CN107326038B - Polyclonal antibody for detecting kiwi canker pathogen PSA3 and application thereof - Google Patents
Polyclonal antibody for detecting kiwi canker pathogen PSA3 and application thereof Download PDFInfo
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Abstract
The invention provides application of a hopZ5 gene in detection of kiwi fruit canker pathogen PSA3, provides a polyclonal antibody for detecting kiwi fruit canker pathogen PSA3 based on a hopZ5 gene, and provides an immune colloidal gold kit containing the polyclonal antibody and application thereof. The PSA3 polyclonal antibody has the characteristics of high detection specificity and wide application range; the kiwi fruit canker pathogen PSA3 point immunoreaction and immune colloidal gold test strip is simple in detection method, can detect a large number of samples simultaneously, and has values of popularization and application in basic level.
Description
[ technical field ] A method for producing a semiconductor device
The invention relates to immunoassay, in particular to a polyclonal antibody for detecting kiwi fruit ulcer pathogen PSA3, an immune colloidal gold test strip containing the antibody, and a detection method of kiwi fruit ulcer pathogen PSA3 by using the antibody.
[ background of the invention ]
Kiwifruit canker, the first disease in kiwifruit producing areas, was first discovered in restat county in japan in 1984, and subsequently reported in several countries such as china, korea, italy, france, portugal, spain, new zealand, and chile. China is mainly distributed in provinces such as Shaanxi, Sichuan, Henan and Guizhou, and at present, diseases are serious in places such as Wei, Yaan and cangxi in Sichuan, and a lot of orchards are damaged. The pathogenic bacteria causing the disease are Pseudomonas syringae kiwi fruit pathogenic variants (PSA), the bacteria have wide adaptability, strong pathogenicity and fierce coming, when the disease is serious, the whole plant withers, and the garden can be damaged within a few years. In recent years, with the continuous expansion of the planting area of kiwifruit canker disease-susceptible varieties, nursery stock breeding, regulation and transportation are not standard, and the influence of factors such as difficulty in prevention and control further expands the occurrence, propagation and spread of kiwifruit canker disease, which becomes the destructive disease of kiwifruit and is one of the major problems to be solved urgently in production.
Recent research reports prove that the kiwifruit canker pathogen has an important characteristic of latent infection. Therefore, the early diagnosis of the field fruit trees with bacteria is the key to preventing and controlling the kiwifruit canker; the accurate detection of the nursery stock for allocation and transportation is the key for preventing and controlling the spread of the nursery stock. The rapid, convenient and accurate detection method is established, and has important significance for preventing and treating kiwifruit canker.
At present, the kiwifruit canker pathogen is mainly detected by molecular biology, wherein PCR detection has the advantages of accuracy and sensitivity. According to the specific bacterium spraying phenomenon of bacteria, a plurality of bacterial diseases are directly subjected to PCR detection through the bacterium-carrying tissue soak solution, and the method is convenient and rapid. For example, chinese patent application CN 201611158141.1 discloses a real-time fluorescence PCR method for quantitatively detecting kiwifruit canker pathogen, which includes specific primer amplification, sequencing and comparing the amplified products, and determining the infection condition of the sample according to the comparison result of the sequences.
However, when the tissues such as branches and leaves of kiwi fruits serving as a sample to be detected are soaked, various secondary metabolites, particularly polysaccharides and polyphenols are soaked out of the tissues, so that interference is caused to PCR reaction and other detection modes, and the simple sample preparation method is not suitable for detection of kiwifruit canker germs. Therefore, the sample preparation mode of the kiwi fruit tissue is the premise of whether the subsequent detection result is stable, the sample preparation process is optimized and simplified, and the method is an important link for improving the current kiwi fruit canker pathogen detection. In addition, since PCR detection is limited by the requirements of instruments and operating environments, and the like, there is still a limit in the basic department, it is necessary to develop other detection methods suitable for different fields and departments.
According to the description, PSA is currently known to be divided into 5 groups. Among them, PSA1 is distributed in japan; PSA2 exists only in korea; PSA3 is widely distributed worldwide, including kiwifruit canker, china, chile, new zealand, korea, etc. in 2008 + 2009 italy, PSA3 is a group that causes significant losses in the kiwifruit industry worldwide; PSA4 is distributed in new zealand and australia. Furthermore, Fujikawa et al identified the PSA5 group using genomics, and were only found in Japan, and PSA4 and PSA5 belong to the low-toxicity group. Aiming at the group PSA3 which is widely distributed and causes serious harm in PSA, the invention expects to find out the specific effector of only PSA3 group by analyzing the difference of pathogenic factors of various groups, including effector difference, so as to prepare a PSA3 polyclonal antibody and provide a simple serological examination method.
[ summary of the invention ]
The invention aims to overcome the defects of the prior art, and provides a specific effector of a PSA3 group, so that a polyclonal antibody for detecting PSA3 of kiwifruit canker pathogen is obtained, a quick, convenient and efficient serological tool of the PSA3 group is realized by the aid of the polyclonal antibody, and a basis is provided for early prevention and control of kiwifruit canker and limitation of spread of nursery stocks with bacteria.
In order to achieve the purpose, the inventor excavates the reported PSA whole genome sequencing result, compares the PSA various population gene difference, particularly the effector difference, and provides the application of the hopZ5 gene in detecting PSA3 of kiwifruit canker pathogen.
Based on the hopZ5 gene, the invention also provides a preparation method of the polyclonal antibody for detecting kiwi canker pathogen PSA3, and the method comprises the following steps: amplifying a hopZ5 gene of Pseudomonas syringae kiwi fruit pathogenic variant (Pseudomonas syringae pv. actindiae, PSA), after the sequencing of the obtained hopZ5 gene fragment is correct, carrying out enzyme digestion by restriction enzymes BamH I and Xho I, connecting the enzyme digestion product to a prokaryotic expression vector PET28 to obtain a recombinant plasmid PET28a-hopZ5 containing a hopZ5 gene, transforming Escherichia coli E.coli BL21 by the recombinant quality, and carrying out IPTG induced protein expression and purification to obtain a protein hopZ 5;
the polyclonal antibody PAb-hopZ5 is prepared by immunizing a New Zealand white rabbit with prokaryotic expression protein hopZ 5.
In the present invention, primers for amplifying the hopZ5 gene were as follows:
Hopz5-BamH I-F：5’-CGCGGATCCATGGGACTTTGTGCATCA-3
Hopz5-Xho I-R：5’-CGGTCTCGAGTTAGGATTCTATCGCTTTTC-3’
the BamHI and XhoI cleavage site sequences are underlined, respectively.
PCR amplification reaction System: PrimeSTAR HS DNA Polymerase 0.3. mu.L, DNA 2. mu.L, upper and lower primers (10. mu.M) each 1. mu.L, dNTP (2.5mM) 2. mu.L, 10 XBuffer 10. mu.L, plus ddH2O to 25. mu.L, and after mixing, PCR was performed.
The PCR amplification conditions were: 94 deg.C for 4min, 94 deg.C for 30sec, 53 deg.C for 30sec, 72 deg.C for 1min for 30sec, 33 cycles, 72 deg.C for 10min, and 12 deg.C.
Wherein the enzyme digestion step comprises:
the target gene double enzyme digestion system is 10 XH Buffer 2 uL; hopz58 μ L; 1 mu L of BamH I; xho I1 μ L; by ddH2O make up to 20 mL. The system is added into a 200 mu L EP tube on ice, mixed evenly and put into a constant-temperature water bath kettle at 37 ℃ for enzyme digestion overnight. Then taking out the DNA gel electrophoresis nucleic acid dye for 1% agarose gel electrophoresis, cutting a target band, and recovering a target fragment according to the DNA gel recovery kit instruction to obtain the target fragment.
Based on the aforementioned hopZ5 gene and DogThe invention also provides an immune colloidal gold test strip containing the polyclonal antibody, which comprises a sample pad, a gold-labeled pad, a nitrocellulose membrane and absorbent paper which are assembled on a bottom plate in sequence, wherein the gold-labeled pad contains a colloidal gold antibody complex, the colloidal gold antibody complex contains the polyclonal antibody obtained by the preparation method of claim 2 and a colloidal gold solution, the concentration of the polyclonal antibody is not less than 50 mu g/ml, and then 0.1mol/L HCl aqueous solution and 0.1mol/L K aqueous solution are used2CO3Adjusting the pH value of the aqueous solution to 7.2 to obtain the colloidal gold antibody complex.
According to a preferred embodiment, the colloidal gold solution is prepared by: a clean bottle is taken, 1g of chloroauric acid is completely dissolved in 100mL of ultrapure water to prepare a 1% (g/mL) chloroauric acid solution, and the chloroauric acid solution is stored at 4 ℃ in the dark. Diluting the 1% chloroauric acid solution into 0.01% (g/mL) chloroauric acid solution, adding 100mL 0.01% chloroauric acid solution into a clean beaker, heating while stirring, adding 1.5mL 1% trisodium citrate preheated at 37 ℃ after boiling, stirring gently, changing the solution from yellow to black, purple and purplish red until wine red, boiling for 2min, cooling to room temperature, adding ultrapure water to 100mL, placing into a clean glass bottle, and storing at 4 ℃ in a dark place. .
Based on the hopZ5 gene and the polyclonal antibody, the invention also provides a method for detecting kiwi canker pathogen PSA3 by using an immune colloidal gold test strip, which comprises the following steps:
(1) crushing a kiwi fruit sample to be detected to pieces with the diameter of 1-2mm, wherein the mass ratio of the sample to be detected to a sample loading buffer solution is 0.05-0.2 g: soaking a sample to be detected in 250-400 mu l of sample adding buffer solution for 2-15 s;
(2) adding 200 mu L of the buffer solution soaked with the sample to be detected in the step (1) on the sample pad of the immune colloidal gold test strip according to claim 5, staying for 5-10min, and observing a T line and a C line in a detection area of the immune colloidal gold test strip;
(3) and when the T line and the C line are both wine red, judging that the sample to be detected is a kiwi fruit canker pathogen PSA3 infected product.
Based on the hopZ5 gene and the polyclonal antibody, the invention also provides a dot immunoassay method for kiwi canker pathogen PSA3, which comprises the following steps:
(1) crushing a kiwi fruit sample to be detected to pieces with the diameter of 1-2mm, wherein the mass ratio of the sample to be detected to the sample loading buffer solution is 0.005-0.01 g: 20-50 mul, soaking the sample to be detected in the sample adding buffer solution for 2-15 s;
(2) dropwise adding the buffer solution soaked with the sample to be detected in the step (1) onto a nitrocellulose membrane, and then drying at room temperature;
(3) sealing the nitrocellulose membrane in the step (2) by using an aqueous solution of 5wt% of skimmed milk powder for 1 h;
(4) incubation with PAb-hopZ5 according to claim 1 for 1h, followed by 3 washes with phosphate buffered PBS;
(5) then commercial alkaline phosphatase (purchased from Beijing China fir Jinqiao biotechnology, Ltd.) is used for marking the goat anti-rabbit IgG for incubation for 1h, and then phosphate buffer PBS is used for washing for 3 times;
(6) and (3) carrying out color reaction by adopting a BCIP/NBT alkaline phosphatase color development kit in a dark environment, and judging the color development sample as the kiwi canker pathogen PSA3 infected product.
Experiments prove that an effector gene hopZ5 of kiwi canker pathogen PSA3 obtains a polyclonal antibody PAb-hopZ5 by cloning a complete sequence, constructing a prokaryotic expression recombinant vector PET28a-hopZ5 and immunizing by prokaryotic expression hopZ5 protein, and the antibody can distinguish pseudomonas and test other pathogenic bacteria, can distinguish PSA3 from other groups, has the characteristic of high specificity and can be applied to detection of kiwi canker pathogen PSA 3.
The polyclonal antibody obtained based on the hopZ5 gene can be used for detection of artificial cultures and bacteria-carrying tissues, comprises preparation of immune colloidal gold test strips or application of point immune detection, has the characteristic of wide application range to samples to be detected, can realize various serological detections of PSA3, is an ideal antibody which can be applied to serological detection and is found for the first time except for molecular detection means for kiwifruit canker pathogen at present and particularly for PSA3 group with extremely strong pathogenicity, and has good development prospect.
The invention also improves the preparation means of the detection sample, and can effectively avoid the release of a large amount of secondary metabolites such as polysaccharide and polyphenol in the sample when the sample to be detected is soaked in the sample adding buffer solution for 2-15 seconds, thereby avoiding influencing the detection result. In addition, the comparison shows that the effect of detecting the sample to be detected after the sample to be detected is soaked in the sample adding buffer solution is more stable than that of detecting the sample to be detected after the sample is soaked in the conventional sterile water.
The detection method has higher detection speed, and is more rapid compared with other traditional Chinese gooseberry canker pathogen detection methods, wherein the immune colloidal gold test strip can complete detection within 15 minutes; the spot immunoassay method also has the characteristic of simple sample preparation.
[ description of the drawings ]
FIG. 1 shows the result of Western blot detection of the hopZ5 protein of Actinidia chinensis planch;
FIG. 2 shows the result of type analysis of PAb-hopZ5 spot immunodetection samples;
FIG. 3 shows the detection results of the PAb-hopZ5 spot immunized field samples;
FIG. 4 shows the specificity test results of the PAb-hopZ5 immune colloidal gold test strip;
FIG. 5 shows the test results of the PAb-hopZ5 immune colloidal gold test strip in field samples.
[ detailed description ] embodiments
The following examples serve to illustrate the technical solution of the present invention without limiting it.
In the present invention, "%" and "parts" used for concentration are weight percentages and parts are parts by weight, unless otherwise specified.
Example 1 preparation of polyclonal antibody containing hopZ5 Gene of PSA3
Collection, isolation and identification of pseudomonas syringae kiwifruit pathogenic variant PSA 3: typical disease-causing leaves of canker disease-causing orchards in Dujiang weir kiwi fruit producing areas are collected, and diseases are separated as original bacteria in laboratories.
The size of the diseased spots of the cut leaves is about 5mm, the diseased spots are firstly washed by sterile water, then the leaves are soaked for 10min by 0.5 percent sodium hypochlorite for surface disinfection, and then the leaves are soaked and rinsed for 3 times by sterile water for 10min each time. The sterilized leaves are smashed and soaked in 200 mul of sterile water, and the inoculating loop is dipped in the sterile water and streaked on an LB culture medium.
Referring to the report of Reese Gorge et al in 2010, a double PCR amplification was performed using a 16s rDNA primer and a HopZ5 primer.
The primers are as follows:
PsaF1：5'-TTTTGCTTTGCA CACCCGATTTT-3'；
PsaR2：5'-CACGCACCCTTCAATCAGGATG-3。
HopZ5-F：5'-TCACTCCTAGACTGGAATAC-3'；
HopZ5-R：5'-GGCTATCATGAAGGCTG TCA-3'。
after double PCR amplification, the amplification results were sent to sequencing, with 99-100% homology to PSA strains CRAFU 14.08, NZ-47 and ICMP1884 (accession numbers CP019732.1, CP017009.1 and CP011972, respectively). Sequencing results show that the isolated and identified bacteria are pseudomonas syringae kiwi fruit pathopoiesia and the group is PSA 3.
Specific primers for the hopZ5 complete gene were designed for cloning based on the sequence of Pseudomonas syringae pv. actinoniae ICMP 18884 strain:
the upstream sequence: hopz 5-BamHI-F: 5' -CGCGGATCCATGGGACTTTGTGCATCA-3;
the downstream sequence: hopz 5-XhoI-R: 5'-CGGTCTCGAGTTAGGATTCTATCGCTTTTC-3' are provided.
PCR amplification reaction System: PrimeSTAR HS DNA Polymerase 0.3. mu.L, DNA 2. mu.L, upper and lower primers (10. mu.M) each 1. mu.L, dNTP (2.5mM) 2. mu.L, 10 XBuffer 10. mu.L, plus ddH2O to 25. mu.L, and after mixing, PCR was performed.
The PCR amplification conditions were 94 ℃ for 4min, 94 ℃ for 30sec, 53 ℃ for 30sec, 72 ℃ for 1min for 30sec, 33 cycles, 72 ℃ for 10min, and 12 ℃ storage.
After the obtained hopZ5 gene fragment is sequenced correctly, carrying out double digestion by two restriction enzymes BamH I and Xho I by using a digestion Buffer solution 10 XH Buffer, connecting the digestion product to a prokaryotic expression vector PET28 to obtain a recombinant plasmid PET28a-hopZ5 containing a hopZ5 gene, transforming Escherichia coli E.coli BL21 by the recombinant mass, and carrying out IPTG induced protein expression and purification to obtain a protein hopZ 5;
the prokaryotic expression protein hopZ5 is sent to Younoke biotechnology limited of Suzhou to immunize a New Zealand white rabbit, and the polyclonal antibody PAb-hopZ5 is prepared.
Example 2 Western-blot detection of hopZ5 protein of PSA3
SDS-PAGE electrophoresis:
(1) cleaning and airing the glass plate, and clamping the glass plate filled with the polyacrylamide gel;
(2) preparation of separation gel: preparing 12% SDS-PAGE separating gel, and sequentially adding the following components in a beaker: 4mL of 30% ACR, 2.5mL of 1.5mol/L Tris-HCl (pH8.8), 100 mu L of 10% SDS, 100 mu L of 10% ammonium persulfate and 5 mu L of TEMED, and sterile deionized water to make up to 10mL, and adding the mixture into the gap between the two glass plates by using a pipette after shaking up;
(3) adding sterile water into the separation gel, standing for 30min, pouring out the sterile water after the separation gel is solidified, and completely sucking residual liquid with a filter paper sheet;
(4) 5% SDS-PAGE concentrated gel is prepared, and the following components are added in a beaker in sequence: 30% ACR 670. mu.l, 1mol/L Tris-HCl (pH6.8)1.25mL, 10% SDS 50. mu.l, 10% ammonium persulfate 50. mu.l, Tetramethylethylenediamine (TEMED) 4. mu.l, sterilized deionized water to make up to 5mL, shaking up, adding onto separation gel, inserting comb;
(5) and (3) processing of a sample: adding the polyclonal antibody PAb-hopZ5 obtained in example 1 into 5 xSDS-PAGE loading buffer according to the volume ratio of 5:1, then putting into hot water at 100 ℃ for boiling for 3min, and taking out for later use;
(6) after the gel in the step (4) is completely solidified, pulling out the comb, fixing the comb in an electrophoresis device, adding electrophoresis buffer solution until the sample hole is covered, and sucking 10 mu l of the sample treated in the step (5) into the gel hole by using a pipette gun;
(7) connecting the electrophoresis device with a power supply, adjusting the voltage to 80V, adjusting the voltage to 120V when the bromophenol blue electrophoresis is indicated to the separation gel, turning off the power supply when the bromophenol blue reaches the bottom of the separation gel, and taking out the polyacrylamide gel.
Film transfer:
(1) after electrophoresis is finished, removing the concentrated gel;
(2) cutting filter paper and a nitrocellulose membrane, completely soaking the filter paper and the nitrocellulose membrane in a membrane transfer solution, and cutting a corner to be used as a mark;
(3) the following placing steps are carried out: the plastic splint (black), the sponge pad, 3 pieces of filter paper, gel, the nitrocellulose filter membrane, 3 pieces of filter paper, the sponge pad and the plastic splint (white) are required to be aligned and air bubbles are discharged when one layer is added. After the ice bags are placed, the clamping plates are fixed in the electric rotary groove, the power supply is connected, and the ice bags are placed in the gap position of the electric rotary groove for electric transfer, 100V and 1 h.
And (3) hybridization:
(1) and (3) sealing of the membrane: after the electrotransfer is finished, taking out the cellulose nitrate filter membrane, putting the cellulose nitrate filter membrane into a clean culture dish, adding a confining liquid (PBS (phosphate buffer solution) containing 5% of skim milk) to completely immerse the membrane, and horizontally placing the membrane on a horizontal shaking table to act for 2 hours at room temperature;
(2) washing the membrane: placing the cellulose nitrate filter membrane into a new culture dish, adding washing solution PBS, flatly placing on a shaker, shaking at room temperature for 5min, and repeatedly washing for 3 times;
(3) adding primary antibody, namely polyclonal antibody PAb-hopZ 5: putting the washed nitrocellulose filter membrane into a new culture dish, immersing the whole membrane into a PAb-hopZ5 antibody solution (the PAb-hopZ5 antibody is diluted to 1:2000 by a PBS solution containing 5% skimmed milk powder), and flatly placing the membrane on a shaking bed to react for 3 hours at room temperature;
(4) washing the membrane: washing the nitrocellulose filter membrane with PBS for 3 times, 5min each time;
(5) adding a commercial alkaline phosphatase-labeled goat anti-rabbit IgG secondary antibody of China fir Jinqiao company: placing the nitrocellulose filter membrane into a culture dish, completely immersing the membrane into a secondary antibody solution (the secondary antibody is diluted to 1:2000 by PBS (phosphate buffer solution) containing 5% skimmed milk powder), and flatly placing the membrane on a shaking bed to react for 2 hours at room temperature;
(6) washing the membrane: washing the nitrocellulose filter membrane with PBS for 3 times, 5min each time;
(7) color development: the nitrocellulose filter was removed and placed in a substrate solution (3.8ml substrate buffer plus 100. mu.l NBT and 100. mu.l BCIP) and reacted for 10-15min in the dark until the positive sample developed color.
(8) And (3) terminating the reaction: the solution was removed, the membrane was washed with distilled water, the reaction was terminated, the results were observed and the membrane sheet was preserved.
The Western blot result is shown in figure 1, the target band can be detected and is single, the size is 39kD, and the prepared antibody is proved to be better and can be used for subsequent experimental requirements.
Example 3 HopZ5 Point immunoassay for PSA3 sample type analysis
Since hopZ5 is an effector of kiwifruit canker pathogen, which is one of the common pathogenic factors of the pathogen, it is necessary to verify whether the hopZ5 antibody can detect PSA cultured on the culture medium and susceptible plants.
The total protein and tissue soak solution extracted from the cultured PSA bacterial suspension and the infected tissue are taken as samples. Wherein the kiwifruit canker standard strain (Pseudomonas syringae pv. actindiae, PSA) is derived from a strain isolated and identified in a plant pathology laboratory. The first generation of the bacterial species stored at-70 ℃ was activated.
The preparation process of the PSA bacterial suspension comprises the following steps: and (3) taking the activated first-generation strain to perform line drawing culture on an LB solid culture medium, then placing the strain in a constant-temperature incubator at 25 ℃ for culture, and eluting the bacterial colony with sterile water to prepare PSA bacterial suspension.
The process for extracting total protein of the infected tissue comprises the following steps: the material was ground well with liquid nitrogen, at 1 g: 2ml of protein extraction buffer (50mmol/L Tris. HCl (pH6.8), 4% SDS, 6% beta-mercaptoethanol, 4mol/L urea, 10% glycerol) was added. Bathing at 100 deg.C for 10min, centrifuging at 12000g for 10min, and collecting supernatant as total protein.
The preparation process of the tissue soak solution comprises the following steps: cutting the tissue with bacteria into small pieces, adding 20 μ l of sterile water into 0.005g of sample pieces, soaking for 10s, and sucking the soaking solution for use.
Each of the three samples was 8. mu.L drop-spotted on nitrocellulose membrane, and the results of the color reaction were shown in FIG. 2 after blocking, primary antibody and secondary antibody incubation and color development. It can be seen that the three samples can develop color on the nitrocellulose membrane, which indicates that the tissue soak solution is the same as the extracted total protein and can be used for DIBA detection, but the sample preparation process of the tissue soak solution is simplified, and only the tissue with bacteria is cut and soaked; in addition, the PSA bacterial suspension can still be detected by using the hopZ5 antibody. The results indicate that the hopZ5 antibody of the invention can be used for the detection of various types of samples.
Example 4 HopZ5 Point Immunity field sample assay of PSA3
Processing field samples collected from a kiwi fruit orchard, cutting off small pieces of the samples, peeling the samples with a scalpel, putting the cut samples into a centrifuge tube, adding 10-20 mu L of sterile water, sucking 8 mu L of soak solution after 10s, dotting the soaked solution on a nitrocellulose membrane, using the remaining samples to extract the total DNA of plant tissues and detect the total DNA by PCR, contrasting the two detection methods, and repeating 15 groups.
The results showed that the results of PCR could not be detected except that sample 3 showed false positive, i.e., DIBA color in the dot immuno binding assay, which indicated that the results obtained by the two detection means were inconsistent. The other samples showed the results of DIBA and PCR, and the results are shown in FIG. 3. The DIBA detection method can be used for detecting field samples, and has high reliability and accuracy rate of more than 90%. In addition, compared with the traditional detection method such as a PCR method, the point immunoassay method using the antibody PAb-hopZ5 of the invention is simple and convenient to operate and is suitable for large-scale detection of samples.
Example 5 HopZ5 Immunocolloidal gold test strip specificity test for PSA3
And sequentially assembling a sample pad, a gold label pad, a nitrocellulose membrane and absorbent paper on the substrate to form the immune colloidal gold test strip. Wherein the gold-labeled pad contains a colloidal gold-antibody complex comprising 50. mu.g/ml of polyclonal antibody PAb-hopZ5 and a colloidal gold solution, followed by 0.1mol/L of aqueous HCl and 0.1mol/L of K2CO3The aqueous solution was adjusted to pH 7.2 to obtain a colloidal gold antibody complex.
Preparing Actinidia canker, Pseudomonas syringae, Bacillus megaterium, Pseudomonas putida, Pseudomonas fluorescens, Bacillus subtilis, PSA1 and PSA2 into 1 × 108cfu/mL bacterial suspension, using clear water as control, and numbering for standby.
Adding 200 μ l of the bacterial suspension onto a sample pad of an immune colloidal gold test strip, standing for 5-10min, and observing a T line and a C line. And when the T line and the C line simultaneously display the red line, the result is positive, only the T line displays the red line, the result is negative, and neither the T line nor the C line displays the red line, and the result is invalid. The color development of the 9 samples is shown in FIG. 4.
As shown in FIG. 4, the hopZ5 immunogold test strip showed simultaneous color development of T-line and C-line only for the bacterial suspension of PSA3 from sample No. 1, and was judged to be positive according to the color development results, and was identical to the sample itself. For the bacterial suspensions not containing PSA3 (samples 2-9), only the T line showed red line, and the color was judged negative according to the color development result, which was consistent with the sample itself. Therefore, the immune colloidal gold test strip has good specificity effect and can specifically detect PSA 3.
Example 6 HopZ5 Immunocolloidal gold test strip for PSA3 field sample testing
Taking field plant tissues, cutting the field plant tissues into small pieces, putting the small pieces into a sterile mortar, adding 1mL of sample adding buffer solution, soaking for 12 seconds, and taking the soaking solution as a sample to be detected. The remaining tissue was used to extract total plant DNA for PCR detection, which was repeated 32 times in comparison with the results of the immune colloidal gold test strips.
The immune colloidal gold test strip is horizontally placed on a table, 200 mu L of field sample soak solution to be detected is absorbed on a sample pad, the sample pad is kept stand for 10min, and the detection result is observed.
The results of the immune colloidal gold test strip detection of the field samples are shown in fig. 5, wherein samples No. 2, 8, 14, 21 and 26 only show a T line, and the results are judged to be negative according to the color reaction, namely the samples are not infected by PSA 3. By contrast with the PCR results, the samples No. 14 and No. 26 were confirmed to be positive by PCR detection, and did not match the color development results, and it was presumed that the reason why the sample No. 14 was not correctly detected by the immune colloidal gold test strip was mainly due to the low amount of bacteria carried by the sample. The reason why sample No. 26 was not detected correctly by the immuno-colloidal gold test strip was that the infection type of this sample was PSA2 group by subsequent identification.
In 32 samples, the detection results of the immune colloidal gold test strips of the other samples are consistent with the detection results of PCR, which shows that the detection results of the immune colloidal gold test strips hopZ5 of PSA3 are stable.
In conclusion, the PSA3 polyclonal antibody has the characteristics of high detection specificity and wide application range; the kiwi fruit canker pathogen PSA3 point immunoreaction and immune colloidal gold test strip is simple in detection method, can detect a large number of samples simultaneously, and has values of popularization and application in basic level.
Sequence listing
<110> Sichuan university of agriculture
<120> polyclonal antibody for detecting kiwi canker pathogen PSA3 and application thereof
<130> 17155.1
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 27
<212> DNA
<213> Hopz5- BamH I -F
<400> 1
cgcggatcca tgggactttg tgcatca 27
<210> 2
<211> 30
<212> DNA
<213> Hopz5-Xho I-R
<400> 2
cggtctcgag ttaggattct atcgcttttc 30
<210> 3
<211> 23
<212> DNA
<213> PsaF1
<400> 3
ttttgctttg cacacccgat ttt 23
<210> 4
<211> 22
<212> DNA
<213> PsaR2
<400> 4
cacgcaccct tcaatcagga tg 22
<210> 5
<211> 20
<212> DNA
<213> HopZ5-F
<400> 5
<210> 6
<211> 20
<212> DNA
<213> HopZ5-R
<400> 6
Claims (6)
1. The application of a polyclonal antibody PAb-hopZ5 in detecting Actinidia kolomikta PSA3 is disclosed, and the preparation method of the polyclonal antibody PAb-hopZ5 comprises the following steps: amplifying a hopZ5 gene of Pseudomonas syringae kiwi fruit pathogenic variant (Pseudomonas syringae pv. actinoniae, PSA), after the sequencing of the obtained hopZ5 gene fragment is correct, carrying out enzyme digestion by restriction enzymes BamH I and Xho I, connecting the enzyme digestion product to a prokaryotic expression vector PET28 to obtain a recombinant plasmid PET28a-hopZ5 containing a hopZ5 gene, transforming escherichia coli E.coli BL21 by the recombinant quality, and carrying out IPTG induced protein expression and purification to obtain a protein hopZ 5; immunizing a New Zealand white rabbit with the hopZ5 protein to prepare and obtain a polyclonal antibody PAb-hopZ 5;
the primers for amplifying the hopZ5 gene are as follows:
Hopz5- BamH I -F：5’-CGCGGATCCATGGGACTTTGTGCATCA -3；
Hopz5-Xho I-R：5’- CGGTCTCGAGTTAGGATTCTATCGCTTTTC -3’；
PCR amplification reaction System: PrimeSTAR HS DNA Polymerase 0.3. mu.L, DNA 2. mu.L, 10. mu.M of each of the upper and lower primers 1. mu.L, 2. mu.L of 2.5mM dNTP, 10 XBuffer 10. mu.L, plus ddH2Supplementing 25 mu L of O, uniformly mixing and carrying out PCR reaction;
the PCR amplification conditions were: 94 deg.C for 4min, 94 deg.C for 30sec, 53 deg.C for 30sec, 72 deg.C for 1min for 30sec, 33 cycles, 72 deg.C for 10min, and 12 deg.C.
2. The use according to claim 1, characterized in that said enzymatic cleavage step comprises:
carrying out double enzyme digestion by using restriction enzymes BamH I and Xho I to share a digestion Buffer solution of 10 XH Buffer, wherein a target gene double digestion system comprises: 10 XH Buffer 2. mu.L; hopz58 μ L; 1 mu L of BamH I; xho I1. mu.L; by ddH2O is complemented to 20 mL; adding the system into a 200 mu LEP tube on ice, uniformly mixing, putting into a 37 ℃ constant-temperature water bath kettle, and performing enzyme digestion overnight; then taking out the DNA gel electrophoresis nucleic acid dye for 1% agarose gel electrophoresis, cutting a target band, and recovering a target fragment according to the DNA gel recovery kit instruction to obtain the target fragment.
3. Use of polyclonal antibody PAb-hopZ5 in preparation of immune colloidal gold test strip for detecting Actinidia kiwii ulcer germ PSA3, the test strip comprises a sample pad, a gold-labeled pad, a nitrocellulose membrane and absorbent paper which are assembled on a bottom plate in sequence, and is characterized in that the gold-labeled pad contains a colloidal gold antibody complex containing the polyclonal antibody obtained by the preparation method according to claim 1 and a colloidal gold solution, wherein the concentration of the polyclonal antibody is not less than 50 [ mu ] g/ml, and then 0.1mol/L HCl aqueous solution and 0.1mol/L K solution are used2CO3Adjusting the pH value of the aqueous solution to 7.2 to obtain the colloidal gold antibody complex.
4. The use according to claim 3, characterized in that the colloidal gold antibody complex is prepared by a method comprising:
(1) 10mL of the colloidal gold solution was added to a clean beaker, and 0.1mol/L HCl and 0.1mol/L K were added with stirring2CO3Adjusting the pH value of the colloidal gold solution to 7.2;
(2) adding 500 μ g of the PAb-hopZ5 antibody according to claim 1 under continuous stirring, and stirring for 1 h;
(3) the gold-labeled antibody with stable binding is subpackaged in a centrifuge tube of 2mL at 4 ℃, 2000r/min for 20 min, and the precipitate formed by the coagulated gold particles is discarded;
(4) transferring the red supernatant to a new centrifuge tube, at 4 ℃, 15000r/min for 30min, and discarding the supernatant;
(5) collecting the dark red precipitate flowing at the bottom of the tube, re-suspending the precipitate in 1/10 with 1 XPBS buffer solution, and storing at 4 deg.c in dark place.
5. A method for detecting Actinidia kolomikta PSA3 by using an immune colloidal gold test strip, comprising the following steps:
(1) crushing a kiwi fruit sample to be detected to pieces with the diameter of 1-2mm, wherein the mass ratio of the sample to be detected to a sample loading buffer solution is 0.05-0.2 g: soaking a sample to be detected in 250-400 mu l of sample adding buffer solution for 2-15 s;
(2) adding 200 mu L of the buffer solution soaked with the sample to be detected in the step (1) on the sample pad of the immune colloidal gold test strip according to claim 3, staying for 5-10min, and observing a T line and a C line in a detection area of the immune colloidal gold test strip;
(3) and when the T line and the C line are both wine red, judging that the sample to be detected is a kiwi fruit canker pathogen PSA3 infected product.
6. A dot immunoassay method for Actinidia kolomikta PSA3, comprising the following steps:
(1) crushing a kiwi fruit sample to be detected to pieces with the diameter of 1-2mm, wherein the mass ratio of the sample to be detected to the sample loading buffer solution is 0.005-0.01 g: 20-50 mul, soaking the sample to be detected in the sample adding buffer solution for 2-15 s;
(2) dropwise adding the buffer solution soaked with the sample to be detected in the step (1) onto a nitrocellulose membrane, and then drying at room temperature;
(3) sealing the cellulose nitrate membrane in the step (2) by using a 5wt% skimmed milk aqueous solution for 1 h;
(4) incubating the polyclonal antibody PAb-hopZ5 of claim 1 for 1h, followed by 3 Phosphate Buffered Saline (PBS) washes;
(5) then, incubating the goat anti-rabbit IgG with alkaline phosphatase for 1h, and washing the goat anti-rabbit IgG with phosphate buffer solution PBS for 3 times;
(6) and (3) carrying out color development reaction by adopting a BCIP/NBT alkaline phosphatase color development kit in a dark environment, and judging the color development sample as a kiwi canker pathogen PSA3 infected sample.
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| Title |
|---|
| Comparative genomics-informed design of two LAMP assays for detection of the kiwifruit pathogen Pseudomonas syringae pv. actinidiae and discrimination of isolates belonging to the pandemic biovar 3;M. Ruinelli et al;《Plant Patholgoy》;20160606;第66卷;第140-149页 * |
| CP011972;Anonymous;《GenBank》;20160916 * |
| Genomic Analysis of the Kiwifruit Pathogen Pseudomonas syringae pv. actinidiae Provides Insight into the Origins of an Emergent Plant Disease;Honour C.McCann et al;《PLOS pathogens》;20130731;第9卷(第7期);e1003503 * |
| 利用胶体金免疫层析技术快速检测柑橘溃疡病菌;李平;《中国优秀硕士论文全文数据库（电子期刊）》;20061215;D046-124 * |
| 应用斑点免疫技术快速检测柑桔溃疡病菌;王中康等;《西南农业大学学报》;19971231;第19卷(第6期);第529-532页 * |
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