Colloidal gold immune test strip for mycoplasma bovis nucleic acid detection and application thereof
阅读说明：本技术 一种牛支原体核酸检测胶体金免疫试纸条及其应用 (Colloidal gold immune test strip for mycoplasma bovis nucleic acid detection and application thereof ) 是由 陈创夫 王震 石峰 王勇 赵阳 宋胜男 吴鹏 于 2021-09-14 设计创作，主要内容包括：本发明属于免疫学检测技术领域,尤其涉及一种牛支原体核酸检测胶体金免疫试纸条及其应用。本申请利用胶体金可以和抗体或生物素等标记的寡核苷酸相结合这一信号放大技术,提高检测灵敏度,构成可显色的检测探针,整合了PCR的特异性、敏感性和金标条快速、简单的特性,建立了M.bovis核酸检测的胶体金免疫试检测纸条,该方法的建立为临床微量牛支原体病原的检测研究提供了研究基础。(The invention belongs to the technical field of immunological detection, and particularly relates to a mycoplasma bovis nucleic acid detection colloidal gold immune test strip and application thereof. The method utilizes a signal amplification technology that colloidal gold can be combined with labeled oligonucleotides such as antibodies or biotin and the like, improves the detection sensitivity, forms a detection probe capable of developing color, integrates the specificity and sensitivity of PCR and the characteristics of rapidness and simplicity of a gold label strip, establishes a colloidal gold immune test detection paper strip for M.bovis nucleic acid detection, and provides a research basis for the detection research of clinical trace mycoplasma bovis pathogens.)
1. A mycoplasma bovis nucleic acid detection colloidal gold immune test strip is characterized in that: from the bottom up, from left to right include PVC board, NC membrane in proper order, go up appearance pad, gold mark pad, sample pad, absorb water and fill up, closely laminate each other and constitute the test paper strip, draw T, C lines with streptavidin and goat anti mouse IgG respectively on the NC membrane, with the coating of gold mark mouse anti digoxin IgG on colloidal gold pad, sample pad and absorb water pad paste respectively in the both ends of test paper strip, are fixed in the plastic casing with the test paper strip.
2. The mycoplasma bovis nucleic acid detection colloidal gold immune test strip of claim 1, wherein: a line T, C was drawn on the NC film using a scriber at a rate of 1. mu.L/cm.
3. The mycoplasma bovis nucleic acid detection colloidal gold immune test strip of claim 2, wherein: the streptavidin concentration used was 2 mg/mL.
4. The mycoplasma bovis nucleic acid detection colloidal gold immune test strip of claim 2, wherein: the dilution ratio of goat anti-mouse IgG used was 1: 100.
5. The mycoplasma bovis nucleic acid detection colloidal gold immune test strip of claim 1, wherein: the colloidal gold solution was prepared at pH 8.5 for the gold-labeled murine anti-digoxin IgG.
6. The use of the colloidal gold immunoassay strip according to claim 1 for detecting mycoplasma bovis nucleic acid in mycoplasma bovis nucleic acid detection.
7. The application of the colloidal gold immune test strip for detecting mycoplasma bovis nucleic acid according to claim 6, wherein the colloidal gold immune test strip is characterized in that: and (3) carrying out PCR reaction by taking the target sample genome DNA as a template, dripping the product on a test paper strip sample pad, and reading the result after 5 min.
8. The application of the colloidal gold immune test strip for detecting mycoplasma bovis nucleic acid according to claim 7 in mycoplasma bovis nucleic acid detection is characterized in that: the sequences of the upstream and downstream primers used for PCR amplification are respectively shown as SEQ ID NO.1 and SEQ ID NO.2, and the 5' ends of the upstream and downstream primers are respectively designed with a labeled biotin probe and a digoxin probe.
Technical Field
The invention belongs to the technical field of immunological detection, and particularly relates to a mycoplasma bovis nucleic acid detection colloidal gold immune test strip and application thereof.
Background
Mycoplasma bovis (m.bovis) is a new important pathogen microorganism that has a great and overlooked impact on cattle farming in the world at present, and can cause various mixed infection chronic diseases such as pneumonia, mastitis, otitis media, reproductive disorders, arthritis and the like of cattle. At present, the research on pathogenic mechanisms and virulence factors of mycoplasma bovis diseases is still in the primary stage, specific drugs, vaccines and rapid diagnosis methods are lacked, and the prevalence of m.bovis diseases in China causes huge economic loss, which becomes a major obstacle for the healthy development of the cattle industry, and the development of efficient, rapid and specific detection methods is increasingly important for enhancing the immunity of mycoplasma bovis and diagnosing and monitoring mycoplasma bovis.
With the increasing maturity of modern biological technology, the current detection method has been mainly changed from the separation and culture of the initial pathogenic strain to the current serological and molecular biological methods. The body produces immune-responsive antibodies after infection of cattle with m.bovis, and this response usually persists for some time after infection, so serological testing is an effective m.bovis diagnostic method. The ELISA is commonly applied in serological detection, the membrane surface variable protein is a main antigen recognized by host body fluid response in the M.bovis infection process, and indirect ELISA based on recombinant proteins coated by VspA, VspB and VspC antigens has higher specificity and sensitivity. With the rapid development of biological technology, the fluorescence quantitative PCR loop-mediated isothermal amplification technology is also widely applied to the detection of M.bovis. Researchers develop a SYBR Green real-time fluorescent PCR detection kit based on M.bovis genes such as uvrC and P81, and the kit has no cross reactivity with other related bacteria and other mycoplasma, good specificity and high sensitivity. In addition, the loop-mediated isothermal amplification technology detection based on the uvrC and P81 genes is successfully used for detecting clinical samples, and the detection method has the characteristics of high sensitivity, strong specificity, rapid and accurate detection and the like. Rapid diagnosis contributes to effective prevention and control of m.bovis-related diseases. The isolation culture method is a gold standard for pathogen detection, but has a long period and is easy to pollute. Serological tests may present common antigens between different mycoplasma and may lead to cross-reactivity, resulting in false positives. The PCR method has high requirements on the operation level of detection personnel and is greatly limited by instruments and equipment.
The biotin-avidin system (Bio-streptavidin, BAS) is the most powerful affinity material currently found in nature, with an affinity at least one million times greater than antigen-antibody binding. The biotin system is widely applied to various detection technologies, is suitable for detecting bacteria, viruses, cells, proteins and nucleic acids, can effectively improve the bioactivity of target corresponding ligand molecules, can obviously improve the binding efficiency and the target molecules, has cascade reaction with detection signals, effectively improves the detection sensitivity, shortens the detection time, realizes quick and sensitive detection and analysis of biomolecules, and brings a new idea for detection of biological and medical researches. The biotin system is gradually becoming an emerging enzyme-linked assay similar to ELISA.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a mycoplasma bovis nucleic acid detection colloidal gold immune test strip and application thereof. It is an object to solve at least some of the problems in the prior art or to at least alleviate some of the problems in the prior art.
The test paper strip comprises a PVC plate, an NC membrane, a sample loading pad, a gold label pad, a sample pad and a water absorption pad which are tightly attached to each other from bottom to top and from left to right to form the test paper strip, wherein the streptavidin and the goat anti-mouse IgG are respectively marked with T, C lines on the NC membrane, the gold-labeled mouse anti-digoxin IgG is coated on the colloidal gold pad, the sample pad and the water absorption pad are respectively attached to two ends of the test paper strip, and the test paper strip is fixed in a plastic shell.
Further, T, C lines were drawn on the NC film using a film drawing instrument in an amount of 1. mu.L/cm.
Further, the streptavidin concentration used was 2 mg/mL.
Further, the dilution ratio of goat anti-mouse IgG used was 1: 100.
Further, the gold-labeled mouse anti-digoxin IgG was prepared at a pH of 8.5 for the colloidal gold solution.
The invention also provides application of the mycoplasma bovis nucleic acid detection colloidal gold immune test strip in mycoplasma bovis nucleic acid detection.
Further, PCR reaction is carried out by taking target sample genome DNA as a template, a product is dripped on a test paper strip loading pad, and a result is read after 5 min.
Furthermore, the sequences of the upstream and downstream primers used for PCR amplification are respectively shown as SEQ ID NO.1 and SEQ ID NO.2, and the 5' ends of the upstream and downstream primers are respectively designed with a labeled biotin probe and a digoxin probe.
The invention aims to provide a rapid and accurate detection and diagnosis technology for mycoplasma bovis basement in field for veterinary clinic, and establishes a colloidal gold immunoassay test strip for M.bovis nucleic acid detection by combining colloidal gold and labeled oligonucleotides such as antibodies or biotin and the like with a signal amplification technology, thereby providing a research basis for clinical detection and research of trace mycoplasma bovis pathogens.
In summary, the advantages and positive effects of the invention are:
the invention designs M.bovis specific primers for marking a streptavidin probe and a digoxin probe, prepares a colloidal gold-marked anti-digoxin monoclonal antibody coated gold-marked pad, lines a T line and a C line for marking streptavidin and goat anti-mouse IgG respectively, assembles a universal colloidal gold immunoassay test strip, integrates a specific and high-sensitivity PCR detection technology and a quick and simple gold-marked test strip technology, indirectly amplifies a detection signal of the M.bovis through a biotin-avidin system, and establishes the colloidal gold immunoassay test strip for detecting M.bovis nucleic acid.
The research utilizes the signal amplification technology that colloidal gold can be combined with labeled oligonucleotides such as antibodies or biotin and the like to improve the detection sensitivityThe characteristic of a chromogenic detection probe is integrated with the specificity and sensitivity of PCR and the characteristic of quick and simple gold label strip, so that the detection can be quickly finished within 2h, the specificity is good, the detection has no cross reaction with other similar pathogenic microorganisms except only producing positive results on M.bovis PG45 and M.bovis Sinkiang isolates, the sensitivity is good, the lower limit of detection is 89 fg/. mu.L of DNA, and the sensitivity is 10-6And the sensitivity of the common agarose gel electrophoresis is generally 10-5The colloidal gold immunochromatography technology is closely combined with a biotin probe in a PCR product by utilizing the amplifiable characteristic of colloidal gold and the extremely strong affinity of a streptavidin-digoxin system, so that the colloidal gold immunochromatography technology is more sensitive and faster, the operation is greatly simplified, the time is saved, and the reaction efficiency is improved.
Drawings
FIG. 1 is a primer specificity test; m: DNA Marker; 1: m.bovis PG 45; 2: bovis Sinkiang isolate; 3: mycoplasma ovipneumoniae; 4: e.coli; 5: pasteurella multocida; 6: b, breeding Brucella melitensis; 7: bovine viral diarrhea virus; 8: mycobacterium bovis; 9: blank control;
FIG. 2 illustrates the assembly and principle of the test strip;
FIG. 3 shows the specificity of the nucleic acid test strip; 1: mycoplasma ovipneumoniae; 2: e.coli; 3: pasteurella multocida; 4: b, breeding Brucella melitensis; 5: bovine viral diarrhea virus; 6: mycobacterium bovis; 7: bovis Sinkiang isolate; 8: bovis PG 45.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the equipment and reagents used in the examples and test examples are commercially available without specific reference. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.
Various modifications to the precise description of the invention will be readily apparent to those skilled in the art from the information contained herein without departing from the spirit and scope of the appended claims. It is to be understood that the scope of the invention is not limited to the procedures, properties, or components defined, as these embodiments, as well as others described, are intended to be merely illustrative of particular aspects of the invention. Indeed, various modifications of the embodiments of the invention which are obvious to those skilled in the art or related fields are intended to be covered by the scope of the appended claims.
For a better understanding of the invention, and not as a limitation on the scope thereof, all numbers expressing quantities, percentages, and other numerical values used in this application are to be understood as being modified in all instances by the term "about". Accordingly, unless expressly indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. In the present invention, "about" means within 10%, preferably within 5% of a given value or range.
In the following examples of the present invention, the temperature is not particularly limited, and all of the conditions are normal temperature conditions. The normal temperature refers to the natural room temperature condition in four seasons, no additional cooling or heating treatment is carried out, and the normal temperature is generally controlled to be 10-30 ℃, preferably 15-25 ℃.
The genes, proteins or fragments thereof involved in the present invention may be naturally purified products, or chemically synthesized products, or produced from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, plants) using recombinant techniques.
The invention discloses a mycoplasma bovis nucleic acid detection colloidal gold immune test strip and application thereof. The bovine mycobacterium tuberculosis positive serum, the pasteurella multocida positive serum, the escherichia coli positive serum, the bovine viral diarrhea virus positive serum, the bovine brucella positive serum and the M.bovis positive serum which are related in the invention are all provided by professor Chen Chufu of institute of animal science and technology, and the mycoplasma pneumoniae positive serum of sheep is provided by professor Shifeng of institute of life science; new Zealand white rabbits were purchased from the Experimental animals center of the university of Stone river. Mouse anti-digoxin IgG monoclonal antibody, goat anti-mouse IgG were purchased from Sigma usa. The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.
Example 1
1. Primer specificity verification
The primer sequences involved in the present application are as follows, and the 5' ends of the upstream and downstream primers are designed with a labeled biotin probe and a digoxin probe, respectively.
TABLE 1 primer sequences
The total reaction system of PCR is shown in Table 2, and the reaction condition is pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 53 ℃ for 30s, and extension at 72 ℃ for 45s for 30 cycles; final extension at 72 ℃ for 10 min. Respectively amplifying 8 test strain templates by utilizing an established PCR reaction system and reaction conditions, mixing 10 mu L of PCR products with 1 mu L of 10 multiplied Loading Buffer, adding 1 mu L of EB (Epstein-Barr) glue in 2% agarose gel (0.9g +45mL of distilled water) for natural drying, carrying out electrophoretic analysis with the voltage of 120V and the time of 30min, observing an amplification result by imaging of a gel imager, and verifying a specific primer for subsequent tests.
TABLE 2 PCR reaction System (25. mu.L)
PCR amplification is carried out according to the reaction system and the reaction conditions, the blank control takes sterile ultrapure water as a template, the result is shown in figure 1, DNA of M.bovis PG45 and M.bovis Sinkiang isolates is amplified to obtain a target band with the size of 448bp, and amplified bands are not detected in DNA amplification products of the blank control and other pathogenic strains, so that the primer has strong specificity and good reaction system and reaction conditions.
2. Search for optimal conditions for gold-labeled murine anti-digoxin IgG
10 clean centrifuge tubes of 1.5mL are divided into two groups and fixed on a supporting plate, 1 mL/tube, the prepared colloidal gold solution is placed in the centrifuge tubes, and 0.1mol/L potassium carbonate (K) is slowly dripped2CO3) The solution is quickly and uniformly mixed while dropwise adding, the pH is respectively adjusted to 7.0, 7.5, 8.0, 8.5 and 9.0, and each pH has two groups with the same pH; diluting the mouse anti-digoxin IgG concentration to 1mg/mL, 2mg/mL, 3mg/mL, 4mg/mL and 5mg/mL by using a 1 XPBS (pH 7.4) buffer solution, dropwise adding the diluted mouse anti-digoxin IgG concentration into a colloidal gold solution, quickly shaking the two groups of solutions for 5min, and standing the uniformly mixed solution for 1h at the temperature of 4 ℃; finally, 10% NaCl solution and 100 mu L/hole are respectively added, fully and uniformly mixed, then the mixture is kept stand for 1h at 4 ℃, the change condition of the solution is observed, and simultaneously, an enzyme-labeling instrument is utilized to read OD450nmValue, select OD450nmThe pH value corresponding to the highest value and the amount of the labeled antibody are used as the optimal conditions for labeling the antibody with the colloidal gold.
After the optimal pH and the optimal labeling amount are determined, centrifuging at 2000r/min for 20min, reserving the supernatant, centrifuging at 14000r/min for 1h at 4 ℃, reserving the supernatant, dropwise adding 2% BSA solution according to the proportion of 1:10, uniformly mixing, and standing for 30min again at 4 ℃; centrifuging at 4 ℃ for 15min at 2000r/min, reserving supernatant into a clean centrifugal tube, and removing unmarked colloidal gold precipitate; centrifuging the supernatant at 4 deg.C for 30min at 10000r/min, discarding the supernatant, retaining the labeled colloidal gold particles, and packaging at 4 deg.C.
The results showed that when the protein concentration was small and insufficient to stabilize the colloidal gold solution, the color changed from red to blue to cause coagulation, and when the amount of protein added reached or exceeded the minimum stabilizing amount, the minimum protein amount at which the red color remained unchanged was the optimum protein amount, and the OD was measured450nmThe value is obtained. The light absorption value is maximum when the pH value is 8.5, which shows that the connection effect with the colloidal gold is optimal, the negatively charged colloidal gold can be stably combined with the positively charged protein through physical properties under the condition of alkalescence, and the light absorption value of the colloidal gold solution is constant under the condition of certain pH valueThe OD was increased gradually with the increase of the amount of protein, when 4mg/mL of mouse anti-digoxin IgG was added450nmThe value was significantly increased, and the absorbance was gradually decreased with the continuous increase, and therefore, it was best when the pH of the colloidal gold solution was 8.5 and the mouse anti-digoxin IgG label was added at 4 mg/mL.
3. Search for optimal dilution factor of streptavidin, gold-labeled mouse anti-digoxin IgG and goat anti-mouse IgG
Streptavidin was coated on the T-line (detection line), diluted to 0.5mg/mL, 1mg/mL, 2mg/mL, and 4mg/mL with 1 XPBS (pH 7.4) buffer, goat anti-mouse IgG was coated on the C-line (quality control line), diluted 100-fold, and C, T lines were drawn on the NC membrane at 1. mu.L/cm with a drawing instrument, and dried overnight at room temperature.
Diluting the colloidal gold labeled mouse anti-digoxin IgG by 3, 5 and 8 times with 1 XPBS (pH 7.4) buffer solution, uniformly coating 30 mu L/cm on a gold label pad, drying overnight at room temperature, combining all parts into a test strip the next day, diluting 10 times, then dropping 100 mu L of negative and positive PCR products and 1 XPBS (pH 7.4)100 mu L on the test strip, analyzing the characteristics of color intensity, stability, developing time and the like of the detection line under different conditions according to the developing result, and selecting the optimal dilution times of streptavidin and the gold labeled mouse anti-digoxin IgG.
On the basis of the above determination conditions, goat anti-mouse IgG is diluted by 40, 80, 100 and 200 times with 1 XPBS (pH 7.4) buffer solution respectively, the characteristics of color intensity, stability, color development time and the like of the detection line are analyzed according to color development results under different conditions, the conditions that the color intensities of the T line and the C line are kept consistent are selected, and the optimal dilution degree of the polyclonal antibody is determined.
The results show that the color development intensity of the C line is weaker and weaker as the dilution factor of the gold-labeled mouse anti-digoxin IgG is increased, the color development intensity of the T line is stronger and stronger as the streptavidin is added, and the best results are determined according to the color development condition of the C, T line until the gold-labeled mouse anti-digoxin IgG is diluted to 5 times and the concentration of the streptavidin is 2mg/mL, and the results are shown in the following Table 3.
TABLE 3 color development results for different dilution factor of streptavidin concentration and gold-labeled murine anti-digoxin IgG
When the concentration of goat anti-mouse IgG is higher, the color intensity of the quality control line is obviously higher than that of the detection lines, when the dilution multiple is 100, the two detection lines are clearly visible, the color intensities are kept consistent, and the result is shown in table 4, so that the optimal dilution ratio of goat anti-mouse IgG 1:100 is realized.
TABLE 4 color development results of different dilution factor of goat anti-mouse IgG
4. Assembling, detecting and result judging of nucleic acid test paper strip
The nucleic acid test strip consists of five parts, namely a PVC plate, a nitrocellulose membrane, a sample loading pad, a gold label pad, a sample pad and a water absorption pad which are tightly attached from bottom to top and from left to right in sequence, wherein a striping instrument scribes T, C lines on an NC membrane according to streptavidin of 1 mu L/cm and goat anti-mouse IgG, the gold label mouse anti-digoxin IgG is evenly coated on the colloidal gold pad and is tightly attached on the PVC plate after being dried overnight at 37 ℃ so as to avoid the influence of bubbles on the chromatographic flow of the sample, the sample pad and the water absorption pad are respectively attached to two ends of the test strip, a strip cutting machine is started to cut the test strip into test strips of 60mm multiplied by 3.7mm, the test strips are fixed in a plastic shell and are sealed, protected from light and are stored for standby at room temperature, and the schematic diagram is shown in figure 2.
And (3) diluting the PCR product, and then dropwise adding the diluted PCR product onto a nucleic acid test strip, and observing whether a red strip is generated or not and a plurality of red strips are generated within about 5 min. Judging the result, wherein clear visible red strips appear in the quality control band and the detection band, and the quality control band and the detection band are positive; only the quality control band appears clear and visible red bands which are negative; a red strip with clear and visible color appears in the quality control band, and a light color band appears in the detection band as a question; the absence of a red band in the control band is ineffective.
5. Specificity test
Carrying out PCR reaction by using genome DNA of mycobacterium bovis, pasteurella multocida, escherichia coli, mycoplasma filiformis, bovine viral diarrhea virus, bovine brucella, mycoplasma ovipneumoniae, M.bovis PG45 and M.bovis Sinkiang isolates as templates, dripping products on a sample pad of a test strip, reading the result after 5min, and simultaneously carrying out agarose gel electrophoresis detection for specificity test.
The results show that: the DNA of mycobacterium bovis, pasteurella multocida, escherichia coli, bovine viral diarrhea virus, bovine brucella, mycoplasma ovipneumoniae, M.bovis PG45 and M.bovis Sinkiang isolates is used as a template to be added into a constructed PCR reaction system, an amplification product is dripped onto a nucleic acid detection test strip, the result is shown in figure 3, the test strip can detect all M.bovis strains in the test strains, and has no cross reaction with other pathogenic bacteria, so that the test strip has good specificity.
6. Sensitivity test
Carrying out 10-fold gradient dilution on the DNA of the M.bovis by using deionized water, taking serial diluent as an amplification template, carrying out a test according to a reaction system, respectively dripping PCR products on sample pads of the same batch of test strips, observing results, and carrying out a sensitivity test.
The results show that: the DNA concentration of extracted M.bovis is 90 ng/. mu.L, and the dilution is 10-1、10-2、10-3、10-4、10-5、10-6、10-7Performing PCR amplification as a template after the amplification, dripping a PCR product after 10-fold dilution on a nucleic acid test strip, observing the detection result after 5min, wherein the test strip has the detection lower limit of 89 fg/mu L DNA for detecting M.bovis DNA and the sensitivity of 10-6。
TABLE 5 sensitivity test of the nucleic acid test strips
Note: + indicates a positive result; -indicating a negative result
7. Test for repeatability and stability
Three different PCR products were randomly diluted 0-fold and 10-fold-210 times of-410 times of-6And (5) carrying out a repeatability test by using the colloidal gold test strip prepared in the same batch, observing the color development condition, and carrying out the repeatability test.
Randomly extracting 36 colloidal gold test strips from three groups of test strips in different batches prepared according to optimized optimal conditions, wherein the first group is the test strips stored at room temperature for 3 months; the second group is test paper stored at room temperature for 6 months, the third group is test paper stored at room temperature for 12 months, and the three groups are verified in parallel and diluted by 0 time and 10 times-210 times of-410 times of-6And performing a stability verification test on the same PCR product, observing the color development condition, and performing a stability test.
The results show that: randomly taking three different PCR products, and respectively diluting by 0 time and 10 times-210 times of-410 times of-6And (2) respectively dropwise adding the complex reagent solution to different prepared colloidal gold test strips in the same batch to perform a repeatability test, observing the color development condition, and obtaining results shown in table 6.
TABLE 6 repeatability test and color development results of nucleic acid test paper
Randomly taking the same PCR product and diluting and releasing by 0 time and 10 times respectively-210 times of-410 times of-6And (2) dropwise adding the test paper strips to different batches of test paper strips stored at room temperature for 3 months, 6 months and 12 months respectively to perform a stability test, observing the color development condition, wherein the results are shown in table 5, the test paper strip detection lines stored at different storage times have small color intensity difference, the detection results are consistent, the stability is good, and the test paper strips can be stored at room temperature for at least 12 months.
TABLE 7 stability verification of nucleic acid test strips
8. Application of nucleic acid detection colloidal gold test strip
Collecting 30 parts of M.bovis positive diseased cow lung and 10 parts of negative sample, respectively extracting DNA, carrying out PCR amplification, carrying out agarose gel electrophoresis and nucleic acid test strip detection on the amplification product, and carrying out parallel comparison on the detection results of the two methods.
The results show that: 30 parts of lung tissue samples of cattle with bovine mycoplasma disease and 10 parts of lung tissue samples of healthy cattle are collected, DNA is respectively extracted and PCR amplification is carried out, amplification products are respectively detected by agarose gel electrophoresis and nucleic acid test paper strips, the detection results of the two methods are compared in parallel, the results are shown in table 8, the two methods have good coincidence rate, and the total number is 40 parts
The coincidence rate of the clinical sample and the agarose gel electrophoresis detection method is 100 percent.
TABLE 8 comparison of the two detection methods
The method utilizes the signal amplification technology that colloidal gold can be combined with labeled oligonucleotides such as antibodies or biotin and the like to improve the detection sensitivity and form a detection probe capable of developing color, integrates the specificity and sensitivity of PCR and the characteristics of rapidness and simplicity of a gold label strip, ensures that the detection can be rapidly finished within 2h, has good specificity, does not have cross reaction with other similar pathogenic microorganisms except only generating positive results on M.bovis PG45 and M.bovis Sinkiang isolates, has good sensitivity, has the detection lower limit of 89 fg/mu L DNA and the sensitivity of 10-6And the sensitivity of the common agarose gel electrophoresis is generally 10-5Good repeatability and stability, and the strong affinity of the streptavidin-digoxin system and the amplifiable property of the colloidal gold are utilized to ensure that the colloidal gold immunochromatographic technique is compatible with PCR productsThe biotin probe is tightly combined, is more sensitive and faster, greatly simplifies the operation, saves the time and improves the reaction efficiency.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
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