CN204287191U - The two-in-one immune colloid gold Rapid detection test strip of a kind of rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola - Google Patents
The two-in-one immune colloid gold Rapid detection test strip of a kind of rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola Download PDFInfo
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- CN204287191U CN204287191U CN201420718740.4U CN201420718740U CN204287191U CN 204287191 U CN204287191 U CN 204287191U CN 201420718740 U CN201420718740 U CN 201420718740U CN 204287191 U CN204287191 U CN 204287191U
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Abstract
The utility model relates to the two-in-one immune colloid gold Rapid detection test strip of a kind of rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola, belongs to technical field of immunoassay.This test strips by PVC base plate, sample pad, coated with gold mark mark mass selection water resistant rice bacterial leaf spot pathogenic bacteria and xanthomonas oryzae pv. oryzicola antibody (3D2) gold mark pad, be coated with mass selection water resistant rice bacterial leaf spot pathogenic bacteria and xanthomonas oryzae pv. oryzicola antibody (4D11) at detection line and be coated with the nitrocellulose filter of sheep anti-mouse igg and adsorptive pads is formed at nature controlling line.This test strips is easy and simple to handle, quick, accurate, and overall process only needs 5min, and not by the interference of environmental baseline, specificity is good, and detection sensitivity is high, is limited to 1 × 10 to rice leaf spot bacteria lowest detection
4cfu/mL, is limited to 1 × 10 to xanthomonas oryzae pv. oryzicola lowest detection
5cfu/mL.The utility model is applicable to customs, silo, enterprise, inspection and quarantine unit etc., can realize the quick detection of this germ in grain or seed specimen.
Description
Technical field
The utility model relate to the two-in-one immune colloid gold Rapid detection test strip of a kind of rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola, the quick detection to rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola can be realized simultaneously, be applicable to the Site Detection of a large amount of sample, the quick detection importing and exporting grain and emergency processing, belong to technical field of immunoassay.
Background technology
Rice leaf spot bacteria (
xanthomonas oryzae pv. oryzae) and xanthomonas oryzae pv. oryzicola (
xanthomonas oryzae pv. oryzicola) be the different pvs oryzae and oryzicola of xanthomonas rice Xanthomonas.Bacterial blight of rice is a kind of worldwide disease, and with Rice In Asian Cultivated Rice, district attaches most importance to, and the harm of bacterial leaf streak of rice is only second to bacterial blight of rice, mainly occurs in south China area, is domestic important quarantine disease.These two kinds of diseases are also classified as quarantine harmful organisms by many countries simultaneously, limit from these two kinds of diseases epidemic-stricken area import rice paddy seed and rice.Therefore, develop a kind of sensitive specialization, accurately can to detect the outlet of Quarantine Techniques to domestic plant quarantine and China's rice paddy seed and rice of these two kinds of bacteriums from paddy rice easily all significant simultaneously.
Comprise field observation, Physiology and biochemistry qualification, immunology detection and qualification and PCR detection technique to the checkout and diagnosis method of rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola, ring mediated isothermal amplification (LAMP) technology and biosensor technique are also used to detect.But biochemistry detection technology is consuming time long, and testing result exists unreliability.PCR detection technique is compared has good sensitivity and accuracy, but owing to depending on the testing staff of expensive instrument and specialty, is not suitable for the quick detection of large-tonnage product.Although ELISA detection technique in detection sensitivity not as PCR method, have detection fast, in batches, cheap advantage.And colloidal gold strip have simple to operate, stability is high, do not need by specialized equipment, be applicable to the advantage of field quick detection
Because rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola can bring grave danger and loss to agricultural, therefore detection method seems particularly important fast and accurately.
Utility model content
The utility model object is to provide a kind of two-in-one immune colloid gold Rapid detection test strip detecting rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola, simple to operate, quick and convenient, highly sensitive, and good stability is with low cost.
The utility model is applicable to customs, silo, enterprise, inspection and quarantine unit etc., can realize the quick detection of rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola in a large amount of grain or seed specimen.
The technical solution of the utility model, the two-in-one immune colloid gold Rapid detection test strip of a kind of rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola, comprises PVC base plate, is respectively equipped with sample pad and adsorptive pads at PVC base plate two ends;
In the middle part of PVC base plate, be provided with nitrocellulose filter detection layers, between nitrocellulose filter detection layers and sample pad, be provided with golden marking pad; Described gold mark pad one end is connected with sample pad and stacks, and the gold mark pad other end is stacked in nitrocellulose filter detection layers;
Described nitrocellulose filter detection layers is disposed with detection line and nature controlling line.
Described gold mark pad is coated with gold mark mark mass selection water resistant rice bacterial leaf spot pathogenic bacteria and xanthomonas oryzae pv. oryzicola antibody (3D2);
Described detection line is coated with mass selection water resistant rice bacterial leaf spot pathogenic bacteria and xanthomonas oryzae pv. oryzicola antibody (4D11);
Described nature controlling line is coated with sheep anti-mouse igg.
The preparation method of the two-in-one immune colloid gold Rapid detection test strip of described rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola, step is as follows:
(1) preparation of the monoclonal antibody of water resistant rice bacterial leaf spot pathogenic bacteria and xanthomonas oryzae pv. oryzicola
Monoclonal antibody adopts rice leaf spot bacteria (NCPPB3002 imports and exports inspection and quarantine bureau from Hunan Province) thalline through specific immune programme for children immunity BALB/c mouse, obtains through hybridoma technology fusion, screening.Rice leaf spot bacteria NCPPB 3002 thalline is as immunogene, and the BALB/c mouse in immune 8 week age, immune programme for children is as follows: within first week, carry out head and exempt from, 1 × 10
8cfu rice leaf spot bacteria NCPPB 3002 thalline/only, subcutaneous multi-point injection after Freund's complete adjuvant emulsification; 4th week is carried out two and is exempted from, and 1 × 10
8cfu rice leaf spot bacteria NCPPB 3002 thalline/only, subcutaneous multi-point injection after incomplete Freund's adjuvant emulsification; Within 6th week, carry out three to exempt from, 1 × 10
7cfu rice leaf spot bacteria NCPPB 3002 thalline/only, subcutaneous multi-point injection after incomplete Freund's adjuvant emulsification; 7th week afterbody blood sampling survey is tired, and selects respectively and to tire the highest mouse to rice leaf spot bacteria NCPPB 3002 thalline; Within 9th week, carry out spurt immunity, 1 × 10
7cfu rice leaf spot bacteria NCPPB 3002 thalline/only, physiological saline solution, lumbar injection; After making a spurt immune 3 days, eye socket carries out merging, screening after taking a blood sample respectively, screens 10 cell lines altogether;
(2) the pairing screening of mass selection monoclonal antibody
10 strain monoclonal antibodies after purifying in step (1) are marked horseradish peroxidase HRP respectively, carry out sandwich method pairing after the success of direct method identification marking, determine the mass selection antibody of water resistant rice bacterial leaf spot pathogenic bacteria required for mass selection immune colloid gold Rapid detection test strip and xanthomonas oryzae pv. oryzicola; Wherein 3D2 is golden labeling antibody, and 4D11 is coated antibody;
(3) preparation of the two-in-one immune colloid gold Rapid detection test strip of rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola:
The preparation of a, blank collaurum:
Adopt citrate reduction legal system for colloid gold particle, in conical flask, add 200mL deionized water and 2mL 1% gold chloride, be heated with stirring to boiling, add 4mL 1% sodium citrate fast, continue heating 15 ~ 20min, to being shiny red, then cool in room temperature, 4 DEG C of stored refrigerated;
The preparation of b, colloidal gold labeled monoclonal antibody:
Blank colloidal gold solution 0.1M K
2cO
3adjust ph to 7.4, drips antibody prepared by appropriate concentration step (2) in stirring, every mL collaurum adds 30 μ g antibody 3D2, continues stirring 30 min, centrifugal, draws supernatant, heavily revolves with golden labeling antibody conserving liquid (0.02mol/L phosphate buffered solution);
The preparation of c, gold mark pad:
Debugging three-dimensional planar point film gold spraying instrument instrument (HM3055), the water resistant rice bacterial leaf spot pathogenic bacteria and the xanthomonas oryzae pv. oryzicola antibody (3D2) that 1mg/mL have been marked collaurum are sprayed on glass fibre membrane uniformly, spouting liquid 0.6 μ L/cm, 37 DEG C of oven dry are spent the night, and envelope is for subsequent use;
The preparation of d, coated film:
Debugging three-dimensional planar point film gold spraying instrument instrument (HM3055), is evenly sprayed on 1mg/mL water resistant rice bacterial leaf spot pathogenic bacteria and xanthomonas oryzae pv. oryzicola antibody (4D11) on nitrocellulose filter, obtains detection line; Evenly be sprayed on nitrocellulose filter by sheep anti-mouse igg antibody, obtain nature controlling line, spouting liquid is 0.6 μ L/cm, and 37 DEG C of oven dry are spent the night, and envelope is for subsequent use.
PVC base plate, sample pad, gold mark pad, the nitrocellulose filter detection layers scribbling detection line and nature controlling line and adsorptive pads are combined, obtains the two-in-one immune colloid gold Rapid detection test strip of product water rice bacterial leaf spot pathogenic bacteria and xanthomonas oryzae pv. oryzicola.
It is applicable to customs, silo, enterprise, inspection and quarantine unit etc., and can realize the quick detection of rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola in grain or seed specimen, it is 1 × 10 that lowest detectable limit is respectively rice leaf spot bacteria
4cfu/mL and xanthomonas oryzae pv. oryzicola 1 × 10
5cfu/mL.。
The principle of work of the utility model test strips: adopt colloidal gold immunochromatographimethod technology, select rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola mass selection antibody as solid formation, utilize double antibody sandwich method principle whether to detect in sample containing rice leaf spot bacteria and/or xanthomonas oryzae pv. oryzicola.When containing rice leaf spot bacteria and/or xanthomonas oryzae pv. oryzicola in measuring samples, antigen mass selection Anti-bacterium antibody (3D2) that is first and colloid gold label combines, because chromatography effect compound moves forward along coated film, when running into the mass selection Anti-bacterium antibody (4D11) on detection line, form antibody-antigene-Jin labeling antibody compound, enrichment on detection line, forms red precipitate line.
Compared with other detection methods, immune colloid gold detection technique has the following advantages: 1, rapid; 2, highly sensitive; 3, with low cost; 4, safe and simple.
The utility model compared with prior art has the following advantages:
1. detection speed is fast, and overall process only needs 5 min, can test the quick detection of batch samples;
2. highly sensitive, lowest detectable limit is respectively 1 × 10
4cfu/mL and 1 × 10
5cfu/mL;
3. easy and simple to handle, without the need to through professional training, be easy to promote;
4. do not need instrument, be applicable to Site Detection;
5. process is simple, and direct sample detection, sample size is few.
Accompanying drawing explanation
The two-in-one immune colloid gold Rapid detection test strip structural representation front elevation of Fig. 1 the utility model rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola;
The two-in-one immune colloid gold Rapid detection test strip structural representation vertical view of Fig. 2 the utility model rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola;
Description of reference numerals: 1, PVC base plate; 2, nitrocellulose filter detection layers; 3, sample pad; 4, gold mark pad; 5, detection line (T line); 6, nature controlling line (C line); 7, adsorptive pads.
The two-in-one immune colloid gold Rapid detection test strip material object of Fig. 3 the utility model rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola detects schematic diagram; Rice leaf spot bacteria (NCPPB 3002 imports and exports inspection and quarantine bureau from Hunan Province)
Description of reference numerals: 1,2 × 10
6cfu/mL NCPPB 3002 cell concentration; 2,3.3 × 10
5cfu/mL NCPPB 3002 cell concentration; 3,1 × 10
5cfu/mL NCPPB 3002 cell concentration; 4,3.3 × 10
4cfu/mL NCPPB 3002 cell concentration; 5,1 × 10
4cfu/mL NCPPB 3002 cell concentration; 6, blank sample.
The two-in-one immune colloid gold Rapid detection test strip material object of Fig. 4 the utility model rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola detects schematic diagram; Xanthomonas oryzae pv. oryzicola (NCPPB 1585 imports and exports inspection and quarantine bureau from Hunan Province)
Description of reference numerals: 1,1 × 10
7cfu/mL NCPPB 1585 cell concentration; 2,1 × 10
6cfu/mL NCPPB 1585 cell concentration; 3,5 × 10
5cfu/mL NCPPB 1585 cell concentration; 4,1 × 10
5cfu/mL NCPPB 1585 cell concentration; 5, blank sample.
Embodiment
Embodiment 1
As shown in Figure 1, the two-in-one immune colloid gold Rapid detection test strip of a kind of rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola, comprises PVC base plate 1, is respectively equipped with sample pad 3 and adsorptive pads 7 at PVC base plate 1 two ends;
In the middle part of PVC base plate 1, be provided with nitrocellulose filter detection layers 2, between nitrocellulose filter detection layers 2 and sample pad 3, be provided with gold mark pad 4; Gold mark pad 4 one end and sample pad 3 superimposed, gold mark pad 4 other end is superimposed upon in nitrocellulose filter detection layers 2;
Nitrocellulose filter detection layers 2 is provided with detection line T line 5 and nature controlling line C line 6 sequentially.
Described gold mark pad 4 is coated with gold mark mark group's property water resistant rice bacterial leaf spot pathogenic bacteria and xanthomonas oryzae pv. oryzicola antibody 3D2; Described detection line T line 5 is coated with mass selection water resistant rice bacterial leaf spot pathogenic bacteria and xanthomonas oryzae pv. oryzicola antibody 4D11; Described nature controlling line 6 is coated with sheep anti-mouse igg.
Embodiment 2
The preparation method of the two-in-one immune colloid gold Rapid detection test strip of described rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola, the steps include:
(1) preparation of the monoclonal antibody of mass selection
Adopt rice leaf spot bacteria (NCPPB 3002 imports and exports inspection and quarantine bureau from Hunan Province) thalline through specific immune programme for children immunity BALB/c mouse, immune programme for children is as follows: within first week, carry out head and exempt from, 1 × 10
8cfu rice leaf spot bacteria NCPPB 3002 thalline/only, subcutaneous multi-point injection after Freund's complete adjuvant emulsification; 4th week is carried out two and is exempted from, and 1 × 10
8cfu rice leaf spot bacteria NCPPB 3002 thalline/only, subcutaneous multi-point injection after incomplete Freund's adjuvant emulsification; Within 6th week, carry out three to exempt from, 1 × 10
7cfu rice leaf spot bacteria NCPPB 3002 thalline/only, subcutaneous multi-point injection after incomplete Freund's adjuvant emulsification; 7th week afterbody blood sampling survey is tired, and selects respectively and to tire the highest mouse to rice leaf spot bacteria NCPPB 3002 thalline; Within 9th week, carry out spurt immunity, 1 × 10
7cfu rice leaf spot bacteria NCPPB 3002 thalline/only, physiological saline solution, lumbar injection; After making a spurt immune 3 days, eye socket carries out merging, screening after taking a blood sample respectively, screens 10 cell lines altogether.
(2) the pairing screening of mass selection monoclonal antibody
10 strain monoclonal antibodies after purifying are marked horseradish peroxidase HRP respectively, carry out sandwich method pairing after the success of direct method identification marking, determine the mass selection Anti-bacterium antibody of water resistant rice bacterial leaf spot pathogenic bacteria required for immune colloid gold Rapid detection test strip and xanthomonas oryzae pv. oryzicola; Wherein 3D2 is golden labeling antibody, and 4D11 is coated antibody;
(3) preparation of the two-in-one immune colloid gold Rapid detection test strip of rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola:
A. the preparation of blank collaurum:
Adopt citrate reduction legal system for colloid gold particle, in conical flask, add 200mL deionized water and 2mL 1% gold chloride, be heated with stirring to boiling, add 4mL 1% sodium citrate fast, continue heating 15 ~ 20min, to being shiny red, then cool in room temperature, 4 DEG C of stored refrigerated;
B. the preparation of colloidal gold labeled monoclonal antibody:
Blank colloidal gold solution 0.1M K
2cO
3adjust ph to 7.4, drips antibody prepared by appropriate concentration step (2) in stirring, every mL collaurum adds 30 μ g antibody (3D2), continue stirring 30 min, centrifugal, draw supernatant, heavily revolve with golden labeling antibody conserving liquid (0.02mol/L phosphate buffered solution);
C. the preparation of gold mark pad:
Debugging three-dimensional planar point film gold spraying instrument instrument (HM3055), the mass selection water resistant rice bacterial leaf spot pathogenic bacteria and the xanthomonas oryzae pv. oryzicola antibody (3D2) that 1mg/mL have been marked collaurum are sprayed on glass fibre membrane uniformly, spouting liquid 0.6 μ L/cm, 37 DEG C of oven dry are spent the night, and envelope is for subsequent use;
D. the preparation of coated film:
Debugging three-dimensional planar point film gold spraying instrument instrument (HM3055), is evenly sprayed on 1mg/mL mass selection water resistant rice bacterial leaf spot pathogenic bacteria and xanthomonas oryzae pv. oryzicola antibody (4D11) on nitrocellulose filter, obtains detection line; Evenly be sprayed on nitrocellulose filter by sheep anti-mouse igg antibody, obtain nature controlling line, spouting liquid is 0.6 μ L/cm, and 37 DEG C of oven dry are spent the night, and envelope is for subsequent use.
PVC base plate, sample pad, gold mark pad, the acid cellulose film detection layers scribbling detection line and nature controlling line nitre and adsorptive pads are combined, obtains the two-in-one immune colloid gold Rapid detection test strip of product water rice bacterial leaf spot pathogenic bacteria and xanthomonas oryzae pv. oryzicola.
The principle of work of the utility model test strips: adopt colloidal gold immunochromatographimethod technology, select rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola mass selection specific antibody as solid formation, utilize double antibody sandwich method principle whether to detect in sample containing rice leaf spot bacteria and/or xanthomonas oryzae pv. oryzicola.When containing rice leaf spot bacteria and/or xanthomonas oryzae pv. oryzicola in measuring samples, mass selection water resistant rice bacterial leaf spot pathogenic bacteria and the xanthomonas oryzae pv. oryzicola antibody (3D2) of antigen elder generation and colloid gold label combine, because chromatography effect compound moves forward along coated film, when the mass selection water resistant rice bacterial leaf spot pathogenic bacteria run on detection line and xanthomonas oryzae pv. oryzicola antibody (4D11), form antibody-antigene-Jin labeling antibody compound, enrichment on detection line, forms red precipitate line.
Detection sample is processed as follows: get 10g seed during detection, first use distilled water flushing to remove relic and surface contamination miscellaneous bacteria, after put into 0.001% tween-phosphate buffer low temperature (5 DEG C ~ 15 DEG C) and soak 4 h ~ 6 h, smash with homogenizer, prepare sample extracting solution.Extract places 4 h ~ 6 h in 22 DEG C ~ 25 DEG C environment, revolves nest mixing suspending liquid 5min, detects.
If sample is negative during detection, then test strips only has nature controlling line C line to develop the color (in Fig. 3 No. 6, in Fig. 4 No. 5); If sample is positive, and rice leaf spot bacteria concentration is greater than 1 × 10
4cfu/mL and/or xanthomonas oryzae pv. oryzicola concentration are greater than 1 × 10
5cfu/mL, then test strips nature controlling line C line, detection line T line all develops the color.If test strips C line does not develop the color, then illustrate that test strips quality goes wrong.
Claims (1)
1. a two-in-one immune colloid gold Rapid detection test strip for rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola, is characterized in that: be respectively equipped with sample pad (3) and adsorptive pads (7) at PVC base plate (1) two ends;
Be provided with at PVC base plate (1) middle part the nitrocellulose filter detection layers (2) comprising detection line (5) and nature controlling line (6), nitrocellulose filter detection layers (2) is provided with the detection line (5) being coated with mass selection water resistant rice bacterial leaf spot pathogenic bacteria and xanthomonas oryzae pv. oryzicola antibody 4D11 and the nature controlling line (6) being coated with sheep anti-mouse igg successively;
Be provided with between nitrocellulose filter detection layers (2) and sample pad (3) and be coated with the gold mark pad (4) that gold marks mass selection water resistant rice bacterial leaf spot pathogenic bacteria and xanthomonas oryzae pv. oryzicola antibody 3D2; Gold mark pad (4) one end and sample pad (3) is superimposed, and gold mark pads (4) other end and overlays in nitrocellulose filter detection layers (2).
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109486970A (en) * | 2018-11-16 | 2019-03-19 | 海南大学 | A kind of ring mediated isothermal amplification detection primer group, detection method and the detection kit of rice Xanthomonas |
| CN111621549A (en) * | 2020-06-05 | 2020-09-04 | 南京农业大学 | LAMP (loop-mediated isothermal amplification) rapid detection method for bacterial blight in rice seeds and application |
-
2014
- 2014-11-26 CN CN201420718740.4U patent/CN204287191U/en active Active
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109486970A (en) * | 2018-11-16 | 2019-03-19 | 海南大学 | A kind of ring mediated isothermal amplification detection primer group, detection method and the detection kit of rice Xanthomonas |
| CN109486970B (en) * | 2018-11-16 | 2021-12-07 | 海南大学 | Loop-mediated isothermal amplification detection primer group, detection method and detection kit for xanthomonas oryzae |
| CN111621549A (en) * | 2020-06-05 | 2020-09-04 | 南京农业大学 | LAMP (loop-mediated isothermal amplification) rapid detection method for bacterial blight in rice seeds and application |
| CN111621549B (en) * | 2020-06-05 | 2022-07-12 | 南京农业大学 | LAMP (loop-mediated isothermal amplification) rapid detection method for bacterial blight in rice seeds and application |
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