CN113278075B

CN113278075B - Inflammation detection kit

Info

Publication number: CN113278075B
Application number: CN202110678903.5A
Authority: CN
Inventors: 马红妙; 张玲
Original assignee: Wuhu Senaichi Biotechnology Co ltd
Current assignee: WUHU SENAICHI BIOTECHNOLOGY CO.,LTD.
Priority date: 2021-06-18
Filing date: 2021-06-18
Publication date: 2021-12-14
Anticipated expiration: 2041-06-18
Also published as: CN113278075A

Abstract

The invention discloses an inflammation detection kit. The invention obtains a monoclonal antibody for specifically recognizing a Clostridium difficile GDH antigen by screening, prepares an antibody fluorescent quantum dot rapid detection test paper by using a nitrocellulose membrane marked by a quantum dot marked by the monoclonal antibody and other antibodies, analyzes the DNA sequence of Clostridium difficile toxin B, selects a conserved region to design an RPA primer and a probe, and prepares the RPA primer and the probe into a corresponding detection test paper; the two detection methods are combined for use, so that the detection accuracy can be further improved, early diagnosis is realized, the patient can be treated as soon as possible, and the method is suitable for large-scale popularization and use.

Description

Inflammation detection kit

Technical Field

The present invention relates to the field of biological detection, and more particularly to an inflammation detection kit.

Background

Difficile (CD), also known as Clostridium difficile or Clostridium difficile, is a sporulating, anaerobic, gram-positive bacterium that is present in the intestinal tract of the environment, animals, and humans. In 1935 Holl et al isolated the bacterium from the stool of healthy newborn and named as Bacillus difficilis (Bacillus difficilis) for the first time, reflecting the difficulty of its culture. In 1978, Larson et al and Bartlett et al identified Clostridium difficile as the causative agent of pseudomembranous enteritis, and the presence of toxins was associated with pathogenicity. Currently, clostridium difficile is already the main causative bacterium of nosocomial infectious diarrhea in developed countries. After clostridium difficile invades a human body, the immune system of the organism interacts with the virulence of the strain, and the clinical manifestations of the clostridium difficile can be asymptomatic carrier, mild to severe diarrhea, colitis, pseudomembranous enteritis, intestinal obstruction, toxic megacolon and fulminant enteritis which are combined with perforation, and the like, and even endanger the life. Infection or diarrhoea caused by Clostridium difficile is known as Clostridium Difficile Infection (CDI) or Clostridium difficile-associated diarrhoea (CDAD).

Difficile includes two classes, toxigenic and non-toxigenic. Its toxigenic strain can produce the following three toxins: toxin a (Tcd a), toxin B (Tcd B), and binary toxin (CDT). Among them, toxin a and toxin B have the greatest influence in the pathogenic process of clostridium difficile and are the main pathogenic factors. Toxin A is enterotoxic and mainly destroys intestinal blood vessels and mucous membranes, and toxin B is cytotoxic and can enter intestinal epithelial cells to destroy the formation of cytoskeletal actin, thereby causing clinical symptoms. A number of clinical studies in recent years have shown that CDI patients may have only toxin B present, with higher severity of clinical symptoms when patients are toxin a negative and toxin B positive.

The currently accepted "gold standard" for the diagnosis of CDI is the cytotoxicity assay and Toxigenic Culture (TC). The cytotoxicity test is not suitable for daily development in clinical laboratories due to complex operation, high price and long time consumption. The toxin-producing culture is to amplify toxin genes by Polymerase Chain Reaction (PCR) after a clostridium difficile culture is obtained by the prior culture so as to further confirm typing, and has the advantages of higher specificity and sensitivity, but the clostridium difficile culture is difficult, takes long time and needs high-precision instrument assistance. Glutamate Dehydrogenase (GDH) is an antigenic protein expressed on the surface of Clostridium difficile, has high stability and sensitivity, but has no capability of distinguishing whether strains have toxicity or not, so that the GDH needs to be combined with other methods for application.

GDH has high sensitivity, but has the defects of false positive and incapability of judging whether Clostridium difficile carries a toxin gene. Nucleic acid detection can accurately detect toxin genotyping, but genetic detection of a large number of samples is expensive, long in period, and expensive due to the need of a high-precision instrument. Therefore, the clinical sensitivity of the existing methods is not very desirable.

Disclosure of Invention

In order to better avoid the defects and provide an early and accurate diagnosis result for a patient, the detection kit is simple to prepare, low in cost, convenient to use, free of a high-precision instrument and more accurate and efficient. The kit detects the clostridium difficile by a nucleic acid-antibody dual detection method, can more effectively detect the clostridium difficile and the genotyping (namely whether the clostridium difficile carries a toxin B gene) of the clostridium difficile, thereby diagnosing as soon as possible, facilitating the treatment of patients as soon as possible and avoiding over-treatment.

The invention provides the following technical scheme:

the invention provides a monoclonal antibody G7-3 specifically binding to Clostridium difficile Glutamate Dehydrogenase (GDH), which comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1 region, a CDR2 region and a CDR3 region, and the amino acid sequences of the heavy chain CDR1 region, the CDR2 region and the CDR3 region are respectively shown as SEQ ID NO:1, 2 and 3; the light chain variable region comprises a CDR1 region, a CDR2 region and a CDR3 region, wherein the amino acid sequences of the light chain CDR1 region, the CDR2 region and the CDR3 region are shown in

SEQ ID NO

4, 5 and 6, respectively.

In another aspect, the present invention provides a monoclonal antibody G7-3 that specifically binds Clostridium difficile GDH, comprising a heavy chain variable region comprising the amino acid sequence SEQ ID NO. 7 and a light chain variable region comprising the amino acid sequence SEQ ID NO. 8.

In some embodiments, the monoclonal antibody against clostridium difficile GDH of the present invention comprises or consists of two heavy chains and two light chains, wherein each heavy chain comprises a heavy chain constant region sequence, a heavy chain variable region sequence, or a CDR sequence as described above, and each light chain comprises a light chain constant region sequence, a light chain variable region sequence, or a CDR sequence as described above. The antibody of the invention may be a full length antibody comprising a constant region, the full length antibody light chain constant region further comprising murine kappa, lambda chain sequences. The full-length antibody heavy chain constant region further comprises murine IgG1, IgG2a, IgG2b, IgG3, IgA or IgM sequences.

In some embodiments, the monoclonal antibody against clostridium difficile GDH of the present invention is a Fab fragment, Fab 'fragment, F (ab')2 fragment, Fv fragment, diabody, linear antibody, single chain antibody molecule or multispecific antibody formed from the anti-clostridium difficile antibody or antibody fragment described above.

The invention also provides a rapid detection test paper for the clostridium difficile fluorescent quantum dots, which is prepared by labeling the quantum dots with the G7-3 monoclonal antibody.

The invention provides another test strip RPA (LFD RPA) detection kit for rapidly detecting clostridium difficile toxin B, which comprises a lateral flow chromatography test strip, a pair of primers and a probe, wherein the sequence of the upstream primer is shown as SEQ ID NO. 9, the sequence of the downstream primer is shown as SEQ ID NO.10, and the sequence of the probe is shown as SEQ ID NO. 11.

Specifically, the upstream primer (SEQ ID NO: 9):

CATTAATACATGATGGTCAATATTATTTTAATG

downstream primer (SEQ ID NO: 10):

Biotin-CTATATTCAACTGCTTGTCCGTAAATATTATC

probe sequence (SEQ ID NO: 11):

FAM-CTGGAGTACAAAACATAGATGATAATTATTTCTATATAGATGAGAAG

in some embodiments, the probes of the invention are modified with a dSpacer at a position 36bp from the middle to the 5' end, thymine (dT) at positions 35bp and 37bp from the 5' end on both sides of the dSpacer molecule are replaced with a fluorophore FAM and a quencher BHQ1, respectively, and are modified at the 3' end of the probe with a blocking group C3 Spacer.

In some embodiments, the fluorescent group can be replaced by TAMARA and the quencher group can be replaced by BHQ 2; the dealkalized site can be replaced by tetrahydrofuran; the C3Spacer modification at the 3' end of the probe can be replaced by phosphorylation design or connection of biotin-TEG.

The test strip is provided with a detection line, and a molecule A is fixed on the detection line;

the primer with the sequence as shown in SEQ ID NO.10 has molecule B capable of combining specifically with the molecule A. The molecule A is a biotin ligand and the molecule B is biotin.

In the test strip RPA detection kit of the present invention, preferably, the kit further includes a hydrolysis buffer solution, magnesium acetate and ddH₂O。

In the test strip method provided by the invention, two thymine nucleotides at the middle position of an RPA probe are respectively marked with a fluorescent group and a fluorescence quenching group, an abasic site (dSpacer) is designed between the two thymine nucleotides, and the abasic site can be identified and cut by exonuclease III with 3'-5' exonuclease activity to free the fluorescent group, so that a fluorescent signal is emitted and then is detected by a fluorescence detector; meanwhile, the extensible 3' -OH is left, the DNA polymerase continues to extend and synthesize DNA by taking the probe as a ' forward primer ', and an amplification product with a double label (a fluorescent group label and an affinity label) is amplified together with a reverse primer (with an affinity label, such as biotin); the product is chromatographed on lateral flow test paper, and when encountering a test paper region (usually a line, i.e., "detection line", with streptavidin) that recognizes the affinity label, it is enriched, exhibiting a linear fluorescent signal. The test strip method does not depend on a fluorescent quantitative PCR instrument, so the cost and the application range are wider.

The invention further provides a method for detecting clostridium difficile, which comprises the steps of amplifying a sample by using the primer and the probe, and detecting an amplification product by using a nucleic acid detection test strip. And (4) detecting a result: and (3) combining with a test strip for color development, sucking 5-25 mu L of the amplification product, diluting 10-50 times with a buffer solution of 1xPBST, and detecting with the test strip marked correspondingly. And (4) interpretation of results: the positive (+) of the T line and the C line occurs at the same time, the negative (-) of the C line occurs only, and the effectiveness of the test strip needs to be considered when the T line occurs only.

In some embodiments, the invention provides a nucleic acid-antibody dual clostridium difficile detection kit comprising an RPA kit for specifically detecting clostridium difficile nucleic acid and a test strip comprising monoclonal antibodies for specifically detecting clostridium difficile; the RPA kit contains primers of SEQ ID NO 9 and 10 and a probe of SEQ ID NO 11; the test strip of the monoclonal antibody is a fluorescent quantum dot rapid detection test strip prepared by labeling quantum dots with an anti-clostridium difficile monoclonal antibody G7-3; wherein the anti-Clostridium difficile monoclonal antibody G7-3 comprises a heavy chain variable region comprising CDR1 shown in SEQ ID NO:1, CDR2 shown in SEQ ID NO:2 and CDR3 shown in SEQ ID NO:3, and a light chain variable region comprising CDR1 shown in SEQ ID NO:4, CDR2 shown in SEQ ID NO:5 and CDR3 shown in SEQ ID NO: 6.

In some embodiments, the invention provides a nucleic acid-antibody dual clostridium difficile detection kit comprising an RPA kit for specifically detecting clostridium difficile nucleic acid and a test strip comprising monoclonal antibodies for specifically detecting clostridium difficile; the RPA kit contains primers of SEQ ID NO 9 and 10 and a probe of SEQ ID NO 11; the test strip of the monoclonal antibody is a fluorescent quantum dot rapid detection test strip prepared by labeling quantum dots with an anti-clostridium difficile monoclonal antibody G7-3; wherein, the heavy chain variable region sequence of the monoclonal antibody G7-3 of the clostridium difficile is shown as SEQ ID NO. 7, and the light chain variable region sequence is shown as SEQ ID NO. 8.

Use of a kit for the detection of clostridium difficile by in vitro determination of the presence and amount of clostridium difficile in a biological sample from a subject.

In some embodiments, the biological sample of the present invention is stool, blood, plasma, serum, urine, saliva, or tissue.

Advantageous effects

The invention analyzes the DNA sequence of the clostridium difficile to obtain the specific RPA primer and probe aiming at the clostridium difficile and prepare the clostridium difficile detection reagent; meanwhile, a monoclonal antibody with a good effect is screened and obtained for clostridium difficile, the clostridium difficile fluorescence quantum dot rapid detection test paper is prepared by using the monoclonal antibody labeled quantum dot and a nitrocellulose membrane labeled by other antibodies, and the two detection methods are combined for diagnosing clostridium difficile patients, so that the detection accuracy can be further improved, early diagnosis is realized, the patients can be treated as soon as possible, and the kit is suitable for large-scale popularization and use.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention.

FIG. 1 mouse antibody subtype identification results

FIG. 2 is a graph showing the results of sensitivity evaluation of the RPA detection method

Detailed Description

The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.

Example 1 preparation of Clostridium difficile GDH antigen

After the C.difficile-630 strain of clostridium difficile is amplified and cultured, a DNA extraction kit (Tiangen) is adopted to extract DNA. The whole genome of the bacterial liquid is taken as a template, GDH gene is amplified by PCR and is connected with a prokaryotic expression vector pET-30a, and the expression vector is named as pET-30 a-GDH. Transforming the recombinant expression plasmid pET-30a-GDH into competent cells, culturing for 12-14 h, selecting positive single bacteria, shaking the positive single bacteria in an LB (kana +) liquid culture medium 2mL/tube at the temperature of 37 ℃ at the speed of 180r overnight, and on the next day, uniformly mixing the bacteria liquid and LB frozen stock solution 1:1 to serve as seed bacteria and storing at the temperature of-80 ℃ for later use. Taking 500 mu L of seed bacteria, recovering, carrying out amplification culture, shaking the bacteria for about 4 hours, adding IPTG (isopropyl thiogalactoside) with the final concentration of 0.8mmol/L for induction after the logarithmic growth period is started and the OD600 is about 0.6-0.8, and continuing shaking the bacteria for 4 hours, and collecting bacterial liquid. Resuspending the precipitate with ultrasonic lysis solution (containing 4M urea), ultrasonically lysing thallus under ice bath condition, centrifuging at 10000rpm after ultrasonic treatment, absorbing supernatant, filtering with 0.22 μ M filter, and standing at 4 deg.C for use. A5 mL size Ni column (pre-packed column) was attached to the purifier path and the column equilibrated with a 25mL volume of a Bingding buffer at a flow rate of 1mL/mL while the collection tray collection volume was set to 2 mL/tube. And (3) loading 5mL of the supernatant subjected to ultrasonic treatment from an instrument loading hole at the flow rate of 0.5mL/min, Washing and eluting the sample by using 25mL of Washing buffer and Elution buffer respectively after the sample flows through the column, collecting the eluent, desalting and concentrating the eluent by using an ultrafiltration tube, measuring the concentration, and subpackaging and storing the eluent at-80 ℃ for later use.

Example 2 preparation of biotinylated Clostridium difficile GDH antigen

The GDH antigen was biotinylated with biotinylated ligase B0101A (GnenCopoia) according to the instructions. The biotinylated GDH antigen was named Biotin-GDH. The GDH antigen was added with BufferA/B and BirA ligase and incubated at 30 ℃ for 2 h. The biotinylation efficiency was determined by ELISA. That is, the initial concentration of Biotin-GDH of 500ng/ml is as follows: diluted 2 fold and coated ELISA plates followed by incubation with SA-HRP. The biotinylation standard was used as a control to finally determine the biotinylation labeling efficiency of the Biotin-GDH as 75%.

Example 3 anti-Clostridium difficile GDH antibody preparation

Balb/c female mice were immunized using the recombinant protein GDH purified in example 1 as an antigen. The recombinant protein is mixed with Freund's adjuvant in the same volume (1 mL: 1mL), and the mixture is fully emulsified by a syringe method, and the mice are immunized by a multi-point abdominal injection method, wherein the immunization dose is 60 mu g/mouse. The second and third immunizations were mixed with Freund's incomplete adjuvant and recombinant protein GDH in equal volumes, and mice were immunized by abdominal multi-site injection at an immunization dose of 30. mu.g/mouse. And taking mouse serum 7 days after the 3 rd boosting immunization to detect titer, injecting the mouse with the highest titer by tail vein for impact immunization, and uniformly mixing the antigen with normal saline, wherein the dosage is 50 mu g/mouse. Taking immunized Balb/c mouse spleen cells, fusing the immunized Balb/c mouse spleen cells with a myeloma Sp2/0 cell line by using a PEG method, re-suspending the fused cells by using a 20% FBS-HAT-DMEM culture medium, then uniformly paving the cells in a 96-well plate at 37 ℃ and 5% CO₂And (5) culturing. And (3) after the fused cells are cultured for about one week, carrying out half-amount liquid change by using a 10% FBS-HT-DMEM culture medium, when the area of the cell colony covering the bottom of the hole reaches 1/3-1/2, taking culture supernatant, and carrying out detection on positive clones by using an indirect ELISA method. Hybridoma cells selected and cultured by cloningCloning and culturing the cell strain by a limiting dilution method. Screening by ELISA method to obtain positive hybridoma cell strain and naming it as G7-3. After expansion culture, the hybridoma cells were cryopreserved.

EXAMPLE 4 purification of monoclonal antibodies

BALB/c mice were injected intraperitoneally with 0.5 ml/mouse, 1 week before hybridoma inoculation. After 1 week, each mouse was inoculated intraperitoneally at about 1X10⁶(ii) individual hybridoma cells; and after 7-10 days, collecting ascites. Centrifuging ascites at 10000 Xg for 30min, removing precipitate, salting out with 50% ammonium sulfate, coarse extracting, dissolving with PBS, and dialyzing with flowing water for 5 hr; dialyzing and equilibrating with 0.1mol/L phosphate buffer (pH8.0) overnight; and (3) loading, eluting the hybrid protein by using 0.1mol/L phosphate buffer solution (pH8.0), eluting by using citrate eluents with different pH values, collecting elution peaks in sections, and concentrating to obtain the purified anti-clostridium difficile antibody G7-3.

Example 5 identification of anti-Clostridium difficile antibody subtypes

The positive mouse monoclonal cell line selected by indirect ELISA was subjected to subclass measurement using a subclass measuring reagent (Sigma). The microplate provided in the kit was already pre-coated with specific antibodies against mouse IgG1, IgG2a, IgG2b, IgG3, IgA, IgM, kappa light chain, lambda light chain, and the sample of anti-clostridium difficile antibody G7-3 purified in example 4 was added to the sample wells at 50 μ l per well without incubation. Adding 1X goat anti-mouse IgA + IgM + IgG-HRP into sample wells, mixing the sample wells with 50 μ l each, and incubating for 1 h. And (4) deducting liquid in the holes, adding 1XPBST to wash the holes for 3 times, and absorbing the excessive moisture by absorbent paper. Adding color development solution, and developing 100 μ l per well in dark at room temperature for 15 min. The color reaction was stopped by adding 100. mu.l of stop solution. As shown in FIG. 1, the monoclonal antibody of the present invention is IgG2b subtype.

EXAMPLE 6 monoclonal antibody sequencing

Taking out the G7-3 hybridoma cell freezing tube from liquid nitrogen, quickly melting at 37 ℃, centrifuging at 1000rpm for 5min to remove the freezing solution, placing the tube in a 100mm pore plate, culturing until the tube accounts for about 80% of the culture plate, adding 1ml of Trizol reagent (Thermo company), and extracting the total RNA of the hybridoma cells according to the instruction. Taking 2.5 mu g ofThe total RNA was added with DECP water to make the volume 11. mu.l, 1.0. mu.l of oligo (dT) (10. mu.M) was added, 1. mu.l of dNTPs (10mM) was added, the mixture was mixed well, incubated at 65 ℃ for 5 minutes and then placed on ice for 1 minute, followed by addition of 4. mu.l of RT buffer (5X), 1.0. mu.l of DTT (100mM), 1. mu.l of Ribonuclose Inhibitor and 1. mu.l of reverse transcriptase (takara Co., Ltd.), and reacted at 50 ℃ for 10 minutes. The reaction was terminated by incubation at 80 ℃ for 10 minutes, and the obtained cDNA was stored at-20 ℃. Designing specific nested PCR primer, the primer sequence used in the amplification reaction is complementary with the first frame region and the constant region of the antibody variable region, and amplifying the target gene by adopting a conventional PCR method. The primer sequences were designed according to the literature (Bodo Brocks. Specifes-Cross active scFv Against the turbine Stroma Marker "fibrous Activation Protein" Selected by phase Display From an amplified FAP)^-/-Knock-Out Mouse).

Sequencing results show that the amino acid sequences of the heavy chain and light chain variable regions of the anti-clostridium difficile antibody G7-3 are respectively shown in SEQ ID NO:7 and SEQ ID NO:8 is shown in the specification; the amino acid sequences of 3 CDRs in the heavy chain variable region of the antibody are respectively shown as SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO 3; the amino acid sequences of 3 CDRs in the light chain variable region are shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, respectively.

Example 7 determination of affinity of antibodies against Clostridium difficile

Biolayer interferometry (biolayer interferometry) was used to measure the interaction between GDH antigens and anti-clostridium difficile GDH antibodies. The Octet QKe instrument (ForteBio) was equipped with a Streptavidin (SA) biosensor. A 40 μ g/m1 Biotin-GDH antigen was coupled to the SA sensor and a serial dilution of clostridium difficile GDH monoclonal antibody from 100nM to 1.56nM was reacted with the GDH antigen coated biosensor for 10min followed by dissociation in PBS buffer for 10 min. The results were then analyzed with ForteBio data analysis software to determine the dissociation constant (Kd), which is a measure used to describe the strength of binding between antibody and antigen, Kon (1/Ms) is the rate of association of antibody-antigen complex formation (on-rate) and Koff (1/s) is the rate of dissociation of antibody-antigen complex dissociation (off-rate). As shown in Table 1, the antibodies of the present invention have high affinity, affinity K_DIs up toTo 3.72x10^-9M。

TABLE 1

Antibodies	Kon(1/Ms)	Koff(1/s)	Kd(M)
				G7-3	1.66x10⁵	6.17x10^-4	3.72x10^-9

Example 8 preparation of polyclonal antibodies against Clostridium difficile

Female rabbits were immunized with the recombinant protein GDH purified in example 1 as an antigen. The recombinant protein is mixed with Freund's adjuvant in the same volume (1m L: 1m L), and the mixture is emulsified fully by a syringe method, and the rabbit is immunized by a multipoint injection method at the back of the neck and the back, wherein the immunization dose is 500 mu g/rabbit. The secondary and tertiary immunizations are mixed with Freund's incomplete adjuvant in equal volume respectively, and are immunized by a multi-point injection method at the back of the neck and the back, and the immunization dose is 400 mu g/mouse. The four-immunization is performed by ear-edge intravenous injection, and the five-immunization is performed by ear-edge intravenous boosting once, and the immunization dose is 100 mu g/mouse. One week after five-immunization, blood is collected from the heart, the collected serum is firstly stood for 2 hours at room temperature, then is transferred to 4 ℃ for standing overnight, is centrifuged at 4000rpm for 15min, and the collected supernatant is respectively packed in a 5mL centrifuge tube and is reserved at-80 ℃ for later use. And taking part of serum to carry out serum titer detection. The residual serum was removed from-80 ℃ and thawed at room temperature, and 0.5m L serum was diluted to 10mL with 1:20 balance, filtered through a 0.45. mu.l filter and placed in a new centrifuge tube. The Protein A pre-column was connected to the purifier channel and 10mL of equilibration solution was equilibrated at a flow rate of 1 mL/min. The treated serum was sampled at a flow rate of 0.5 mL/min. Click open the collection tray at the same time, set the collection volume to 1.5 mL/tube. The mixture was washed and eluted with 10mL of the equilibration and elution solutions, and 300. mu.L/tube of the neutralization solution was added to the collection tray, so as to prevent the purified antibody from being denatured and degraded in the acidic elution solution. After purification, collecting the eluates, ultrafiltering, desalting and concentrating by an ultrafiltration tube to obtain the polyclonal antibody against the GDH of the clostridium difficile, detecting the concentration of the antibody, subpackaging and storing at-80 ℃ for later use.

Example 9 preparation and verification of Clostridium difficile fluorescent quantum dot rapid detection test paper

The G7-3 monoclonal antibody purified in example 4 is used for labeling quantum dots, and the anti-Clostridium difficile polyclonal antibody purified in example 8 is used for coating a nitrocellulose membrane and detecting the mixture. After the test paper is prepared into rapid test paper, the specificity of the test paper is detected. And (3) respectively detecting recombinant antigens diluted by Clostridium perfringens (Clostridium perfringens), salmonella enteritidis, staphylococcus aureus, pseudomonas aeruginosa, vibrio cholerae, Clostridium difficile and PBS by using the Clostridium difficile fluorescent quantum dot rapid detection test paper, and observing whether cross reaction exists. (+) represents positive result, and (-) represents negative result. The detection results are shown in table 2, and the results show that the reaction results of the clostridium difficile fluorescent quantum dot rapid detection test paper with clostridium perfringens, salmonella enteritidis, staphylococcus aureus, pseudomonas aeruginosa and vibrio cholerae are negative, and the reaction results with clostridium difficile and a recombinant antigen are positive, so that the test paper strip can be preliminarily determined to have good specificity.

TABLE 2

Sample name	Results
		Clostridium perfringens	-
Salmonella enteritidis	-
		Staphylococcus aureus	-
Pseudomonas aeruginosa	-
		Vibrio cholerae	-
Clostridium difficile	+
		Recombinant GDH antigen	+

Example 10 design of RPA-specific detection primers

Designing an RPA primer group for detecting the clostridium difficile toxin B aiming at the gene sequence of the clostridium difficile toxin B, wherein the sequence of the RPA primer group is shown as follows:

upstream primer (SEQ ID NO: 9):

CATTAATACATGATGGTCAATATTATTTTAATG

downstream primer (SEQ ID NO: 10):

Biotin-CTATATTCAACTGCTTGTCCGTAAATATTATC

probe sequence (SEQ ID NO: 11):

FAM-CTGGAGTACAAAACATAGATGATAATTATTTCTATATAGATGAGAAG, the probe is modified by dSpacer at the position 36bp away from the 5' end, thymine (dT) at the positions 35bp and 37bp away from the 5' end on both sides of dSpacer molecule are respectively replaced by a fluorescent group FAM and a quenching group BHQ1, and the 3' end of the probe is modified by a blocking group C3 Spacer.

Example 11 Clostridium difficile DNA extraction

The operation is carried out according to the instruction of a bacterial genome DNA extraction kit (Tiangen), and the specific operation steps are that 1-5ml of clostridium difficile culture solution with positive toxin B is taken, centrifuged for 1min at 10,000rpm (11,500 Xg), and supernatant is sucked up as far as possible. To the pellet of the cells, 110. mu.l of a buffer (20mM Tris, pH 8.0; 2mM Na 2-EDTA; 1.2% Triton) and 70. mu.l of a lysozyme solution (50mg/ml) were added and the mixture was treated at 37 ℃ for 30 minutes. Mu.l RNase A (100mg/ml) solution was added thereto, shaken for 15s, and left at room temperature for 5 min. Add 20. mu.l of protease K solution to the tube and mix well. Add 220. mu.l buffer GB, shake for 15s, and leave at 70 ℃ for 10 min. Add 220. mu.l of absolute ethanol, shake well and mix for 15 s. The resulting solution and flocculent precipitate were added to an adsorption column CB3 (adsorption column placed in collection tube), centrifuged at 12,000rpm (-13,400 Xg) for 30s, the waste stream was decanted, and adsorption column CB3 was placed in collection tube. To adsorption column CB3 was added 500. mu.l of buffer GD, centrifuged at 12,000rpm (. about.13,400 Xg) for 30s, the waste solution was discarded, and adsorption column CB3 was placed in the collection tube. 600. mu.l of the rinsing solution PW was added to the adsorption column CB3, and centrifuged at 12,000rpm (. about.13,400 Xg) for 30 seconds to discard the waste liquid, and the adsorption column CB3 was put into the collection tube. Transferring the adsorption column CB3 into a clean centrifuge tube, suspending and dripping 50-200 mu l of elution buffer TE into the middle part of the adsorption membrane, standing for 2-5min at room temperature, centrifuging for 2min at 12,000rpm (13,400 Xg), collecting the solution into the centrifuge tube, wherein the solution is the extracted Clostridium difficile genome DNA, and storing at-20 ℃ for later use.

Example 12 sensitivity of RPA reaction detection

Taking Clostridium difficile DNA positive to toxin B extracted in example 11 as a template, performing RPA test, performing RPA amplification by using a screened primer, setting ultrapure water as a negative control, setting the reaction temperature to 35 ℃, the reaction time to be 18min, and setting the RPA reaction system to be 50 ul, wherein 2 ul of forward and reverse primers (10 uM), 2 ul of reverse primers (10 uM), 0.6 ul of probe, 25 ul of buffer solution containing recombinase, DNA polymerase, single-strand binding protein and endonuclease IV, 1 ul of template and 17.9 ul of ddH2O, fully oscillating, uniformly mixing and instantaneously separating, finally adding 2.5 ul of 280mM magnesium acetate, and placing the reaction tube in a real-time fluorescence PCR instrument for constant temperature reaction at 36 ℃ for corresponding time; the results are shown in FIG. 2. As shown in FIG. 2, when the template concentrations were 50pg, 5pg, and 0.5pg, distinct amplification curves appeared, but when the template concentration was less than 0.5pg, no distinct amplification curve appeared, which means that the detection limit of RPA was 0.5pg, and the detection precision was better.

EXAMPLE 13 preparation and detection of Clostridium difficile RPA test strip

Streptavidin-coated gold nanoparticles were prepared by adding 200mM borax solution to 1mL gold nanoparticle solution (0.15pmol/mL) and adjusting the pH to 9.5. At the same time, 2. mu.l of streptavidin (2mg/ml) was mixed with 398. mu.l of borax solution (2mM) in another tube; the diluted streptavidin was added to the aforementioned gold nanoparticle solution in an amount of 50. mu.l, and the solution was stirred while adding. The mixture was left at room temperature for 45 minutes, 155.6. mu.l of a 2mM borax solution containing 10% BSA was added, the solution was left at room temperature for another 10 minutes, 4500g was centrifuged for 15 minutes, the liquid was aspirated, the precipitate was resuspended in 1ml of a washing solution (2mM borax solution containing 10g/L BSA), 4500g was centrifuged for 15 minutes, the liquid was aspirated, and the red precipitate was resuspended in 250. mu.l of a buffer containing 5% BSA, 137mM NaCl and 0.025% Tween-200. Sufficient streptavidin-coated gold nanoparticles were prepared in this ratio, 250. mu.l of the coated gold nanoparticles were dropped onto a 7mm by 300mm laser-cut glass fiber pad, allowed to spread evenly, and dried overnight on the bench top.

The test strip for colloidal gold lateral flow immunochromatography was prepared by diluting an anti-carboxyfluorescein antibody and biotinylated anti-mouse IgG to final concentrations of 0.5mg/ml and 1.0mg/ml, respectively, with 100mM sodium bicarbonate buffer containing 5% methanol, 2% sucrose. All antibodies were dispensed using a lateral flow reagent dispenser with a dispenser head speed set at 1-3 cm/min, preferably 2 cm/min, and a syringe pump flow rate set at 0.1-0.3 ml/min, preferably 0.1 ml/min. After completion of the spotting of both antibodies on the strip, the strip was dried at 37 ℃ for 1 hour. The test card was then assembled by first placing a 17mm x 300mm absorbent pad on the right hand downstream end of a plastic-supported nitrocellulose membrane, the two being superposed by 2 mm; a 7mm x 300mm glass fibre mat containing dried gold nanoparticles was then placed on the left hand upstream end of the nitrocellulose membrane, overlapping by 2 mm; finally, a 12mm by 300mm glass fiber sample pad was placed on the left hand end of the gold nanoparticle pad, with the two overlapping by 2 mm. After the assembly is completed, the test paper card is immediately cut into test paper strips with the width of 3mm, and the colloidal gold lateral flow immunochromatographic test paper strips are obtained, sealed, dried and stored.

In addition, the RPA kit of Clostridium difficile comprises the primers and probes designed in example 10, a hydrolysis buffer, an enzyme mixture, magnesium acetate (280mM), nuclease-free pure water, and a lateral chromatography strip with a detection line on which the molecule streptavidin is immobilized, which is capable of specifically binding to biotin at the end of primer SEQ ID NO: 10.

The test strip was used to detect the genomic DNA of clostridium difficile, salmonella enteritidis, staphylococcus aureus, pseudomonas aeruginosa, vibrio cholerae extracted in example 11 (the method is as described in example 11) respectively, so as to determine the specificity of the RPA detection method of the present invention. The RPA reaction system was 50. mu.l, with 2. mu.l forward primer (10. mu.M), 2. mu.l reverse primer (10. mu.M), 0.6. mu.l probe, 25. mu.l containing recombinase, DNA polymerase, single-strand binding protein, endonuclease IV, 1. mu.l sample and 17.9. mu.l ddH2O, mixed well with shaking and flash separated, and finally 2.5. mu.l of 280mM magnesium acetate was added. The reaction was carried out in a water bath at 35 ℃ for 18 min. The result shows that the T line and the C line of the DNA sample of the clostridium difficile are positive (+) while the C line of the DNA sample of other pathogens is negative (-) only, which indicates that the test strip kit can effectively detect the clostridium difficile, and the result is shown in the following table 3.

TABLE 3

Example 14 actual sample detection

200 samples of excrement of a clostridium difficile toxin patient are taken, and 200 negative control samples are taken at the same time, wherein the samples are all from people hospitals in Tianjin.

Total DNA of feces was extracted according to the instructions of the fecal genomic DNA extraction kit (tiangen). The method comprises the following specific steps: the stool sample 180-220mg was weighed into a 2ml centrifuge tube and the tube was placed on ice. To the sample, 500. mu.l of buffer SA, 100. mu.l of buffer SC, 15. mu.l of protease K, and 0.25g of beads were added and shaken intermittently for 1min until the sample was well mixed. Incubate at 70 ℃ for 15min, shake 2-3 times during incubation. Vortex for 15s, centrifuge at 12,000rpm (-13,400 Xg) for 3min, transfer the supernatant to a new centrifuge tube, add 10. mu.l RNase A, mix well with shaking, and then stand at room temperature for 5 min. Add 200. mu.l buffer SH, shake and mix well, put on ice for 5 min. Centrifuge at 12,000rpm (. about.13,400 Xg) for 3 min. The resulting supernatant was transferred to a new 1.5ml centrifuge tube and an equal volume of buffer GFA was added. The resulting solution was loaded into an adsorption column CR2 (adsorption column placed in collection tube), centrifuged at 12,000rpm (. about.13,400 Xg) for 30s, the waste liquid was decanted, and adsorption column CR2 was placed in collection tube. To the adsorption column CR2 was added 500. mu.l of buffer GD, centrifuged at 12,000rpm (. about.13,400 Xg) for 30s, the waste solution was decanted, and the adsorption column CR2 was placed in a collection tube. 700. mu.l of the rinsing solution PW was added, centrifuged at 12,000rpm (. about.13,400 Xg) for 30s, the waste liquid was decanted, and an adsorption column CR2 was placed in the collection tube. The adsorption column CR2 was returned to the collection tube and centrifuged at 12,000rpm (. about.13,400 Xg) for 2min to discard the waste. The adsorption column CR2 was left at room temperature for several minutes to thoroughly dry the residual rinse solution from the adsorption material. Transferring the adsorption column CR2 into a clean centrifuge tube, suspending and dripping 50 μ l of elution buffer TB into the middle part of the adsorption membrane, standing at room temperature for 2-5min, centrifuging at 12,000rpm (13,400 Xg) for 2min, and collecting the solution into the centrifuge tube. The resulting solution was fecal genomic DNA.

And (3) taking the excrement extracting solution and the excrement genome DNA, and respectively adopting fluorescent quantum dot rapid detection test paper and test paper strip RPA for detection, wherein the detection results are shown in Table 4. The results in table 4 show that the antibody fluorescent quantum dot test strip and the RPA test strip can be used for better detecting positive patients with clostridium difficile, the positive rate of the fluorescent quantum dot rapid detection test strip is 99%, the positive rate of the test strip RPA test strip is 83.5%, the positive rate of the fluorescent quantum dot rapid detection test strip and the test strip RPA combined detection of clostridium difficile reaches 100%, and the combined detection of the fluorescent quantum dot rapid detection test strip and the test strip RPA is higher than that of the single detection of the fluorescent quantum dot rapid detection test strip and the test strip RPA. In addition, when a healthy patient sample is detected, the misdiagnosis rate of the antibody fluorescence quantum dot test strip is 3%, but the accuracy of the test strip RPA is 100%, so that the diagnosis accuracy is further improved by the combined application of the fluorescence quantum dot rapid detection test strip and the test strip RPA. The combination of the detection of the GDH antigen of the clostridium difficile and the DNA detection of the toxin B can provide good supplement and enhance the accuracy of the detection result.

TABLE 4

Sequence listing

<110> Beijing Bao Picture Biotechnology Ltd

<120> an inflammation detection kit

<160> 11

<170> SIPOSequenceListing 1.0

<210> 1

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Ser Tyr Gly Asp His

1 5

<210> 2

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Asn Ile Gly Ala Arg Met Thr Asp Glu Ser Met Lys Val Cys Phe Lys

1 5 10 15

Gly

<210> 3

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Gly Ala Ser Arg Gly Asn Thr Glu Val Phe Asp Ile

1 5 10

<210> 4

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Glu Ala Arg Gly Pro Thr Ser Val Gln Gln Tyr Leu Ala

1 5 10

<210> 5

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Arg Ala Ala Ser Leu Gln Ser

1 5

<210> 6

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Gln Gln Arg Ala Ser Tyr Pro Tyr Thr

1 5

<210> 7

<211> 121

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Tyr Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Gly Asp His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Asn Ile Gly Ala Arg Met Thr Asp Glu Ser Met Lys Val Cys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Gly Ala Ser Arg Gly Asn Thr Glu Val Phe Asp Ile Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ala

115 120

<210> 8

<211> 109

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Asp Ile Val Met Thr Gln Ser Pro Ala Ser Leu Ala Met Ser Leu Gly

1 5 10 15

Lys Arg Ala Thr Ile Ser Cys Glu Ala Arg Gly Pro Thr Ser Val Gln

20 25 30

Gln Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu

35 40 45

Leu Ile Tyr Arg Ala Ala Ser Leu Gln Ser Gly Val Pro Ala Arg Phe

50 55 60

Ser Gly Ser Gly Ser Gly Ala Asp Phe Thr Leu Thr Ile Asp Pro Val

65 70 75 80

Glu Gly Asp Asp Val Ala Leu Tyr Tyr Cys Gln Gln Arg Ala Ser Tyr

85 90 95

Pro Tyr Thr Phe Gly Gly Gly Thr Lys Met Glu Ile Lys

100 105

<210> 9

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

cattaataca tgatggtcaa tattatttta atg 33

<210> 10

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ctatattcaa ctgcttgtcc gtaaatatta tc 32

<210> 11

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ctggagtaca aaacatagat gataattatt tctatataga tgagaag 47

Claims

1. A monoclonal antibody specifically binding to Clostridium difficile GDH, characterized in that the monoclonal antibody comprises a heavy chain variable region comprising CDR1 shown in SEQ ID NO:1, CDR2 shown in SEQ ID NO:2 and CDR3 shown in SEQ ID NO:3, and a light chain variable region comprising CDR1 shown in SEQ ID NO:4, CDR2 shown in SEQ ID NO:5 and CDR3 shown in SEQ ID NO: 6.

2. An antibody specifically binding with a Clostridium difficile GDH monoclonal, which is characterized in that the heavy chain variable region sequence of the monoclonal antibody is shown as SEQ ID NO. 7, and the light chain variable region sequence is shown as SEQ ID NO. 8.

3. A kit for detecting clostridium difficile, which is characterized by comprising a fluorescent quantum dot rapid detection test strip prepared from the monoclonal antibody labeled quantum dot of claim 1 or 2.

4. A kit for nucleic acid-antibody dual detection of clostridium difficile comprises the kit of claim 3 and a test strip RPA detection kit, wherein the test strip RPA detection kit comprises a pair of primers and a probe, the sequences of the pair of primers are shown as SEQ ID NO. 9 and 10, and the sequence of the probe is shown as SEQ ID NO. 11.

5. The kit of claim 4, wherein the probe is labeled with a fluorophore, a fluorescence quencher, an abasic site, and a blocking group.

6. The kit according to claim 5, wherein the fluorescent group is FAM, the fluorescence quenching group is BHQ1, the abasic site is dSpacer modification and the blocking group is C3Spacer modification.

7. The kit according to claim 5, wherein the fluorescent group is TAMARA, the fluorescence quenching group is BHQ2, the abasic site is modified with tetrahydrofuran, and the blocking group is modified with C3 Spacer.

8. The kit of any one of claims 4-7, wherein the test strip RPA detection kit further comprises a hydrolysis buffer, magnesium acetate, and ddH₂O。

9. The kit according to claim 8, wherein the RPA amplification reaction is carried out in a water bath set at 35 ℃ for 18 min.

10. Use of the kit of claim 4 in the preparation of a reagent for the detection of Clostridium difficile and genotyping thereof.