A group rotavirus bacterium standard items
| Concentration (titre) μ g/ml | 0.5 | 1 | 2.5 | 5 | 10 | 20 |
| Measured value for the first time | 0.18 | 0.33 | 0.55 | 0.90 | 1.32 | 2.01 |
| Second time measured value | 0.12 | 0.35 | 0.57 | 0.84 | 1.38 | 1.95 |
| Mean value | 0.15 | 0.34 | 0.56 | 0.87 | 1.35 | 1.98 |
C () adopts and adds recovery test method, get the strawberry sample 75g not detecting A group rotavirus, divide 3 parts, work, pulverize, add the PBS damping fluid dilute sample of 225mL containing 5%BSA, 0.1%Tween 20 respectively, then add a certain amount of A group rotavirus capsid protein antigen, concuss mixes, and gets supernatant 1ml stand-by.Draw 50 μ L also in pretreated sample instillation well, to be absorbed completely after drip 50 μ L chromatographic solutions again, room temperature leaves standstill 10 minutes, and test strips is put into Portable near infrared Fluorescence Scanner reading, result is as shown in the table.According to the concentration of the A group rotavirus in typical curve calculation sample.Result is as shown in table 2.
Table 2 is according to the concentration of the rotavirus in typical curve calculation sample.
Above table data are known, and the present invention adopts the concentration of the A group rotavirus measured based on fluorescent marker method comparatively accurate, can be used for the use of the detection of daily rotavirus.
Obviously, above-described embodiment is only for clearly example being described, and the restriction not to embodiment.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here exhaustive without the need to also giving all embodiments.And thus the apparent change of extending out or variation be still among the protection domain of the invention.
Claims (10)
1., based on a low noise excitation formula fluorescently-labeled A group rotavirus immuno-chromatographic test paper strip, it is characterized in that, comprising:
Base plate (7) and along the sample pad (1) length direction of described base plate (7) sticked to successively on described base plate, pad (2), antibody carrier film (3) and adsorptive pads (6), and described sample pad (1), pad (2), between antibody carrier film (3) and adsorptive pads (6), successively with and only contact with adjacent regions and partly overlap; Described antibody carrier film (3) adheres to the middle part of described base plate (7), be provided with detection line (4) separately and nature controlling line (5), described detection line (4) is near described pad (2), and described nature controlling line (5) is near described adsorptive pads (6);
Described pad (2) is coated with low noise excitation formula fluorochrome label can with the monoclonal antibody of described A group rotavirus capsid protein specific binding;
Described detection line (4) by can with the polyclonal antibody coating formation of described A group rotavirus specific binding; Described nature controlling line (5) is formed by with described rotavirus and the described antibody coating that all can have nothing to do with the polyclonal antibody of A group rotavirus specific binding.
2. according to claim 1 based on low noise excitation formula fluorescently-labeled A group rotavirus immuno-chromatographic test paper strip, it is characterized in that, upper the described of spraying of described pad (2) can be mouse-anti A group rotavirus capsid protein monoclonal antibody with the monoclonal antibody of A group rotavirus specific binding.
3. according to claim 1 and 2 based on low noise excitation formula fluorescently-labeled A group rotavirus immuno-chromatographic test paper strip, it is characterized in that, forming the described of described detection line (4) can be goat-anti A group rotavirus polyclonal antibody with the polyclonal antibody of A group rotavirus specific binding.
4. arbitrary described based on low noise excitation formula fluorescently-labeled A group rotavirus immuno-chromatographic test paper strip according to claim 1-3, it is characterized in that, the antibody forming described nature controlling line (5) is sheep anti-mouse igg polyclonal antibody.
5. arbitrary described based on low noise excitation formula fluorescently-labeled A group rotavirus immuno-chromatographic test paper strip according to claim 1-4, it is characterized in that, the fluorescence molecule of described low noise excitation formula fluorescent dye to be emission wavelength be 650-1000nm.
6. according to claim 5ly it is characterized in that based on low noise excitation formula fluorescently-labeled A group rotavirus immuno-chromatographic test paper strip, described low noise excitation formula fluorescent dye is the low noise excitation formula fluorescent dye DyLight800 of NHS activation.
7. prepare the arbitrary described method based on low noise excitation formula fluorescently-labeled A group rotavirus immuno-chromatographic test paper strip of claim 1-6, it is characterized in that, comprise the steps:
A) get dilute 10 times with PBS damping fluid described low noise excitation formula fluorescent dye DyLight800 with described A group rotavirus capsid protein monoclonal antibody in mass ratio for 1:4 mixes, under room temperature condition, lucifuge reacts 2 hours, subsequently the marked product of gained is put into the bag filter containing PBS damping fluid, 4 DEG C of dialysed overnight, in the antibody-solutions marked product that described mark is good, add final concentration is 1.5%BSA, 0.1%Tween20 and sodium azide 0.15 ‰, 4 DEG C of preservations, for subsequent use;
B) the above-mentioned steps A after the upper spraying of described pad (2) dilutes 4000 times with chromatography buffer) in described marked product, then room temperature is air-dry, for subsequent use;
C) the PBS damping fluid containing 5%BSA, 0.1%Tween 20 is used to be sprayed in described sample pad (1), after room temperature is air-dry, for subsequent use;
D) get respectively described A group rotavirus polyclonal antibody and described with A group rotavirus and the antibody pen machine that all can have nothing to do with the polyclonal antibody of A group rotavirus specific binding draw described detection line (4) and a described nature controlling line (5) described antibody carrier film (3) is upper, room temperature is air-dry, for subsequent use;
E) described sample pad (1), described pad (2), described antibody carrier film (3) and described adsorptive pads (6) that above-mentioned steps obtains are adhered to successively along on the length direction of described base plate (7), then cut into test strips, to obtain final product.
8. claim 1-6 is arbitrary described based on the purposes of low noise excitation formula fluorescently-labeled A group rotavirus immuno-chromatographic test paper strip in qualitative and quantitative detection rotavirus field.
9. utilize the arbitrary described method detecting rotavirus based on low noise excitation formula fluorescently-labeled A group rotavirus immuno-chromatographic test paper strip of claim 1-6, it is characterized in that, comprise the steps:
A) with pH be 7.2 PBS damping fluid dilution process testing sample, getting supernatant as treating sample, separately getting described PBS damping fluid as negative control, respectively to utilize the arbitrary described test strips of claim 1-6 to carry out immunochromatography detection;
B) detection line described in fluorescent scanning (4) and described nature controlling line (5), measure the fluorescence intensity in described detection line (4) and described nature controlling line (5) region respectively, if there is fluorescence emission peak in described nature controlling line (5) region, then show that this test strips is effective, on the contrary then invalid; If described detection line (4) region occurs fluorescence emission peak simultaneously, be positive findings, otherwise be then negative.
10. the method for detection A group rotavirus according to claim 9, is characterized in that, the step also comprising preparation standard curve and quantitatively detect: wherein
The step of described preparation standard curve comprises: get the serial standards that A group rotavirus capsid protein antigen is mixed with 1-100 times of gradient concentration; And above-mentioned concentration series standard items are carried out immunochromatography by described test strips respectively, detection line described in fluorescent scanning (4) and described nature controlling line (5), measure the fluorescence intensity in described detection line (4) and described nature controlling line (5) region respectively, make the typical curve of relation between antigen concentration and fluorescence intensity ratio, and fit equation;
The step of described quantitative detection comprises: get testing sample and carry out immunochromatography detection with described test strips, utilizes above-mentioned typical curve and equation to calculate described A group rotavirus concentration.
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Cited By (3)
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| CN109406780A (en) * | 2018-12-21 | 2019-03-01 | 中国检验检疫科学研究院 | A kind of rotavirus Test paper card and its preparation method and application |
| CN110018313A (en) * | 2019-04-10 | 2019-07-16 | 江苏硕世生物科技股份有限公司 | A kind of A group wheel virus antigen test strip, kit and preparation method thereof |
| CN111983225A (en) * | 2020-05-07 | 2020-11-24 | 中国人民解放军军事科学院军事医学研究院 | Preparation method of rotavirus quantum dot immunochromatography test strip |
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