WO2019179174A1 - Bandelette d'examen à l'or colloïdal et kit de détection de clostridium difficile - Google Patents

Bandelette d'examen à l'or colloïdal et kit de détection de clostridium difficile Download PDF

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Publication number
WO2019179174A1
WO2019179174A1 PCT/CN2018/119838 CN2018119838W WO2019179174A1 WO 2019179174 A1 WO2019179174 A1 WO 2019179174A1 CN 2018119838 W CN2018119838 W CN 2018119838W WO 2019179174 A1 WO2019179174 A1 WO 2019179174A1
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colloidal gold
dcas9
test strip
line
grna
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PCT/CN2018/119838
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English (en)
Chinese (zh)
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李程
丁秋蓉
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杭州观梓健康科技有限公司
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Publication of WO2019179174A1 publication Critical patent/WO2019179174A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/33—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Clostridium (G)

Definitions

  • the present disclosure relates to the field of biomedicine, and in particular to a colloidal gold test strip and kit for detecting C. difficile.
  • Clostridium difficile is one of the main pathogens causing intestinal infection in hospitalized patients. It is a drug-resistant bacterium that can cause fatal infections in hospitals and nursing institutions. The US Centers for Disease Control and Prevention listed it as the "most urgent drug-resistant threat list in the United States”. Clinically, about 30% of antibiotic-associated diarrhea (AAD) and 50% to 75% of antibiotics are related. Colitis and 95% to 100% of pseudomembranous colitis (PMC) are caused by Clostridium difficile infection (CDI). CDI is mainly caused by overproduction of the toxin-producing C. difficile, leading to intestinal flora imbalance and release of toxins. The main clinical symptoms are fever, abdominal pain, and watery stool diarrhea. Severe cases cause pseudomembranous colitis, and often accompanied by toxic megacolon, intestinal perforation, septic shock and other complications, and eventually lead to death.
  • AAD antibiotic-associated diarrhea
  • CDI Clostridium difficile infection
  • the C. difficile strain mainly secretes toxin A and toxin B, which are encoded by the toxin genes tcdA and tcdB, respectively.
  • toxin A-negative, toxin B-positive strains have been found; toxin A-positive, toxin-B-negative strains have not been found, and toxin B is considered to be pathogenic alone.
  • toxin B is 10 times more toxic than toxin A.
  • Toxin B mainly destroys the cytoskeleton by depolymerizing actin, causing cell necrosis and directly damaging intestinal wall cells.
  • GDH glutamate dehydrogenase
  • EIAs toxin enzyme immunoassay
  • TC toxin-producing culture
  • NAATs nucleic acid amplification test
  • EIAs detection is specific and rapid, but the sensitivity is poor; GDH is relatively fast, but this method has cross-reaction, some non-toxin C. difficile can also secrete GDH and therefore has false positives, high detection cost, need to be tested in batches to reduce personnel Operation and reagent costs, but at the same time extend the detection cycle.
  • the widely used PCR detection methods at home and abroad are high in requirements of experimental instruments, cumbersome and time-consuming. After the completion of amplification, the products need to be identified by electrophoresis and development, which is not suitable for the requirements of basic hospitals and on-site rapid detection. Therefore, the development of a C. difficile detection kit method that can be applied to the rapid screening of clinical disease control, and has high sensitivity, specificity and low cost is particularly important for the control and clinical diagnosis and treatment of disease infection.
  • the inventors of the present disclosure have unexpectedly discovered that the dCas9 protein which loses DNA cleavage activity in the CRISPR/Cas9 gene editing technology can be provided by specific binding of the dCas9 protein to the C. difficile toxin B gene under specific gRNA-mediated A C. difficile test kit with high sensitivity, high specificity, low cost and capable of screening a large number of DNA samples to be tested in a few minutes, and a preparation method thereof, thereby obtaining the technical solution of the present disclosure.
  • the present disclosure provides a colloidal gold test strip for detecting C. difficile, the colloidal gold test strip comprising an absorbent pad, a base film, a gold standard pad and a sample pad connected in sequence; a C line and a T line are disposed on the membrane, wherein the C line is coated with a dCas9 antibody, and the T line is coated with a tcdB gene fragment-bovine serum albumin complex; the gold standard pad contains a colloid Gold-labeled dCas9-gRNA protein nucleic acid complex.
  • the present disclosure provides a kit for detecting C. difficile, wherein the kit includes a sample extract and a colloidal gold test strip for detecting C. difficile as described above.
  • the present disclosure utilizes the ability of dCas9-gRNA to accurately recognize a target DNA sequence, and selects a gRNA which specifically targets a conserved gene sequence fragment of tcdB and has strong affinity, and is destructively replaced by dCas9-gRNA and DNA specific recognition.
  • the commonly used colloidal gold assay developed by antigen-antibody binding avoids differences in detection results due to differences in antibody sensitivity and specificity, and is equally effective against C. difficile variants in which antibodies are not found.
  • Pretreatment of the glass fiber membrane with a reasonable formula ensures the stability of the dCas9-gRNA colloidal gold paper dCas9-gRNA protein nucleic acid complex, and effectively improves product stability and reaction sensitivity.
  • Fig. 1 is a schematic view showing the assembly structure of a colloidal gold test paper.
  • the present disclosure provides a colloidal gold test strip for detecting C. difficile, the colloidal gold test strip comprising an absorbent pad, a base film, a gold standard pad and a sample pad connected in sequence; the base film is provided with C line and T line, wherein the C line is coated with a dCas9 antibody, and the T line is coated with a tcdB gene fragment-bovine serum albumin conjugate; the gold standard pad contains a colloidal gold label dCas9-gRNA protein nucleic acid complex.
  • the tcdB gene fragment-bovine serum albumin complex is prepared by adding 400-600 ng of the synthesized tcdB gene fragment to 8-12 wt% of bovine serum albumin for 30 minutes at room temperature to obtain a tcdB gene fragment-bovine serum albumin. Complex.
  • the dCas9-gRNA protein nucleic acid complex can be prepared by incubating the dCas9 protein with the gRNA obtained by in vitro transcription in a buffer containing 3 mol/L NaAc and having a pH of 5.2 for 60 minutes at 37 ° C to obtain dCas9-gRNA. Complex.
  • gRNA sequence in the dCas9-gRNA protein nucleic acid complex is CATATACAATTGAGACTGGA, as shown in SEQ ID NO.
  • the tcdB gene fragment sequence is GCAGTTGAATATAGTGGTTTAGTTAGAGTTGGTGAAGATGTATATTATTTTGGAGAAACATATACAATTGAGGGYGGATAGGATATATGATATGGAAAATGAAAGTGATAAATATTATTTCAATCCAGAAACTAAAAAAGCATG, as shown in SEQ ID NO.
  • the amount of DNA of the tcdB gene fragment is 400-600 ng and the amount of bovine serum albumin is 400-600 ng with respect to 1 cm of the T line.
  • dCas9 antibody is an antibody available from Abcam under the trade number Ab204448.
  • the colloidal gold-labeled dCas9-gRNA protein nucleic acid complex contained in the gold standard pad is a colloidal gold-labeled dCas9-gRNA protein nucleic acid complex solution labeled with 3-8 ⁇ g/mL colloidal gold.
  • the spraying speed of 0.5-5 ⁇ L/cm is obtained by spraying on a gold standard pad by a gold spray film machine; the colloidal gold-labeled dCas9-gRNA protein nucleic acid complex solution has a colloidal gold content of 3-8 ⁇ g/mL.
  • the content of dCas9 protein is 10-50 ng/mL
  • the content of gRNA nucleic acid is 10-50 ng/mL.
  • the TcdB gene fragment-bovine serum albumin complex coated on the T line is composed of 20-50 mg/mL of the tcdB gene fragment-bovine serum albumin conjugate solution at 1-10 ⁇ L/cm
  • the spraying speed is obtained by spraying a gold spray machine on a T line
  • the tcdB gene fragment-bovine serum albumin conjugate solution has a tcdB gene fragment content of 20-50 ng/mL
  • bovine serum white The protein content is from 0.1 to 0.3 w/v%.
  • the C-line coated dCas9 antibody is obtained by spraying 0.1-mg/mL of dCas9 antibody solution at a spraying speed of 1-10 ⁇ L/cm through a spray gold film machine on a C line. of.
  • the present disclosure also provides a kit for detecting C. difficile, wherein the kit includes a sample extract and a colloidal gold test strip for detecting C. difficile as described above.
  • the composition of the sample extract may be a 2.5-3.5 mol/L NaAc solution.
  • the extraction operation may include boiling in a boiling water bath for 30 minutes.
  • the method for performing the colloidal gold labeling of the monoclonal antibody may be a conventional method in the art, and for example, may include: heating and boiling a 0.01% by weight solution of HAuCl 4 and adding a 1% by weight solution of trisodium citrate until the solution When the color is completely transparent red, the heating is stopped after 10 minutes of reflux, and the mixture is cooled to room temperature to obtain colloidal gold; 1 mL of the prepared colloidal gold is taken, and the pH is adjusted to 8.0 with 1% by weight of K 2 CO 3 .
  • the method for preparing the immunocolloidal gold test paper may be a conventional method in the art, and for example, may include: spraying a first monoclonal antibody and a goat anti-mouse IgG onto a nitrocellulose membrane by a gold spray machine to form a mutual Parallel detection of the T line and the quality control C line, and then drying; the second monoclonal antibody labeled with colloidal gold is evenly sprayed on the gold standard pad by a gold spray film machine; then the sample pad, the gold standard pad, and the spray are The base film (nitrocellulose membrane) of the T-line and the C-line and the absorbent paper are sequentially connected and assembled, and then the package is ready for use.
  • the spacing between the C line and the T line is 2.0-7.0 mm.
  • dCas9 protein was purchased from Shanghai Nearshore Technology Co., Ltd., item number E368-01A
  • dCas9 antibody was purchased from Abcam
  • product number is Ab204448
  • tcdB gene fragment and gRNA were produced by Bioengineering (Shanghai) Co., Ltd. synthesized
  • RNase inhibitor was purchased from Shanghai Nearshore Technology Co., Ltd.
  • gRNA in vitro transcription kit was purchased from Beijing Yingmao Shengye Biotechnology Co., Ltd.
  • Nitrocellulose membrane, glass fiber membrane purchased from SARTORIUS.
  • gRNA was in vitro transcribed according to the procedure in the gRNA in vitro transcription kit.
  • the dCas9 protein was incubated with gRNA obtained by in vitro transcription in 3M NaAc pH 5.2 buffer for 60 minutes at 37 ° C to obtain the dCas9-gRNA complex. During the incubation, the RNase-free consumables must be used for experiments to prevent RNase contamination. .
  • dCas9-gRNA complex was diluted to a concentration of 1 mg/ml with 0.1 M phosphate buffer of pH 7.8.
  • the dCAS9 antibody was diluted to 0.5 mg/ml with a phosphate buffer to prepare a quality control line (C line) solution.
  • the tcdB gene fragment-bovine serum albumin conjugate is prepared by adding 400-600 ng of the synthesized tcdB gene fragment to 10 wt% bovine serum albumin for 30 minutes at room temperature to obtain a tcdB gene fragment-bovine serum albumin complex;
  • the tcdB gene fragment-bovine serum albumin conjugate was diluted to 0.5 mg/ml with a phosphate buffer solution, and the tcdB gene fragment DNA content was 500 ng/ml to prepare a detection line (T line) solution.
  • the filter paper, dCas9-gRNA colloidal gold paper, nitrocellulose membrane, and absorbent paper were sequentially adhered to the rubber sheet, and cut into strips having a width of 4 mm.
  • a colloidal gold test strip was prepared as in Example 1, except that the gRNA sequence was changed to TATAGTGGTTTAGTTAGAGT (SEQ ID NO. 5).
  • the cross-test samples were phosphate solutions containing the following common intestinal bacterial DNA concentrations of 200 ng/ml: rumen bacteria, Bifidobacterium, Rhizobium septicum, Brevibacterium M. serrata, Salmonella, Shigella infection, Campylobacter.
  • test reagent strip is placed in the sample of the cross-reaction experiment to be tested, and about 150 ul is taken, and the result is judged after 5-8 minutes. Each strain was tested 3 times.
  • Test strip prepared in Example 1
  • Test strip prepared in Example 2 Ruminococcus negative negative Bifidobacterium negative negative Rhizobium negative negative Brevibacterium methane negative Negative/positive salmonella negative negative Shigella infection negative negative Campylobacter negative Negative/positive
  • test strips prepared by the method of Example 1 were not detected when the intestinal bacterial DNA concentration was 200 ng/ml or less, and the test strip prepared by the method of Example 1 was specific. The test strips were significantly greater than those prepared by the method of Example 2.
  • Test samples Samples containing C. difficile genomic DNA concentrations of 50 ng/ml, 100 ng/ml, 120 ng/ml, 150 ng/ml, 180 ng/ml, 200 ng/ml were prepared.
  • Test reagent strip is placed in the sample to be tested to take about 150 ul of the sample, and the result is judged after 5-8 minutes, and each concentration is detected 3 times.
  • the experimental results are shown in Table 2.
  • the detection sensitivity of the test strip prepared by the method of Example 1 was 100 ng/ml, which was significantly greater than the detection sensitivity of the test strip prepared by the method of Example 2, 150 ng/ml.
  • Detection method Different types of samples to be tested are tested with three different batches of test kits, and each sample is tested three times, and the overall positive rate detection and repeatability of the samples to be tested are compared.

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Abstract

L'invention concerne une bandelette d'examen à l'or colloïdal destinée à la détection de Clostridium difficile, comprenant un tampon absorbant, un film de base, un tampon marqué à l'or et un tampon d'échantillon, reliés en séquence. Le film de base est pourvu d'une ligne C et d'une ligne T. La ligne C est revêtue d'un anticorps anti-dCas9 et la ligne T est revêtue d'un complexe de fragment de gène tcdB-sérum albumine bovine, et le tampon marqué à l'or contient un complexe d'acide nucléique de protéine dCas9-gARN marqué à l'or colloïdal. L'invention concerne également un kit de détection de Clostridium difficile, le kit comprenant la bandelette d'examen à l'or colloïdal destinée à la détection de Clostridium difficile telle que décrite ci-dessus.
PCT/CN2018/119838 2018-03-21 2018-12-07 Bandelette d'examen à l'or colloïdal et kit de détection de clostridium difficile WO2019179174A1 (fr)

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CN201810236481.4A CN108445212B (zh) 2018-03-21 2018-03-21 一种用于检测艰难梭菌的胶体金试纸条和试剂盒
CN201810236481.4 2018-03-21

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