CN107907680B - Colloidal gold immune test paper for detecting bacillus larvae of bees and preparation and application thereof - Google Patents
Colloidal gold immune test paper for detecting bacillus larvae of bees and preparation and application thereof Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- 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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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/32—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Bacillus (G)
Abstract
The invention discloses colloidal gold immune test paper for detecting bacillus larvae, and preparation and application thereof, and particularly comprises a sample pad, a gold label pad, a nitrocellulose membrane, a water absorption pad and a bottom plate, wherein the gold label pad contains a PLMP protein polyclonal antibody marked by colloidal gold, the nitrocellulose membrane is provided with a detection line pre-coated by the PLMP protein polyclonal antibody, and a quality control line pre-coated by goat anti-rabbit IgG solution, the prepared colloidal gold immune test paper has strong specificity, no detection condition on negative bacteria liquid, the specificity can reach 100%, the sensitivity is good, and the minimum detection concentrations of bacteria liquid of the bacillus larvae and positive samples are respectively 105CFU/mL and 106CFU/mL, good detection repeatability, stable performance, and a shelf life of 3 months.
Description
Technical Field
The invention mainly relates to the technical field of molecular biology, in particular to the technical field of polyclonal antibody preparation.
Background
American Foulbrood (AFB), an acute, virulent infectious disease of bee larvae and pupae caused by bacillus larvae (paenibacilsulvarae, p.larvae). The main pathogenic factors of the larval bacillus are some extracellular secreted proteases, and the research shows that the proteases are zinc-containing metalloproteases (PLMP) and participate in larval degeneration. The disease is serious and has strong destructiveness, and only the bee larvae are infected, the body color of the larvae is changed from normal pearl white to brown, brown or even black brown in the early stage of infection, the house cover is sunken, and the moist color is dark; in the later stage of infection, the dead body of the larva can pull out 2cm-3cm of filament, give off fishy smell, and finally cling to the ovary wall with black brown scale-shaped objects. The spore of Larvae has strong resistance, can resist heat and chemical substances, and can survive for many years in severe environments such as high-temperature drying and the like. Once the disease occurs, the bee colony is easy to weaken and die. The disease occurs in the United kingdom initially and then is transmitted to Europe and America, and now all over the world, the world animal health Organization (OIE) classifies the disease as a legal report animal epidemic disease, and China classifies the disease as a second type animal epidemic disease and an entry animal quarantine disease. China is a big bee-keeping country and is the biggest bee product producing and exporting country, and the disease has serious influence on the development of the AFB countryside bee-keeping industry and the quality of bee products.
The current identification method for AFB mainly comprises pathogen separation identification and PCR method, and the methods are not favorable for on-site rapid diagnosis due to time consumption, special instruments and equipment, professional technicians and the like. The method is accurate and sensitive, and is beneficial to on-site rapid diagnosis of AFB, and has important significance in solving the practical difficulty brought to bee disease diagnosis, prevention and control due to the characteristics of fluidity, dispersibility, field operation and the like in bee-keeping production. The report about AFB detection by adopting an immunological method is not available, and the immunoassay method comprises an enzyme-linked immunosorbent assay (ELISA) and a colloidal Gold Immunochromatography (GICA), has the characteristics of high sensitivity, good specificity, low cost, convenient operation and the like, and is suitable for screening large-batch samples. As a new immunological detection method, GICA has the specificity of the immunological method, has the characteristics of simplicity, convenience, rapidness and no need of instruments, and is widely applied to the fields of medicine, animal and plant quarantine, food detection and the like. The colloidal gold test strip for detecting the bacillus larvae is beneficial to the rapid and accurate detection of the bacillus larvae.
Disclosure of Invention
The invention aims to provide colloidal gold immunity test paper for detecting the bacillus larvae of bees, preparation and application thereof, and establishes a method for accurately and sensitively detecting the bacillus larvae of bees on site.
The invention is realized by the following technical scheme:
the invention specifically provides colloidal gold immune test paper for detecting bacillus larvae of bees, which specifically comprises a sample pad, a gold-labeled pad, a nitrocellulose membrane, a water absorption pad and a bottom plate, wherein the gold-labeled pad contains a PLMP protein polyclonal antibody labeled by colloidal gold, the nitrocellulose membrane is provided with a detection line (T line) pre-coated by the PLMP protein polyclonal antibody, and a quality control line (C line) pre-coated by goat anti-rabbit IgG solution.
Preferably, the amount of gold labeled for the colloidal gold labeled PLMP protein polyclonal antibody is 30. mu.g/mL.
Preferably, the detection line has an antibody coating concentration of 1.2mg/mL and an antibody coating volume of 1.5. mu.L.
Preferably, the antibody coating concentration of the quality control line is 1.0mg/mL, and the antibody coating volume is 1.5. mu.L.
Meanwhile, the invention provides a preparation method of the colloidal gold immunity test paper for detecting the bacillus larvae of the bees, which specifically adopts the following technical steps:
(1) preparation of colloidal gold labeled PLMP protein polyclonal antibody: sucking 10mL of colloidal gold solution into a centrifuge tube which is well pickled and siliconized, placing the centrifuge tube on a vortex oscillator, adjusting the speed to 1500r/min, and dropwise adding 0.2mol/L of K2CO3150 mu L of the solution is simultaneously shaken and mixed uniformly to adjust the pH value to 9.0, 240 mu g of PLMP protein polyclonal antibody is added drop by drop, shaking and mixing uniformly is carried out at 1500r/min for 10min, then 10 percent BSA is added to ensure that the final concentration is 1 percent, and standing for 30min is carried out after shaking and mixing uniformly; centrifuging the marked colloidal gold antibody solution at 4 ℃ and 1000r/min for 10min, taking supernatant, centrifuging the supernatant at 4 ℃ and 5000r/min for 10min, separating the supernatant from the precipitate, collecting the precipitate, centrifuging the supernatant at 4 ℃ and 10000r/min for 30min, collecting the twice precipitates, namely the purified and marked colloidal gold antibody compound, taking 1/10, the volume of which is the volume of the original unpurified gold-marked antibody solution, as the dosage of the colloidal gold heavy suspension, adding the colloidal gold heavy suspension into the twice collected precipitates, and obtaining the purified gold-marked antibody by heavy suspension precipitation, and storing the gold-marked antibody at 4 ℃.
(2) Preparing a gold label pad: cutting the gold label pad into strips with the size of 0.7cm multiplied by 10cm by using clean scissors, soaking the cut gold label pad in a gold label pad treatment solution for 30min, drying overnight at 4 ℃, uniformly dripping a purified gold label antibody solution on the treated gold label pad, suspending and flatly placing the gold label pad, naturally drying the gold label pad at room temperature to remove excessive moisture, then placing the gold label pad at 4 ℃ for air drying, and sealing and storing the gold label pad.
(3) Preparation of detection line and quality control line: taking two glass sheets which are subjected to acid washing and silicification treatment, sucking a PLMP protein polyclonal antibody and a goat anti-rabbit IgG secondary antibody by a liquid transfer machine, uniformly coating the side surfaces of the glass sheets, printing the side surfaces of the glass sheets on an NC membrane after coating, coating the detection line and the quality control line at an interval of 0.5cm, coating the PLMP protein polyclonal antibody and the goat anti-rabbit IgG secondary antibody on the detection area and the quality control area on a nitrocellulose membrane, and naturally drying at room temperature after coating.
(4) Assembling the test strip: cutting the bottom plate into strips with the width of 3.2cm, firstly pasting the bottom surface of the nitrocellulose membrane on the bottom plate, cutting the gold label pad coated by the antibody into 6.0cm multiplied by 0.7cm pressed nitrocellulose membrane 2mm, flatly pasting the gold label pad coated by the antibody on the bottom plate, pasting the gold label pad coated by the sample pad with the width of 6.0cm multiplied by 1.8cm pressed nitrocellulose membrane 2mm on the bottom plate, pasting the gold label pad coated by the sample pad with the width of 6.0cm multiplied by 1.7cm pressed nitrocellulose membrane 2mm on the bottom plate, pasting the gold label pad and the sample pad by using a joint adhesive tape, compacting each layer, cutting the assembled test paper strip into small strips with the width of 3mm by using scissors, sealing, drying and storing at 4 ℃.
Preferably, the polyclonal antibody against PLMP protein used in the invention is prepared by injecting PLMP protein into immunized rabbit.
Further, the invention also provides an application method of the colloidal gold immunity test paper for detecting the bacillus larvae in the honey, the beeswax, the adult bees and the bacterial liquid, which specifically adopts the following technical steps:
(1) sample pretreatment:
(a) honey: fully dissolving 5g of honey into 40mL of deionized water, sucking 100 mu L of the solution, and dropwise adding the solution onto a test strip sample pad for detection.
(b) Beeswax or adult bees: placing Cera flava or adult bee into the wall-breaking tube, adding appropriate amount of double distilled water, fixing on wall-breaking instrument, and crushing. After disruption, 100. mu.L of the supernatant was collected and examined.
(c) Bacterial liquid: and directly taking 100 mu L of bacterial liquid sample without treatment, and dropwise adding the bacterial liquid sample to a test strip sample pad for detection.
(2) And (3) detecting by using a colloidal gold chromatography test strip:
dripping the liquid onto the sample pad, and allowing to act at room temperature for 10-15 min.
(3) Determination of results
(a) Positive: the quality control line and the detection line both present red bands, which indicates that the bacillus larvae PLMP protein exists in the sample.
(b) Negative: the detection line shows no red band, and the quality control line shows a red band, which indicates that no bacillus larvae PLMP protein exists in the sample.
(c) And (3) failure: and if the detection line and the quality control line do not have a red strip or only the detection line has a red strip, the test strip is invalid.
By implementing the technical scheme of the invention, the following beneficial effects can be achieved:
(1) the colloidal gold immune test paper for detecting the bacillus larvae provided by the invention has strong specificity, no detection condition on negative bacteria liquid, high specificity reaching 100%, high sensitivity, and the lowest detection concentrations of the bacteria liquid of the bacillus larvae and the positive sample being 10 respectively5CFU/mL and 106CFU/mL。
(2) The colloidal gold immune test paper for detecting the bacillus larvae of the bees has good detection repeatability, stable performance, a storage life of 3 months, simple, convenient, rapid, visual and accurate use method, wide application range, low cost and easy popularization and use.
Drawings
FIG. 1 shows a schematic diagram of a colloidal gold immunochromatographic strip for detecting Bacillus larvae in bees.
Fig. 2 is a schematic diagram showing the result judgment of the colloidal gold immunochromatographic test strip in the fifth embodiment.
FIG. 3 shows the test strip specificity test results. Wherein the test strip 1 is a bacillus larvae bacterial liquid, the test strip 2 is a streptococcus alvei bacterial liquid, the test strip 3 is a septicemia melitensis bacterial liquid, the test strip 4 is a bacillus halofoenii bacterial liquid, and the test strip 5 is double distilled water.
Detailed Description
The present invention will be described below by way of examples, but the present invention is not limited to the following examples.
The goat anti-rabbit IgG, the colloidal gold solution and the tetrachloroauric acid adopted by the invention, the filtration sample pad, the colloidal gold pad, the water absorption pad, the nitrocellulose membrane (Millipore135), the PVC base plate, the plastic card and the splicing adhesive tape are purchased from Shanghai Jiening Biotech Co., Ltd; BSA, from Sigma; other reagents are domestic analytical pure reagents which are purchased from conventional sources, and the PLMP protein polyclonal antibody is rabbit antibody serum prepared by injecting PLMP protein into immunized rabbits by adopting conventional technical means in the field.
All materials, reagents and equipment selected for use in the present invention are well known in the art, but do not limit the practice of the invention, and other reagents and equipment well known in the art may be suitable for use in the practice of the following embodiments of the invention.
The first embodiment is as follows: purification of PLMP Multi-antibody
1. Serum pretreatment
6mL of rabbit serum was drawn using a clean syringe and filtered through a 0.45 μm filter.
2. Caprylic acid precipitated non-IgG proteins
A50 mL autoclaved centrifuge tube was added with 24mL of acetic acid buffer and 6mL of rabbit serum filtered through a 0.45 μm filter, shaken and mixed, and the pH was adjusted to 4.5 with 1mol/L NaOH. Adding 2.25mL of n-octanoic acid, stirring at room temperature for 30min, centrifuging at 10000rpm for 20min, and discarding the precipitate. The remaining liquid portion was aspirated by a syringe, filtered through a 0.45 μm filter, added with 1/10 volumes of 0.01mol/L PBS (pH 7.4), adjusted to pH 7.4 with 1mol/L NaOH, and placed in an ice bath for further experiments.
3. Ammonium sulfate precipitation of IgG
And adding saturated ammonium sulfate with the same volume into the solution in an ice bath environment, uniformly stirring and mixing for 30min, and standing for 2 h. Centrifuging at 4 deg.C and 10000rpm for 20min, and discarding supernatant. The remaining precipitate was dissolved in 0.0L mol/L PBS solution. After the pellet had dissolved, it was placed in a dialysis bag and dialyzed against ddH2O for 2h and then against 0.01mol/L PBS at 4 ℃ overnight, during which the dialysate was changed every 4 h. Finally, centrifugation is carried out at 10000rpm for 10min at 4 ℃, and supernatant is collected.
Example two: preparation of gold label pad
1. Determination of optimum pH value of colloidal gold
This test was carried out with the addition of 0.2mol/L K2CO3The amount of (c) is a standard. 10 1.5mL centrifuge tubes were numbered and 1mL of the gold colloid solution was added to each centrifuge tube. Adding a certain amount of 0.2mol/L K into each centrifuge tube respectively2CO3Mix well (as shown in Table 3-2), then add 30. mu.l to each tubeg purified polyclonal antibody, mixing well again, standing at room temperature for 30 min. To each tube slowly add 20 u L10% NaCl solution, fully mixing, and then the room temperature standing for 2 hours. The 10 tubes were observed for color change and the presence of black particles with sediment accumulation. K for selecting the tube which starts to be free of aggregates2CO3The addition amount of the solution is the most suitable 0.2mol/L K for the colloidal gold in the test2CO3The optimum pH value is the addition amount of (1). The values were determined using precision pH paper and are given in Table 1.
Table 1: determination of optimum pH of colloidal gold-labeled antibody
As can be seen by observing the sequence listed in Table 1, the 1 st to 3 rd tubes have the phenomenon of bluing and coagulating to different degrees, the 4 th to 9 th tubes are transparent wine red, the 4 th tube is slightly deeper than the 10 th tube (contrast), which shows that the adsorption of colloidal gold and polyclonal antibody is better, so that the 4 th tube is selected to correspond to 0.2mol/LK2CO3Amount of the compound (A). Detecting with a precise pH test paper, and adding 0.2mol/LK2CO3When the amount of (2) is 20. mu.L, the pH of the colloidal gold solution is 9.0. Therefore, the optimal pH for the colloidal gold label is 9.0.
2. Determination of the optimal amount of antibody labeling
10 autoclaved 1.5mL centrifuge tubes were added to 1.0mL colloidal gold solution per tube. According to the optimum pH of the colloidal gold determined in 3.1.8.1, 5. mu.g, 10. mu.g, 15. mu.g, 20. mu.g, 25. mu.g, 30. mu.g, 35. mu.g, 40. mu.g, and 45. mu.g of PLMP polyclonal antibody (shown in Table 2) was added to each centrifuge tube, mixed well, and allowed to stand at room temperature for 10 min. Slowly adding 10% NaCl 20 μ l into each centrifuge tube, mixing, standing for 30min, and observing color change of each tube. The addition amount of the polyclonal antibody without changing the color of the colloidal gold solution is taken as the minimum dosage of the stable colloidal gold, and on the basis, the polyclonal antibody amount is increased by 20 percent, namely the optimal antibody labeling amount of 1mL of the colloidal gold solution.
Table 2: determination of optimum amount of colloidal gold-labeled antibody
Colloidal gold was adjusted to pH9.0 and diluted with 0.01mol/L PBS (pH 7.4) to 0.2 mg/mL. The tubes with low antibody content appeared to be bluish and coagulated, while the solution in the centrifuge tube remained red when the antibody was added to or above the minimum stable amount. As seen from the sequence listed in Table 2, the antibody amount in the 5 th tube was 25. mu.g/mL as the minimum antibody addition amount, and 20% was further added as the optimum labeling amount for stabilizing the colloidal gold antibody. Therefore, the optimal amount of the standard gold for the antibody is 30. mu.g/mL.
3. Preparation of gold-labeled antibody solution
Sucking 10mL of colloidal gold solution into a centrifuge tube which is well pickled and siliconized, placing the centrifuge tube on a vortex oscillator, adjusting the speed to 1500r/min, and dropwise adding 0.2mol/L of K2CO3150 mu L of the solution is simultaneously shaken and mixed uniformly to adjust the pH value to 9.0, 240 mu g of PLMP protein polyclonal antibody is added drop by drop, shaking and mixing uniformly is carried out at 1500r/min for 10min, then 10 percent BSA is added to ensure that the final concentration is 1 percent, and standing for 30min is carried out after shaking and mixing uniformly; centrifuging the marked colloidal gold antibody solution at 4 ℃ and 1000r/min for 10min, taking supernatant, centrifuging the supernatant at 4 ℃ and 5000r/min for 10min, separating the supernatant from the precipitate, collecting the precipitate, centrifuging the supernatant at 4 ℃ and 10000r/min for 30min, collecting the twice precipitates, namely the purified and marked colloidal gold antibody compound, taking 1/10, the volume of which is the volume of the original unpurified gold-marked antibody solution, as the dosage of the colloidal gold heavy suspension, adding the colloidal gold heavy suspension into the twice collected precipitates, and obtaining the purified gold-marked antibody by heavy suspension precipitation, and storing the gold-marked antibody at 4 ℃.
4. Preparation of gold label pad
Cutting the gold label pad into strips with the size of 0.7cm multiplied by 10cm by using clean scissors, soaking the cut gold label pad in a gold label pad treatment solution for 30min, drying overnight at 4 ℃, uniformly dripping a purified gold label antibody solution on the treated gold label pad, suspending and flatly placing the gold label pad, naturally drying the gold label pad at room temperature to remove excessive moisture, then placing the gold label pad at 4 ℃ for air drying, and sealing and storing the gold label pad.
Example three: preparation of detection line and quality control line
1. Determination of detection line coating antibody concentration
1. mu.L PLMP polyclonal antibody diluted with PBS at concentrations of 0.4mg/mL, 0.6mg/mL, 0.8mg/mL, 1.0mg/mL, 1.2mg/mL, 1.4mg/mL, 1.6 mg/mL and 1.8mg/mL was dropped on the NC membrane of the detection line, and 1.0. mu.L goat anti-rabbit IgG antibody diluted with PBS at a concentration of 1.0mg/mL was dropped on the NC membrane of the quality control line. The detection is carried out by using 10 mu g/mL purified PLMP protein, and the color development of the detection line is observed to determine the optimal concentration of the coating antibody.
When 1.5 mu L of goat anti-rabbit IgG antibody with the concentration of 1mg/mL is coated on the C line, 8 concentration gradients are set for the PLMP protein polyclonal antibody, and the PLMP protein is used for detection, and the color is observed to be lightened when the concentration of the PLMP protein polyclonal antibody is diluted from 1.0mg/mL to 0.4mg/mL, and gradually darkened when the concentration of the PLMP protein polyclonal antibody is from 1.2mg/mL to 1.8 mg/mL. Therefore, the PLMP protein polyclonal antibody with the concentration of 1.2mg/mL is selected as the optimal antibody coating concentration of the T line.
2. Determination of volume of test line coated antibody
On the basis of determining the concentration of the detection line coated antibody, 0.75 muL, 1.0 muL, 1.25 muL, 1.5 muL and 2.0 muL of PLMP multi-antibody are respectively dripped on an NC membrane of the detection line. The detection with PLMP protein shows that 1.5. mu.L of PLMP protein polyclonal antibody is the optimal antibody coating volume of the T line.
3. Determination of optimal coating condition of quality control line
(1) Determination of concentration of quality control line coated antibody
Under the best condition of the determined detection line, goat anti-rabbit IgG antibodies with the concentrations of 0.2mg/mL, 0.4mg/mL, 0.6mg/mL, 0.8mg/mL, 1.0mg/mL, 1.2mg/mL and 1.4mg/mL diluted by PBS are respectively dropped on the NC membrane of the quality control line by 1.0. mu.l. The PLMP protein was used for detection to determine the optimal concentration of coating antibody.
(2) Determination of volume of control line coated antibody
The goat anti-rabbit IgG antibody was coated on the NC membrane of the quality control line in a volume of 0.75. mu.L, 1.0. mu.L, 1.25. mu.L, 1.5. mu.L, 2.0. mu.L, respectively. Detection was performed with PLMP protein and the optimal volume of coated antibody was determined.
The goat anti-rabbit IgG antibody is diluted in proportion and then coated with a C line, and test paper strip detection is carried out, and the result shows that the detection effect is best when the coating concentration of the C line is 1.0mg/mL and the volume of the antibody is 1.5 muL.
4. Pretreatment of nitrocellulose membranes
Taking two glass sheets which are subjected to acid washing and silicification treatment, sucking a PLMP protein polyclonal antibody and a goat anti-rabbit IgG secondary antibody by a liquid transfer machine, uniformly coating the side surfaces of the glass sheets, printing the side surfaces of the glass sheets on an NC membrane after coating, coating the detection line and the quality control line at an interval of 0.5cm, coating the PLMP protein polyclonal antibody and the goat anti-rabbit IgG secondary antibody on the detection area and the quality control area on a nitrocellulose membrane, and naturally drying at room temperature after coating.
Example four: test strip assembly
Cutting the bottom plate into strips with the width of 3.2cm, firstly pasting the bottom surface of the nitrocellulose membrane on the bottom plate, cutting the gold label pad coated by the antibody into 6.0cm multiplied by 0.7cm pressed nitrocellulose membrane 2mm, flatly pasting the gold label pad coated by the antibody on the bottom plate, pasting the sample pad 6.0cm multiplied by 1.8cm pressed gold label pad 2mm on the bottom plate, pasting the water absorption pad 6.0cm multiplied by 1.7cm pressed nitrocellulose membrane 2mm on the bottom plate, sticking the joint of the gold label pad and the sample pad by a linking adhesive tape, pressing each layer tightly, cutting the assembled test paper strip into small strips with the width of 3mm by scissors, sealing, drying, storing at 4 ℃, and storing the colloidal gold immunochromatography test paper strip pattern as shown in figure 1.
Example five: application method of colloidal gold immune test paper for detecting bacillus larvae of bees
(1) Sample pretreatment:
(a) honey: fully dissolving 5g of honey into 40mL of deionized water, sucking 100 mu L of the solution, and dropwise adding the solution onto a test strip sample pad for detection.
(b) Beeswax or adult bees: placing Cera flava or adult bee into the wall-breaking tube, adding appropriate amount of double distilled water, fixing on wall-breaking instrument, and crushing. After disruption, 100. mu.L of the supernatant was collected and examined.
(c) Bacterial liquid: and directly taking 100 mu L of bacterial liquid sample without treatment, and dropwise adding the bacterial liquid sample to a test strip sample pad for detection.
(2) And (3) detecting by using a colloidal gold chromatography test strip:
dripping the liquid onto the sample pad, and allowing to act at room temperature for 10-15 min.
(3) Determination of results
(a) Positive: the quality control line and the detection line both present red bands, which indicates that the bacillus larvae PLMP protein exists in the sample.
(b) Negative: the detection line shows no red band, and the quality control line shows a red band, which indicates that no bacillus larvae PLMP protein exists in the sample.
(c) And (3) failure: and if the detection line and the quality control line do not have a red strip or only the detection line has a red strip, the test strip is invalid. The result of the colloidal gold immunochromatographic test strip shown in the attached figure 2 is judged.
Example six: testing of test strip Performance
1. Test strip specificity test
Dripping 100 μ L of Streptococcus nidulans solution, herba Patriniae solution, Enterobacter Hafnensis solution, and double distilled water onto the sample adding pad of the colloidal gold test strip, and collecting the test strip with the result of L0 min as shown in figure 3, wherein the test strip for detecting Bacillus larvae positive bacteria solution sample has red line on T line and C line, and the result is correct; all negative samples only appeared red on the line C of the quality control line, and the result was correct.
2. Test strip sensitivity test
Using colloidal gold test paper prepared in the same batch to make the larval bacillus liquid into a test paper strip according to the ratio of 108CFU/mL、107CFU/mL、106CFU/mL、105CFU/mL、104CFU/mL、 103And (3) detecting after CFU/mL dilution, and observing that no red line appears on the test strip T line No. 1-2 within 10-15min and red appears on the test strip T line No. 3-6 within 10-15 min. Thereby determining that the test strip has a PLMP protein detection limit of 105CFU/mL, can be used for detecting Bacillus larvae.
3. Test strip stability test
Detecting the positive bacteria liquid of the larval bacillus by using a test strip stored at 4 ℃ and room temperature, and performing data arrangement on 7d, 15d, 30d, 60d and 90d respectively, wherein the results are shown in table 3, and the test strip can be stored for 3 months when being stored at 4 ℃; storing at room temperature for at least 1 month.
Table 3: test strip stability test results
Note: "+ ++" indicates that the color development is obvious and the bands are clear; "+ + + +" indicates that the color development is clear, the band is clear but the color is slightly lighter; the color of the T line is lightened by "+ +" indicating that the detection result is accurate; "+" indicates that T, C lines were all lighter in color, but that the test samples were positive and negative.
4. Test strip repeatability test
And (3) respectively detecting positive and negative bacteria liquid of the bacillus larvae by using test strips of different batches, and performing batch repeatability tests. In addition, the bacterial liquid is detected by using test strips of the same batch, and an in-batch repeatability test is carried out. The obtained results are consistent, and the repeatability is better.
5. Detection of clinical samples
In the test, 40 parts of bee larvae, 52 parts of honey, 22 parts of beeswax and 243 adult bees collected from 10 bee fields in Xinyuan county of Ili are detected by using a PCR method in SN/T1681-.
Table 4: clinical sample test results
| Detection method | Positive sample | Negative sample | |
| PCR | |||
| 0 | 357 | 0 | |
| Colloidal |
0 | 357 | 0 |
6. Detection of artificially simulated contaminated samples
Respectively taking 1mL of honey diluted by double distilled water, ground larva and beeswax, and mixing with 1mL of 109CFU/mL、108CFU/mL、107CFU/mL、106CFU/mL、 105And (3) fully mixing the CFU/mL of the bacillus larvae liquid to prepare an artificial simulated pollution sample. The colloidal gold test strip prepared in the research is used for detecting the samples, the colloidal gold test strip prepared in the research is used for respectively adding the larval bacillus bacteria liquid with different concentrations after treatment to carry out artificial simulation pollution sample detection, and the result shows that the concentration of the minimum detected larval bacillus in honey is 106CFU/mL, the lowest detected larval bacillus concentration of bee larva is 105CFU/mL, the lowest detected Bacillus larvae concentration of beeswax is 105CFU/mL。
In conclusion, the colloidal gold immune test paper for detecting the bacillus larvae provided by the invention has strong specificity, no detection condition on negative bacteria liquid, high specificity reaching 100 percent and high sensitivity, and the minimum detection concentrations of the bacteria liquid of the bacillus larvae and the positive sample are respectively 105CFU/mL and 106CFU/mL; the colloidal gold immunity test paper has good detection repeatability, stable performance, long storage life up to 3 months, and simple and convenient use methodThe method is rapid, visual and accurate, has wide application range, low cost and easy popularization and use.
As described above, the present invention can be preferably implemented, and the above-mentioned embodiments only describe the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various changes and modifications of the technical solution of the present invention made by those skilled in the art without departing from the design spirit of the present invention shall fall within the protection scope defined by the present invention.
Claims (9)
1. The colloidal gold immune test paper for detecting the bacillus larvae in the bees is characterized by specifically comprising a sample pad, a gold-labeled pad, a nitrocellulose membrane, a water absorption pad and a bottom plate, wherein the gold-labeled pad contains a PLMP protein polyclonal antibody labeled by colloidal gold.
2. The colloidal gold immunoassay strip for detecting bacillus larvae in claim 1, wherein the nitrocellulose membrane has a detection line pre-coated with PLMP protein polyclonal antibody and a quality control line pre-coated with goat anti-rabbit IgG solution.
3. The colloidal gold immunoassay strip for detecting bacillus larvae in claim 1, wherein the amount of gold labeled with the PLMP protein polyclonal antibody is 30 μ g/mL.
4. The colloidal gold immunoassay strip for detecting bacillus larvae in claim 2, wherein said test line has an antibody coating concentration of 1.2mg/mL and an antibody coating volume of 1.5 μ L.
5. The colloidal gold immunoassay strip for detecting Bacillus larvae in claim 2, wherein the antibody coating concentration of the quality control line is 1.0mg/mL and the antibody coating volume is 1.5. mu.L.
6. The method for preparing colloidal gold immunoassay paper for detecting Bacillus larvae in claim 1, wherein the following steps are adopted:
(1) preparation of colloidal gold labeled PLMP protein polyclonal antibody: sucking 10mL of colloidal gold solution into a centrifuge tube which is well pickled and siliconized, placing the centrifuge tube on a vortex oscillator, adjusting the speed to 1500r/min, and dropwise adding 0.2mol/L of K2CO3150 mu L of the solution is simultaneously shaken and mixed uniformly to adjust the pH value to 9.0, 240 mu g of PLMP protein polyclonal antibody is added drop by drop, shaking and mixing are carried out at 1500r/min for 10min, then 10 percent BSA is added to ensure that the final concentration is 1 percent, and standing is carried out for 30min after shaking and mixing are carried out; centrifuging the marked colloidal gold antibody solution at 4 ℃ and 1000r/min for 10min, taking supernatant, centrifuging the supernatant at 4 ℃ and 5000r/min for 10min, separating the supernatant from the precipitate, collecting the precipitate, centrifuging the supernatant at 4 ℃ and 10000r/min for 30min, collecting the twice precipitates, namely the purified and marked colloidal gold antibody compound, taking 1/10, the volume of which is the volume of the original unpurified gold-marked antibody solution, as the dosage of the colloidal gold heavy suspension, adding the colloidal gold heavy suspension into the twice collected precipitates, and obtaining the purified gold-marked antibody by heavy suspension precipitation, and storing the gold-marked antibody at 4 ℃;
(2) preparing a gold label pad: cutting the gold label pad into strips with the size of 0.7cm multiplied by 10cm by using clean scissors, soaking the cut gold label pad in a gold label pad treatment solution for 30min, drying overnight at 4 ℃, uniformly dripping a purified gold label antibody solution on the treated gold label pad, suspending and flatly placing the gold label pad in the air, naturally drying the gold label pad at room temperature to remove redundant moisture, then placing the gold label pad at 4 ℃ for air drying, and sealing and storing the gold label pad;
(3) preparation of detection line and quality control line: taking two glass sheets which are subjected to acid washing and silicification treatment, sucking a PLMP protein polyclonal antibody and a goat anti-rabbit IgG secondary antibody by a pipettor, uniformly coating the side surfaces of the glass sheets, printing the side surfaces of the glass sheets on an NC membrane after coating, coating the detection line and the quality control line at an interval of 0.5cm, coating the PLMP protein polyclonal antibody and the goat anti-rabbit IgG secondary antibody on the detection line and the quality control line on a nitrocellulose membrane, and naturally drying at room temperature after coating;
(4) assembling the test strip: cutting the bottom plate into strips with the width of 3.2cm, firstly pasting the bottom surface of the nitrocellulose membrane on the bottom plate, cutting the gold label pad coated by the antibody into 6.0cm multiplied by 0.7cm pressed nitrocellulose membrane 2mm, flatly pasting the gold label pad coated by the antibody on the bottom plate, pasting the gold label pad coated by the sample pad with the width of 6.0cm multiplied by 1.8cm pressed nitrocellulose membrane 2mm on the bottom plate, pasting the gold label pad coated by the sample pad with the width of 6.0cm multiplied by 1.7cm pressed nitrocellulose membrane 2mm on the bottom plate, pasting the gold label pad and the sample pad by using a joint adhesive tape, compacting each layer, cutting the assembled test paper strip into small strips with the width of 3mm by using scissors, sealing, drying and storing at 4 ℃.
7. The method of claim 6, wherein the PLMP protein polyclonal antibody is prepared from rabbit immunized by injecting PLMP protein.
8. The use of the colloidal gold immunoassay strip of claim 1 for detecting Bacillus larvae in honey, bees wax, adult bees, and bacterial solutions.
9. The application of the colloidal gold immunity test paper for detecting the bacillus larvae in the honey, the beeswax, the adult bees and the bacillus larvae in the bacterial liquid according to claim 8, which specifically adopts the following technical steps:
(1) sample pretreatment:
(a) honey: fully dissolving 5g of honey into 40mL of deionized water, sucking 100 mu L of solution, dropwise adding the solution onto a test strip sample pad, and detecting;
(b) beeswax or adult bees: putting beeswax or adult bee into a wall breaking tube, adding appropriate double distilled water, fixing on a wall breaking instrument, breaking, and collecting supernatant 100 μ L for detection;
(c) bacterial liquid: directly taking 100 mu L of a bacterial liquid sample without treatment, and dropwise adding the bacterial liquid sample to a test strip sample pad for detection;
(2) and (3) detecting by using a colloidal gold chromatography test strip:
dripping the liquid onto the sample pad, and allowing to act at room temperature for 10-15 min;
(3) and (4) judging a result:
(a) positive: the quality control line and the detection line both present red strips, which indicates that the bacillus larvae PLMP protein exists in the sample;
(b) negative: the detection line shows no red strip, and the quality control line shows a red strip, which indicates that no bacillus larvae PLMP protein exists in the sample;
(c) and (3) failure: and if the detection line and the quality control line do not have a red strip or only the detection line has a red strip, the test strip is invalid.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103412121A (en) * | 2013-07-20 | 2013-11-27 | 福建农林大学 | Colloidal gold immune test strip for rapid detection of Papaya ringspot virus |
| CN106872697A (en) * | 2017-02-10 | 2017-06-20 | 江西农业大学 | Apis cerana sac brood virus immunity colloidal gold strip |
| CN107012251A (en) * | 2017-05-19 | 2017-08-04 | 福建出入境检验检疫局检验检疫技术中心 | Bee larva bacillus fluorescent quantificationally PCR detecting kit and its application |
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|---|---|---|---|---|
| CN103412121A (en) * | 2013-07-20 | 2013-11-27 | 福建农林大学 | Colloidal gold immune test strip for rapid detection of Papaya ringspot virus |
| CN106872697A (en) * | 2017-02-10 | 2017-06-20 | 江西农业大学 | Apis cerana sac brood virus immunity colloidal gold strip |
| CN107012251A (en) * | 2017-05-19 | 2017-08-04 | 福建出入境检验检疫局检验检疫技术中心 | Bee larva bacillus fluorescent quantificationally PCR detecting kit and its application |
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