AU2021104199A4 - Rapid immunoassay test strip for Ralstonia solanacearum and preparation method and application thereof - Google Patents
Rapid immunoassay test strip for Ralstonia solanacearum and preparation method and application thereof Download PDFInfo
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Abstract
The present invention provides a rapid immunoassay test strip for Ralstonia
solanacearum and a preparation method and application thereof, belonging to the
technical field of biological detection. The test strip contains an anti-Ralstonia
5 solanacearum monoclonal antibody, wherein a heavy chain variable region of the
anti-Ralstonia solanacearum monoclonal antibody contains an amino acid sequence
shown in SEQ ID NO. 1, and a light chain variable region thereof contains an amino
acid sequence shown in SEQ ID NO. 2. By improving and optimizing the existing
colloidal gold test strips for detecting Ralstonia solanacearum, the present invention
10 successfully prepares a rapid immunoassay test strip for Ralstonia solanacearum with
the advantages of strong specificity, high sensitivity, simple operation, rapid response,
etc., which has good practical application value accordingly.
Description
Field of the Invention The present invention belongs to the technical field of biological detection, and specifically relates to a rapid immunoassay test strip for Ralstonia solanacearum and a preparation method and application thereof.
Background of the Invention The information disclosed in the Background of the Invention is merely intended to increase the understanding on the general background of the present invention, and should not be construed as acknowledgment or hint in any form that the information constitutes the prior art known by those skilled in the art.
Bacterial wilt caused by Ralstonia solanacearum is a devastating soil-borne disease.
In addition to solanaceae crops, R.solanacearum can also infect more than 200 plants
in more than 50 families. It is an important limiting factor on the yield and quality of
crops such as tobacco, tomato, pepper, eggplant, ginger, potato, peanut, strawberry
and banana. Because of its wide host range and difficult control, it is called "cancer"
of plants. Due to the lack of effective control agents, agricultural streptomycin sulfate
has been used for the prevention and treatment of bacterial wilt in China. As China
stopped using the agricultural streptomycin sulfate, the control of plant bacterial wilt
is facing a huge challenge.
Accurate and rapid detection of Ralstonia solanacearum is an important prerequisite
for the implementation of prevention and control measures against Ralstonia
solanacearum. At present, this pathogen is mainly detected at a genetic molecular
level, including PCR and the like. The detection technology at the genetic molecular
level has the advantages of accuracy and sensitivity, but requires relatively strict
experimental conditions, measures and operations, and cannot fully meet the existing
requirements for rapid inspection and quarantine. Therefore, the establishment of a
rapid and accurate on-site detection method for Ralstonia solanacearum is of great
practical significance for the strict implementation of prevention and control measures.
Colloidal gold immunochromatographic test strips have attracted much attention due
to their rapidness, convenience, simple operation, low cost, stable performance, etc. A
target analyte can be detected through a color reaction visible to the naked eye, using
colloidal gold particles as a marker and using a specific antigen-antibody reaction.
Therefore, the colloidal gold immunochromatographic test strips are of great
significance for the rapid detection of pathogens on site. The inventor found that there
are already colloidal gold test strips for detecting Ralstonia solanacearum in the prior
art. However, they generally have defects such as low detection sensitivity, poor
specificity, long detection time and the like, which affect their practical application.
Summary of the Invention Aiming at the problems in the prior art, the present invention provides a rapid immunoassay test strip for Ralstonia solanacearum and a preparation method and application thereof. By improving and optimizing the existing colloidal gold test strips for detecting Ralstonia solanacearum, the present invention successfully prepares a rapid immunoassay test strip for Ralstonia solanacearum with the advantages of strong specificity, high sensitivity, simple operation, rapid response, etc., which has good practical application value accordingly.
Specifically, the present invention relates to the following technical solutions:
The first aspect of the present invention provides a rapid immunoassay test strip for
Ralstonia solanacearum, the test strip being configured according to a conventional
colloidal gold test strip, the test strip containing an anti-Ralstonia solanacearum
monoclonal antibody, wherein a heavy chain variable region of the anti-Ralstonia
solanacearum monoclonal antibody contains an amino acid sequence shown in SEQ
ID NO. 1, and a light chain variable region thereof contains an amino acid sequence
shown in SEQ ID NO. 2.
Specifically, the amino acid sequence of the heavy chain variable region of the
anti-Ralstonia solanacearum monoclonal antibody has at least 80% of homology
compared with SEQ ID NO. 1; more preferably, at least 90% of homology; most preferably, at least 95% of homology; for example, at least 96%, 97%, 98%, or 99% of homology.
The amino acid sequence of the light chain variable region of the anti-Ralstonia
solanacearum monoclonal antibody has at least 80% of homology compared with
SEQ ID NO. 1; more preferably, at least 90% of homology; most preferably, at least
95% of homology; for example, at least 96%, 97%, 98%, or 99% of homology.
In another specific embodiment of the present invention, the rapid immunoassay test
strip for Ralstonia solanacearum includes a bottom board on which an absorbent pad,
a detection pad, a gold-labeled pad and a sample pad are arranged in sequence, and
the adjacent pads are overlapped and connected at the junction.
Wherein,
the detection pad is based on a nitrocellulose membrane, the nitrocellulose membrane
is provided with a quality control line and a detection line thereon in sequence, the
detection line is coated with an anti-Ralstoniasolanacearumpolyclonal antibody, and
the quality control line is coated with a goat anti-mouse IgG;
Wherein, the polyclonal antibody may be obtained by a conventional polyclonal
antibody preparation method, the immunogen is Ralstonia solanacearum, and the
immunized animal may be a rabbit, such as a New Zealand white rabbit.
The gold-labeled pad is sprayed with the anti-Ralstonia solanacearum monoclonal
antibody labeled by nano-gold.
The absorbent pad may be an absorbent board, and the bottom board may be a
cardboard.
The specification of the rapid immunoassay test strip for Ralstonia solanacearum can
be set in a conventional manner. In a specific embodiment of the present invention,
the length of the absorbent pad is controlled to 15-20 mm, and the width is controlled
to 3-5 mm; the length of the detection pad is controlled to 20-25 mm, and the width is
controlled to 3-5 mm; the length of the gold-labeled pad is controlled to 3-7 mm, and
the width is controlled to 3-5 mm; the length of the sample pad is controlled to 20-25
mm, and the width is controlled to 3-5mm; the overlapping length of the adjacent pads
is controlled to 3-6 mm;
In another specific embodiment of the present invention, the distance between the quality control line and the detection line on the detection pad is controlled to 3-6 mm, and the distance between the detection line and the upper edge of the nitrocellulose membrane is 13-18 mm. By controlling the length, width and distance of each component, the prepared rapid immunoassay test strip for Ralstonia solanacearumhas a more suitable specification, is convenient to carry and use, and is also beneficial to improving the detection efficiency. In another specific embodiment of the present invention, the particle size of the nano-gold used in the gold-labeled pad is controlled to 30-50 nm; per centimeter of spraying length on the gold-labeled pad requires 80-120 ng of nano-gold-labeled anti-Ralstonia solanacearum monoclonal antibody; and per centimeter of spraying length on the detection line of the detection pad requires 80-120 ng of anti-Ralstonia solanacearumpolyclonal antibody. By controlling the size of nano-gold particles and the dosages of the monoclonal antibody and the polyclonal antibody, the detection sensitivity and specificity can be effectively improved. The second aspect of the present invention provides a preparation method of the rapid immunoassay test strip for Ralstonia solanacearum.The preparation method includes: adding an anti-Ralstoniasolanacearummonoclonal antibody into nano-gold to obtain a nano-gold-labeled anti-Ralstoniasolanacearum monoclonal antibody, and spraying the monoclonal antibody onto a gold-labeled pad; The preparation method further includes: pasting an absorbent pad, a detection pad, the gold-labeled pad and a sample pad on a bottom board in sequence to form the test strip; The detection pad is based on a nitrocellulose membrane, the nitrocellulose membrane is provided with a quality control line and a detection line thereon in sequence, the detection line is coated with an anti-Ralstoniasolanacearumpolyclonal antibody, and the quality control line is coated with a goat anti-mouse IgG. The third aspect of the present invention provides application of the test strip in the detection of Ralstonia solanacearum. The fourth aspect of the present invention provides a method for rapidly detecting
Ralstonia solanacearum, the method including: detecting a sample to be tested with the test strip, and analyzing the detection result. In another specific embodiment of the present invention, the sample to be tested only needs to be simply pretreated, specifically, a plant sample to be tested is ground and then added with a sample buffer to obtain the sample to be tested, which is dropped onto the sample pad, and the result is determined on the following basis after 5-10 minutes: (1) the quality control line (C) shows a color, and the test line (T) shows a red band, indicating positive; (2) the C line shows a color, and the T line does not show a red band, indicating negative; (3) the C line does not show any color or only the T line shows a color, indicating an incorrect operation process or an invalid test strip. Beneficial technical effects of one or more of the above technical solutions: (1) The rapid immunoassay test strip for Ralstonia solanacearum provided in the above technical solution is cheap and easy to obtain, which greatly reduces the cost of rapid screening of Ralstonia solanacearum in the field; meanwhile, the test strip has a short detection time, and the detection can be completed within 5-10 minutes, which greatly improves the rapid screening efficiency of Ralstonia solanacearum; the monoclonal antibody used has a subtype IgG2b, which greatly improves the stability of product batches and reduces the production cost. (2) The rapid immunoassay test strip for Ralstonia solanacearum provided in the above technical solution has high sensitivity, and has a detection limit of 1x104
cfu/mL for the Ralstonia solanacearum; the test strip has good stability, and can be stored for 18 months at room temperature; the field test requires a low sampling amount, and only a leaf of 10 mm*10 mm is required for a single test, which reduces the field sampling amount. (3) The rapid immunoassay test strip for Ralstonia solanacearum provided in the above technical solution has strong specificity and broad detection spectrum, can recognize Ralstonia solanacearum strains collected in nationwide major tobacco planting provinces such as Yunnan, Guizhou, Sichuan, Chongqing, Hunan, Hubei,
Henan and Shandong, does not have any cross reaction with other common bacteria
such as Escherichia coli, Bacillus and Pseudomonas, and therefore has good practical
application value.
Brief Description of the Drawings
The accompanying drawings constituting a part of the present invention are used for
providing a further understanding of the present invention, and the schematic
embodiments of the present invention and the descriptions thereof are used for
interpreting the present invention, rather than constituting improper limitations to the
present invention.
Fig. 1 illustrates sensitivity test of a rapid immunoassay test strip for Ralstonia
solanacearumaccording to the present invention, 108, 107, 106, 105, 104, and 0 cfu/mL
from left to right in thefigure.
Detailed Description of Embodiments
It should be noted that the following detailed descriptions are exemplary and are
intended to provide further descriptions of the present application. All technical and
scientific terms used herein have the same meanings as commonly understood by
those ordinary skilled in the art to which the present application belongs, unless
specified otherwise.
It should be noted that terms used herein are intended to describe specific
embodiments only rather than to limit the exemplary embodiments according to the
present application. As used herein, the singular form is also intended to comprise the
plural form unless otherwise indicated in the context. In addition, it should be
understood that when the terms "contain" and/or "comprise" are used in the
description, they are intended to indicate the presence of features, steps, operations,
devices, components and/or combinations thereof. In the following specific
embodiments, the experimental methods with specific conditions that are not
indicated usually follow the conventional methods and conditions of molecular biology in the art. Such techniques and conditions are fully explained in the literature.
See, for example, Sambrook et al., Molecular Cloning: Laboratory Manual for the
techniques and conditions, or follow the conditions recommended by the
manufacturer.
In a specific embodiment of the present invention, a rapid immunoassay test strip for
Ralstonia solanacearum is provided. The test strip includes a bottom board, one side
of the bottom board is sequentially pasted with an absorbent pad, a detection pad, a
gold-labeled pad and a sample pad from top to bottom, the adjacent pads are
overlapped and connected at the junction, the detection pad is based on a
nitrocellulose membrane, the nitrocellulose membrane is provided with a quality
control line and a detection line horizontally from top to bottom, the detection line is
located below the quality control line, the detection line is coated with an
anti-Ralstonia solanacearum polyclonal antibody, and the quality control line is
coated with a goat anti-mouse IgG; the gold-labeled pad is sprayed with a
nano-gold-labeled anti-Ralstonia solanacearum monoclonal antibody; and the
anti-Ralstonia solanacearum monoclonal antibody is secreted by a hybridoma cell
strain.
In another specific embodiment of the present invention, a heavy chain variable
region of the anti-Ralstonia solanacearum monoclonal antibody includes an amino
acid sequence shown in SEQ ID NO. 1, and a light chain variable region thereof
includes an amino acid sequence shown in SEQ ID NO. 2.
In another specific embodiment of the present invention, the amino acid sequence of
the heavy chain variable region of the anti-Ralstonia solanacearum monoclonal
antibody has at least 80% of homology compared with SEQ ID NO. 1; more
preferably, at least 90% of homology; most preferably, at least 95% of homology; for
example, at least 96%, 97%, 98%, or 99% of homology.
In another specific embodiment of the present invention, the amino acid sequence of
the light chain variable region of the anti-Ralstonia solanacearum monoclonal
antibody has at least 80% of homology compared with SEQ ID NO. 1; more
preferably, at least 90% of homology; most preferably, at least 95% of homology; for example, at least 96%, 97%, 98%, or 99% of homology.
In another specific embodiment of the present invention, in the rapid immunoassay
test strip for Ralstonia solanacearum, the absorbent pad has a length of 18 mm and a
width of 4 mm; the detection pad has a length of 25 mm and a width of 4 mm; the
gold-labeled pad has a length of 5 mm and a width of 4 mm; the sample pad has a
length of 22 mm and a width of 4 mm, and the overlapping length of the adjacent pads
is 5 mm; the absorbent pad is an absorbent board; and the bottom board is cardboard.
In another specific embodiment of the present invention, the distance between the
quality control line and the detection line on the detection pad is 5 mm, and the
distance between the detection line and the upper edge of the nitrocellulose membrane
is 15 mm.
In another specific embodiment of the present invention, nano-gold used in the
gold-labeled pad has a particle size of 40 nm; per centimeter of spraying length on the
gold-labeled pad requires 100 ng of nano-gold-labeled anti-Ralstonia solanacearum
monoclonal antibody; and per centimeter of spraying length on the detection line of
the detection pad requires 100 ng of anti-Ralstoniasolanacearumpolyclonal antibody.
In another specific embodiment of the present invention, a preparation method of the
rapid immunoassay test strip for Ralstonia solanacearumis provided. The preparation
method includes: adding an anti-Ralstonia solanacearum monoclonal antibody into
nano-gold to obtain a nano-gold-labeled anti-Ralstonia solanacearum monoclonal
antibody, and spraying the monoclonal antibody onto a gold-labeled pad;
The preparation method further includes: pasting an absorbent pad, a detection pad,
the gold-labeled pad and a sample pad on a bottom board in sequence to form the test
strip;
The detection pad is based on a nitrocellulose membrane, the nitrocellulose membrane
is provided with a quality control line and a detection line thereon in sequence, the
detection line is coated with an anti-Ralstoniasolanacearumpolyclonal antibody, and
the quality control line is coated with a goat anti-mouse IgG.
In another specific embodiment of the present invention, the anti-Ralstonia
solanacearum monoclonal antibody can be prepared by a conventional monoclonal antibody preparation method. In a specific embodiment of the present invention, the preparation method includes: animal immunization, cell fusion, cell strain screening, and identification of an antibody subtype secreted by hybridoma cells.
The immunogen in the animal immunization is Ralstonia solanacearum, and the
immunized animal is a mouse, such as a BALB/C mouse.
In the identification of the antibody subtype secreted by the hybridoma cells, the
antibody is an IgG2b antibody. Experiments have shown that the monoclonal antibody
obtained by the present invention can not only be paired with the anti-Ralstonia
solanacearum polyclonal antibody 0334, but also has high stability (thus prolonging
the shelf life of the test strip), broad detection spectrum and high sensitivity, and
therefore, is very suitable for rapid detection of Ralstoniasolanacearum.
The preparation method further includes preparation of the anti-Ralstonia
solanacearum polyclonal antibody. The anti-Ralstonia solanacearum polyclonal
antibody is obtained by a conventional polyclonal antibody preparation method. The
immunogen is Ralstonia solanacearum, and the immunized animal may be a rabbit,
such as a New Zealand white rabbit.
In another specific embodiment of the present invention, application of the above test
strip in the detection of Ralstoniasolanacearum is provided.
In another specific embodiment of the present invention, a method for rapidly
detecting Ralstonia solanacearum is provided. The method includes: detecting a
sample to be tested on the test strip, and analyzing the detection result.
In another specific embodiment of the present invention, the detecting method
includes: cutting an appropriate amount of plant sample into a grinding bag, folding a
frosted surface of the single-sided grinding bag in half along the middle, fully
grinding the sample in the double-folded curved surface, adding 2 mL of sample
buffer, mixing well to obtain a sample solution to be tested; sucking the test solution
by a plastic dropper, dropping into a sample end of the test strip, laying flat, reacting
for 5-10 minutes, and determining the result according to the colors of the test line
and the quality control line.
In another specific embodiment of the present invention, the appropriate amount of plant sample refers to a sample of about 10 mm*10 mm cut at a disease and health junction. In another specific embodiment of the present invention, the dosage of the sample solution to be tested is 150 pL, and the detection time is 5-10 minutes. Experiments have proved that the test strip prepared by the present invention has strong specificity and broad detection spectrum, can recognize Ralstonia solanacearum strains collected in nationwide major tobacco planting provinces such as Yunnan, Guizhou, Sichuan, Chongqing, Hunan, Hubei, Henan and Shandong, and does not have any cross reaction with other common bacteria such as Escherichia coli, Bacillus and Pseudomonas. The sensitivity of the test strip can reach xI104 Cfu/mL.
The present invention will be further explained below by embodiments, which do not constitute limitations to the present invention. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. It should be noted that the Ralstonia solanacearum RS-10 used in the following embodiments was actually disclosed in the patent CN108624543B (that is, the Ralstonia solanacearum in Embodiment 1), and the public can obtain the strain from Tobacco Research Institute, Chinese Academy of Agricultural Sciences. Embodiment 1: Screening of anti-Ralstonia solanacearum monoclonal antibody 1. Animal immunization Ralstonia solanacearumRS-10 was selected, and an LB plate was streaked and placed in a 28°C incubator to culture the Ralstonia solanacearum for 24 to 72 h; a single Ralstonia solanacearumcolony that grew better on the LB plate was picked, placed in an LB liquid medium, then put into a 28°C, 200 r/min shaker and cultured for about 10 to 12 h, an OD of the bacterial solution was tested, and if the OD value was >1.0, the medium was transferred; if the OD value was not >1.0, the colony was continuously cultured until the OD value was >1.0; the transferred medium was put into the 28°C, 200 r/min shaker and continuously cultured for about 5 to 6 h, and when 1.0<OD<2.0, a bacterial suspension was obtained; the bacterial suspension was centrifuged under the conditions of 4°C, 6000 rpm/min and 10 min, and the supernatant was discarded; the bacterial suspension was suspended with 0.9% sterilized physiological saline and centrifuged under the conditions of 4°C, 6000 rpm/min and 10 min, the supernatant was discarded, and this step was repeated once; the bacterial suspension was suspended with 0.9% physiological saline to prepare a bacterial suspension with OD=1.0; the bacterial suspension was charged into a pre-treated dialysis bag, treated in 2% glutaraldehyde solution for 4 h, and then dialyzed in 0.9% NaCl solution for 72 h, and the dialysate was changed every 6 h. The dialyzed bacterial suspension was dispensed with 1 ml per tube and stored at -20°C. Ten 6-week-old female BALB/C mice were purchased and immunized with the above bacterial suspension. For the first immunization, the bacterial suspension was mixed and emulsified with the same volume of Freund's complete adjuvant, and then the mice were injected intraperitoneally. The second immunization was carried out 3 weeks after the first immunization, Freund's incomplete adjuvant was mixed and emulsified with the same volume of bacterial suspension, and the mice were injected intraperitoneally. The interval between the third immunization and the second immunization was 2 weeks, and the immunization method and dosage were the same as those of the second immunization. The fourth immunization was carried out 2 weeks after the third immunization, the method and dosage were the same as those of the second immunization, and the immunization dosage was 200 pl per mouse each time. After the third immunization, 1 week after each immunization, blood was collected from the tail vein, and the serum titer of the mice was monitored by an indirect ELISA method. When the titer no longer rose, the mice corresponding to the serum with relatively high titer were selected for booster immunization, and the immunization dosage was 200 pl of bacterial suspension. 2. Cell fusion Three days after the booster immunization, mice spleens were collected under aseptic conditions, the obtained spleen cell suspension was transferred to a 50 ml sterile centrifuge tube, the volume was adjusted to 35 ml, and the obtained SP2/0 cells were centrifuged at 1000 rpm for 10 min. The supernatant was discarded. If there was still a lot of fat in the precipitate, the precipitate can be re-suspended and washed once.
The SP2/0 cells and the spleen cells were mixed according to a certain ratio, 1:5 in
this experiment, the volume of the centrifuge tube was adjusted to 35 ml, the cells
were centrifuged at 1000 rpm for 10 min, and the supernatant was discarded; the
centrifuge tube was tilted about 45, the surface of a super clean bench was gently and
continuously tapped until cell clusters were completely loose, and then the centrifuge
tube was placed in a 37°C water bath kettle in the super clean bench for warm bath; 1
ml of 50% PEG1500 was sucked, preheated at 37°C and completely dropped into the
cell clusters within 1 min, with 100 1 added uniformly at the first 15 s, 200 1 at the
second 15 s, 300 [ at the third 15 s, and 400 [ at the final 15 s, the centrifuge tube
was rotated while adding, followed by standing for 1 minute; a preheated DMEM
medium was added with an elbow dropper to terminate PEG reaction, with 3 s/drop at
the first 1 min, 2 s/drop at the second 1 min, and 1 s/drop at the third 1 min, and the
volume was fixed to 30 ml at the last minute, followed by standing for 10 min;
centrifugation was carried out at 800 rpm for 8 min, the supernatant was discarded, 6
mL of cells was suspended at the bottom of the centrifuge tube, and blowing by force
was forbidden, otherwise, the fusion cells dispersed easily; the cell liquid was
dispensed into two 50 mL centrifuge tubes, each with a volume of 35 mL, the cell
liquid was dropped into a 96-well cell plate containing feeder cells with the elbow
dropper, 2 drops/well, and edges were sealed; culture was carried out at 37°C in a 5%
C02 incubator; and 24 h later, two drops of 37°C preheated HAT medium was added
to each well.
3. Screening of cell strains
The screening of fusion cells was mainly to screen out positive clones that can secrete
antibody by using the HAT medium. Under the HAT medium, only hybridoma cells
that secreted a predetermined specific antibody can grow, while other fusion cells
cannot grow. The cell culture supernatant was taken 7 to 8 days after cell fusion as a
primary antibody, tested for the first time by an indirect ELISA method, and tested for
the second time after 10 to 11 days. If both test results were positive, it indicated there
were hybridoma cells that can secrete antibodies in such wells, and a second step of
monoclonal screening can be carried out. If colors appeared in only one test, the growth of the cells should be continuously monitored. Whether these wells were subjected to third indirect ELISA test was determined as required. After screening by the HAT medium, cells that can secrete antibodies were in positive cell wells. However, these positive cell wells cannot guarantee only one clone in one well. Monoclonalization of cells was required to separate these polyclonal cells, and the common method was a limiting dilution method. (1) The suspension in the wells that were positive in the two ELISA tests was transferred with a pipette to a 24-well plate with wells denoted as A wells, the original wells were supplemented with the HAT medium, and the A wells were supplemented with 2 mL of HAT medium, denoted as B, C and D wells; (2) The cells in the A wells were tapped uniformly with the pipette, and 200 1 was sucked and added into the B, C and D wells for the same dilution treatment; (3) After 3-4 days, adherent cells were counted, about 100 hybridoma cells were suspended and transferred to a 50 mL sterile centrifuge tube, the HAT medium was fixed to a volume of 30 mL, and the cells were uniformly spread in the 96-well plate, sealed, marked with date and time, and cultured in the 37°C, 5% C02 incubator; (4) After 6-9 days, when clonal cells visible to the naked eye appeared, the cell supernatant was taken for indirect ELISA detection and observed under an inverted microscope, positive wells with only single clone growth were marked, and the above operation was repeated; (5) 3-5 times of operation was repeated until the positive rate was 100%. 4. Identification of antibody subtype secreted by hybridoma cells (1) Coating: the prepared Ralstonia solanacearum coating detection agent and a coating buffer were diluted by 1:(8-10) times, added to a 96-well ELISA plate with 100 1l/well, and incubated at 37°C for 2 h; (2) Washing the plate: the plate was washed five to six times with a prepared washing solution PBST, and patted dry with absorbent paper to reduce experimental errors; (3) Blocking: a 1% gelatin blocking solution was prepared and added with 200 1t/well, the solution was shaken at room temperature for 1.5 h, and the plate was washed;
(4) Adding a primary antibody: the culture supernatant of positive hybridoma cells
can be added as a primary antibody with 100 l/well, and shaken at room temperature
for 1 h, and the plate was washed;
(5) Adding a secondary antibody: HRP-labeled goat anti-mouse IgA, IgG1, IgG2a,
IgG2b, or IgM was diluted with 0.15 M PBS (pH=7.4) at a ratio of 1:500, 100 l/well,
followed by shaking at room temperature for 1 h, and the plate was washed;
(6) Matching with a color development solution: the color development solution was
mainly prepared from an ABTS solution, a citric acid solution with a pH value of 4,
and 30% H 2 0 2 at a ratio of 200 l:10 mL:10 l;
ABTS was powder, 15 mg of the powder was weighed and dissolved in 1.0 mL of
triple-distilled water, and the solution was stored at 4°C in the dark; the citric acid
solution with a pH value of 4 was obtained by weighing 525 mg of citric acid powder,
dissolving in 50.0 mL of triple-distilled water, stirring, and adjusting the pH value to
4;
(7) Color development: the prepared color development solution was added, 100
1t/well; the antibody will appear light green or dark green in corresponding subtype
wells, and appear colorless in other wells;
(8) Measurement of absorbance: the absorbance of each well was measured by a
microplate reader at a wavelength of 450 nm.
Embodiment 2: Sequencing of variable region of anti-Ralstonia solanacearum
monoclonal antibody
1. Extraction of total RNA from positive monoclonal hybridoma cell strains
A Tiangen total RNA extraction kit was used for experiment. After the monoclonal
cells in a cell culture flask grew to a logarithmic phase, the medium was discarded,
1xPBS was added for washing once, then PBS was added, cells were blown, and the
cell suspension was collected; the cell suspension was centrifuged at 8000 rpm for 5
min, and the supernatant was discarded. According to the number of cells, 250 1 of
RNA-easy can be added to digest and lyse 104 to 106 cells. The cell suspension was
repeatedly blown by the tip of the pipette till no particles existed; 2/5 RNA-easy
volume of RNase-free ddH2 0 was added, and the cell suspension was shaken vigorously for 15 s, and stood for 5 min at room temperature; the cell suspension was centrifuged at 12000 rpm for 15min, the supernatant was transferred to a clean centrifuge tube, and an equal volume of isopropanol was added, followed by mixing upside down and standing at room temperature for 1 h; the cell suspension was centrifuged at 12000 rpm for 10 min, usually a white gelatinous precipitate can be seen, the supernatant was discarded, and the precipitate was retained; 500 1 of 75% ethanol (prepared with RNase-free ddH 20) was added, and the precipitate was inverted several times, fully washed and suspended; the suspension was centrifuged at
8000 rpm for 3 min, the previous step was repeated, and the supernatant was
discarded; 70 1 of RNase-free ddH 20 was added to dissolve the precipitate, and the
suspension was repeatedly blown and mixed uniformly by the tip of the pipette and
transfered to a clean centrifuge tube to obtain an RNA solution; an A280 value of the
RNA solution was measured by a Nano Drop TM 2000 spectrophotometer, the purity
and concentration of RNA were tested, and the RNA solution was stored at -20°C for
later use.
2. Identification on total RNA
Sample preparation: 1 pg of RNA was diluted with RNase-free ddH 20 to 8 pl in a
PCR tube, and then 1 l of RNA Loading Buffer was added, followed by uniform
mixing; an electrophoresis tank was prepared: a nucleic acid gel electrophoresis tank
was washed with tap water and rinsed with 1xTAE solution 2 to 3 times, and 1xTAE
electrophoresis buffer was poured; sample application and gel running: gel was taken
out and put into the electrophoresis tank, the buffer completely submerged the gel, the
sample was added to sample wells by the pipette, and parameters 150 V and 12 min
were set; and result analysis: after the gel running, power was turned off, and the gel
was taken out, exposed and photographed.
3. RNA reverse transcription cDNA
A TaKaRa reverse transcription kit was used for experiment. Removal of genomic
DNA: 4 1 of RNA obtained as a template, 8 1 of RNase-free ddH20, and 4 1 of
4xgDNA wiper Mix were prepared into a mixture in a PCR tube, 42°C water bath, 2
min; PCR amplification of the reverse transcription reaction system: 4 1 of
5xHiScriptIII qRT SuperMix was added to the mixture after water bath treatment,
followed by uniform mixing. The program was set at 37°C for 15 min, and
amplification was carried out 85°C for 5 sec; an amplified product cDNA was
obtained, its concentration was tested, the cDNA was used as a template for next PCR
amplification and sequencing, and the cDNA was dispensed and stored at -20°C.
Genes in the variable region gene were cloned by PCR and sent to the company for
sequencing to obtain a variable region sequence.
Embodiment 3: Preparation of anti-Ralstonia solanacearum polyclonal antibody
The immunogen in Embodiment 1 was used to immunize New Zealand white rabbits,
over 6 months old, weighing about 2.5 kg. The dosage of the immunogen for each
immunization was 200 1. After emulsification, the rabbits were immunized with
multiple subcutaneous injections on the back and neck.
The initial immunization and the second immunization were separated by 2 weeks, the
remaining two immunizations were separated by 3 to 4 weeks, and serum titers were
measured one week after the third immunization. According to the results of serum
titers, the whole blood of rabbits was collected after the fifth immunization, and serum
was prepared to obtain an anti-Ralstoniasolanacearumpolyclonal antibody, named as
anti-Ralstoniasolanacearumpolyclonal antibody 0334.
Embodiment 4: Comparison of characteristics of screened monoclonal antibody
Through the method of Embodiment 1, 10 strains of anti-Ralstonia solanacearum
were screened, of which 3 strains were subtype IgM and 7 strains were IgG2b. After
paired with the anti-Ralstonia solanacearum polyclonal antibody 0334, 2 strains of
IgM and 5 strains of IgG2b monoclonal antibody can be paired as a sandwich
immunoassay test strip method.
1. Test on stability
7 strains of monoclonal antibody can be paired with 0334 to form sandwich
immunoassay test strips for detecting Ralstonia solanacearum. The test strips were
stored in vacuum, dry, and room temperature, and their stability was tested after 7
days, 14 days, 30 days, and 60 days. It was found that the test strip paired with IgM
antibody showed different degrees of color reduction after 14 days; and the test strip paired with IgG2b antibody still maintained a stable color effect after 60 days. This phenomenon was expected to be due to the higher stability of IgG2b antibody than
IgM antibody, larger molecular weight of IgM antibody, difficult operation, etc.
2. Test on broad spectrum
Five sandwich immunoassay test strips that passed the stability test were tested for
broad spectrum of Ralstonia solanacearum collected from all over the country. A total
of 15 Ralstonia solanacearumsfrom all over the country were collected for testing. It
was found that the test strip on which the monoclonal antibody 3H3 (an amino acid
sequence of its heavy chain variable region was shown in SEQ ID NO. 1, and an
amino acid sequence of its light chain variable region was shown in SEQ ID NO. 2)
was paired with 0334 can identify all the 15 solanacearums. The remaining 4 types of
test strips cannot identify all the 15 solanacearums. Relevant content of the test on the
broad spectrum of Ralstonia solanacearumby the 3H3 and 0334 paired test strip was
shown in Embodiment 5.
Embodiment 5: Test on broad spectrum by rapid immunoassay test strip for
Ralstonia solanacearum
1. Identification of test strains using genetic molecular technology (PCR)
A total of 90 strains for testing were collected from crops with suspected symptoms
across the country. Ralstonia solanacearum specific detection primers RsolfliC
(Rsol_fliC-F:5'-GAACGCCAACGGTGCGAACT-3', SEQ ID NO.3;Rsol-fliC-R:5'-GGCGGCCTTCAGGGAGGTC-3', SEQ ID NO.4) were used for PCR amplification of the 90 strains. 25 L of PCR system contained 5.0 L of
5xPCR buffer, 1.0 L of DNA template (100 ng L- 1), 0.75 L of dNTPs (10 mM),
0.75 L of forward primer (10 M), 0.75 L of reverse primer (10 M). 2.5 pL of
MgCl2 (25 mM), 0.25 pL of Taq DNA polymerase (5 U pL-1 Promega), and the
balance of the ddH 20. A standard Ralstonia solanacearum strain was used as a
positive control.
PCR reaction conditions: in a PCR amplifier, pre-denaturation at 95°C for 5 min,
denaturation at 95°C for 30 s, annealing at 55°C for 40 s, extension at 72°C for 30 s,
30 cycles, heat preservation at 72°C for 10 min, and storage at 4°C.
The amplified product was electrophoresed in a 1% agarose gel (1xTAE Buffer) for
15 min (voltage 220 V), and the gel was taken out and put into a digital image
analyzer for analysis (see Table 1 for results).
2. Measurement of tested strains by rapid immunoassay test strip for Ralstonia
solanacearumstrains
90 strains were taken for testing, streaked on an NA plate medium, and cultured in a
28°C thermostatic incubator. After colonies grew 1-3 days, single colonies were
picked, inoculated in the NA medium and cultured on a shaker (28°C, 200 rpm/min).
When the strains grew to an OD of 0.3, the cultured bacterial solution was taken,
centrifuged at 4°C and 6000 r/min for 5 min, centrifuged with a test strip
chromatographic solution, washed twice and then suspended to obtain a 108 cfu/mL
bacterial suspension for later use.
150 1 of the bacterial suspension was dropped onto the sample pad of the rapid
immunoassay test strip for Ralstonia solanacearum. After 5-10 minutes of
chromatography, the results were determined on the following basis. The
determination results were shown in Table 1.
(1) The quality control line (C) showed a color, and the test line (T) showed a red
band, indicating positive;
(2) The C line showed a color, and the T line did not show a red band, indicating
negative;
(3) The C line did not show any color or only the T line showed a color, indicating an
incorrect operation process or an invalid test strip.
Table 1 Summary table of specific test with rapid immunoassay test strips for
Ralstonia solanacearum
Serial PCR result Test result of test strip Number of strains number Positive Negative Positive Negative
14 suspected strains
1 collected from all over 9 5 9 5
the country
30 suspected strains 2 collected from all over 23 7 23 7
the country
46 suspected strains
3 collected from all over 32 14 32 14
the country
Embodiment 6: Test on field samples by rapid immunoassay test strip for
Ralstonia solanacearum
Field sampling and processing
Diseased tissues with suspected tobacco symptoms were collected in the field,
samples of about 10 mmx10 mm (at disease and health junctions) were tore off and
placed in single-sided frosted grinding bags, frosted surfaces of the grinding bags
were folded in half along the middle, the samples were placed in the double-folded
curved surfaces and fully ground, and 2 mL of sample buffer was added, followed by
uniform mixing for testing.
100-200 pL of the test solution was sucked with a plastic dropper, dropped into a
sample region of the test strip, and reacted for 5-10 minutes. The results were
determined on the following basis.
(1) The quality control line (C) showed a color, and the test line (T) showed a red
band, indicating positive;
(2) The C line showed a color, and the T line did not show a red band, indicating
negative;
(3) The C line did not show any color or only the T line showed a color, indicating an
incorrect operation process or an invalid test strip.
Finally, it should be noted that the above descriptions are only preferred embodiments
of the present invention and are not intended to limit the present invention. Although
the present invention is described in detail with reference to the foregoing
embodiments, the technical solutions described in the foregoing embodiments can still
be modified or some of them can be equivalently substituted by those skilled in the
art. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention shall fall into the protection scope of the present invention.
SEQUENCE LISTING.txt Jul 2021
<110> Tobacco Research Institute of Chinese Academy of Agricultural(Qingzhou Tobacco Research Institute of China National Tobacco Company)
<120> Rapid immunoassay test strip for Ralstonia solanacearum and preparation method and application thereof <130> 2021104199
<160> 4
<170> PatentIn version 3.3
<210> 1 <211> 60 <212> PRT <213> Artificial sequence
<400> 1
Leu Pro Phe Tyr Phe Ser Ala Trp Ser Gln His Arg Thr Phe Glu Glu 1 5 10 15
Cys Tyr Asn Val Ala Asp Arg Ser Asn Leu Pro Gly Leu Gln Pro Ala 20 25 30
His Cys Asn Trp Leu Glu Glu Asn Leu Ser Gln Ile His Cys Leu Gln 35 40 45
Ser Asp Gln Gly Pro Gln Ile Pro Asn Trp Met Gln 50 55 60
<210> 2 <211> 56 <212> PRT <213> Artificial sequence
<400> 2
Ser Ile Pro Glu Val Asn Glu Thr Val Leu Val Ser Ala Gly Thr Arg 1 5 10 15
Pro Asn Ser Ser Phe Asp Cys Tyr Tyr Leu Lys Gly Ser Asp Trp Thr 20 25 30
Cys Ser Ser Asn Asn Pro Ser Val Leu Leu Thr Asn Pro Gly Arg Met Page 1
SEQUENCE LISTING.txt Jul 2021
35 40 45
Glu Thr Gly Glu Leu Gln Cys Gln 50 55
<210> 3 <211> 20 2021104199
<212> DNA <213> Artificial sequence
<400> 3 gaacgccaac ggtgcgaact 20
<210> 4 <211> 19 <212> DNA <213> Artificial sequence
<400> 4 ggcggccttc agggaggtc 19
Page 2
Claims (10)
1. A rapid immunoassay test strip for Ralstonia solanacearum, wherein the test strip
contains an anti-Ralstoniasolanacearummonoclonal antibody, a heavy chain variable
region of the anti-Ralstonia solanacearum monoclonal antibody contains an amino
acid sequence shown in SEQ ID NO. 1, and a light chain variable region thereof
contains an amino acid sequence shown in SEQ ID NO. 2.
2. The test strip according to claim 1, wherein the rapid immunoassay test strip for
Ralstonia solanacearum comprises a bottom board on which an absorbent pad, a
detection pad, a gold-labeled pad and a sample pad are arranged in sequence, and the
adjacent pads are overlapped and connected at the junction.
3. The test strip according to claim 2, wherein the detection pad is based on a
nitrocellulose membrane, the nitrocellulose membrane is provided with a quality
control line and a detection line thereon in sequence, the detection line is coated with
an anti-Ralstonia solanacearum polyclonal antibody, and the quality control line is
coated with a goat anti-mouse IgG; and
the gold-labeled pad is sprayed with the anti-Ralstonia solanacearum monoclonal
antibody labeled by nano-gold.
4. The test strip according to claim 2, wherein the length of the absorbent pad is
controlled to 15-20 mm, and the width is controlled to 3-5 mm; the length of the
detection pad is controlled to 20-25 mm, and the width is controlled to 3-5 mm; the
length of the gold-labeled pad is controlled to 3-7 mm, and the width is controlled to
3-5 mm; the length of the sample pad is controlled to 20-25 mm, and the width is
controlled to 3-5 mm; the overlapping length of the adjacent pads is controlled to 3-6
mm; or,
the distance between the quality control line and the detection line on the detection
pad is controlled to 3-6 mm, and the distance between the detection line and the upper
edge of the nitrocellulose membrane is 13-18 mm.
5. The test strip according to claim 2, wherein the particle size of the nano-gold used
in the gold-labeled pad is controlled to 30-50 nm; per centimeter of spraying length on
the gold-labeled pad requires 80-120 ng of nano-gold-labeled anti-Ralstonia solanacearum monoclonal antibody; and per centimeter of spraying length on the detection line of the detection pad requires 80-120 ng of anti-Ralstonia solanacearum polyclonal antibody.
6. A preparation method of the rapid immunoassay test strip for Ralstonia
solanacearum according to any one of claims 1 to 5, wherein the preparation method
comprises: adding an anti-Ralstonia solanacearum monoclonal antibody into
nano-gold to obtain a nano-gold-labeled anti-Ralstonia solanacearum monoclonal
antibody, and spraying the monoclonal antibody onto a gold-labeled pad.
7. The preparation method according to claim 6, wherein the preparation method
further comprises: pasting an absorbent pad, a detection pad, the gold-labeled pad and
a sample pad on a bottom board in sequence to form the test strip.
8. The preparation method according to claim 7, wherein the detection pad is based on
a nitrocellulose membrane, the nitrocellulose membrane is provided with a quality
control line and a detection line thereon in sequence, the detection line is coated with
an anti-Ralstonia solanacearum polyclonal antibody, and the quality control line is
coated with a goat anti-mouse IgG.
9. Application of the test strip according to any one of claims 1 to 5 in the detection of
Ralstonia solanacearum.
10. A method for rapidly detecting Ralstonia solanacearum, wherein the method
comprises: detecting a sample to be tested with the test strip according to any one of
claims 1 to 5, and analyzing the detection result.
Fig. 1
Page 1/1
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| CN202110407113.3A CN113189331B (en) | 2021-04-15 | 2021-04-15 | Test strip for rapid immunodetection of bacterial wilt in tobacco, preparation method and application thereof |
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| CN101988924B (en) * | 2009-07-31 | 2014-06-11 | 上海科新生物技术股份有限公司 | Test strip for detecting anti-cyclic citrullinated peptide antibody in blood and preparation method |
| CN109030823B (en) * | 2018-10-15 | 2020-10-27 | 中国烟草总公司郑州烟草研究院 | Colloidal gold test strip for detecting ralstonia solanacearum and preparation method and application thereof |
| CN109406778B (en) * | 2018-10-15 | 2021-06-29 | 国家烟草质量监督检验中心 | Time-resolved fluorescence quantitative test strip for detecting ralstonia solanacearum in tobacco leaves and preparation method and application thereof |
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