WO2019179174A1 - Colloidal gold test strip and kit for detecting clostridium difficile - Google Patents
Colloidal gold test strip and kit for detecting clostridium difficile Download PDFInfo
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- 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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- 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
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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
- G01N33/569—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
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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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
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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
- G01N33/577—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
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- 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
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- 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
Provided is a colloidal gold test strip for detecting clostridium difficile comprising an absorbent pad, a base film, a gold-labeled pad and a sample pad connected in sequence, wehrein the base film is provided with a C line and a 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 complex,and the gold-labeled pad contains a colloidal gold-labeled dCas9-gRNA protein nucleic acid complex. Also provided is a kit for detecting the clostridium difficile, wherein the kit comprises the colloidal gold test strip for detecting the clostridium difficile as described above.
Description
本公开涉及生物医药领域,具体地,涉及一种用于检测艰难梭菌的胶体金试纸条和试剂盒。The present disclosure relates to the field of biomedicine, and in particular to a colloidal gold test strip and kit for detecting C. difficile.
艰难梭菌是引起住院病人肠道感染的主要致病菌之一,是一种在医院和护理机构可引起致命性传染的耐药性细菌。美国疾控中心将其列为“美国最紧急抗药性威胁榜单”的榜首,临床上,约30%的抗菌药物相关性腹泻(antibiotic associated diarrhea,AAD)、50%~75%的抗菌药物相关性结肠炎和95%~100%的伪膜性肠炎(pseudomembranouscolitis,PMC)是由艰难梭菌感染(Clostridium difficileinfection,CDI)引起。CDI主要是由产毒素艰难梭菌过度繁殖导致肠道菌群失调并释放毒素所引起,主要临床症状为发热、腹痛、水样便腹泻。严重者引发伪膜性肠炎,且常伴有中毒性巨结肠、肠穿孔、感染性休克等并发症,甚至最终导致死亡。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.
艰难梭菌产毒株主要分泌毒素A和毒素B,分别由毒素基因tcdA和tcdB编码。目前,已发现毒素A阴性、毒素B阳性的菌株;而毒素A阳性,毒素B阴性的菌株尚未被发现,认为毒素B可单独致病。另外,毒素B的毒力较毒素A强10倍。毒素B主要通过解聚肌动蛋白,破坏细胞骨架,导致细胞坏死,直接损伤肠壁细胞。The C. difficile strain mainly secretes toxin A and toxin B, which are encoded by the toxin genes tcdA and tcdB, respectively. At present, 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. In addition, 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.
目前,粪便中艰难梭菌检测主要通过三步法进行:首先使用谷氨酸脱氢酶(GDH)试验进行初筛,GDH阳性进行毒素酶免疫方法(EIAs)试验,二者结果不一致使用细胞毒性试验(CCTA)、产毒素培养(TC)或核酸扩增试验(NAATs)确证。CCTA和TC目前是检测CDI的“金标准”,但因培 养条件苛刻、技术要求高、操作复杂及耗时,难以用于临床艰难梭菌的常规检测。EIAs检测则特异、快速,但灵敏性差;GDH较为快速,但该方法存在交叉反应、一些无毒素艰难梭菌也可分泌出GDH因此已出现假阳性,检测费用高,需要成批检测以降低人员操作及试剂成本,但同时使得检测周期延长。国内外广泛应用的PCR检测方法则因实验仪器要求高、操作繁琐、耗时,扩增完成后需要通过电泳、显影等步骤对产物进行判别,不适合基层医院及现场快速检测的要求。因此,开发一款能够适用于临床疾控快速筛查要求,且灵敏性高、特异性强且价格低廉的艰难梭菌检测试剂盒方法对于疾病感染的控制和临床诊治显得尤其重要。At present, the detection of C. difficile in feces is mainly carried out by a three-step method: first, glutamate dehydrogenase (GDH) test is used for primary screening, and GDH is positive for toxin enzyme immunoassay (EIAs) test. The results are inconsistent with cytotoxicity. Test (CCTA), toxin-producing culture (TC) or nucleic acid amplification test (NAATs) confirmation. CCTA and TC are currently the “gold standard” for detecting CDI, but due to harsh culture conditions, high technical requirements, complicated operation and time-consuming, it is difficult to use for routine detection of C. difficile. 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.
发明内容Summary of the invention
本公开的目的是提供一种灵敏性高、特异性强且价格低廉的艰难梭菌检测试剂盒。It is an object of the present disclosure to provide a C. difficile assay kit that is highly sensitive, specific, and inexpensive.
本公开的发明人出乎意料地发现:利用CRISPR/Cas9基因编辑技术中失去DNA切割活性的dCas9蛋白,通过dCas9蛋白在特定gRNA介导下与艰难梭菌毒素B基因的特异结合,就能够提供一种灵敏性高、特异性强、价格低廉且可在数分钟内对大量待测DNA样本进行筛查的艰难梭菌检测试剂盒及其制备方法,由此得到了本公开的技术方案。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.
为了实现上述目的,本公开提供一种用于检测艰难梭菌的胶体金试纸条,该胶体金试纸条包括依次相连接的吸水垫、基膜、金标垫和样品垫;所述基膜上设置有C线和T线,其中,所述C线上包被有dCas9抗体,所述T线上包被有tcdB基因片段-牛血清白蛋白复合物;所述金标垫中含有胶体金标记的dCas9-gRNA蛋白核酸复合物。In order to achieve the above object, 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.
另一方面,本公开提供了一种用于检测艰难梭菌的试剂盒,其中,该试剂盒包括样品提取液以及如上所述的用于检测艰难梭菌的胶体金试纸条。In another aspect, 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.
通过上述技术方案,本公开利用了dCas9-gRNA精确识别目标DNA序列的能力,筛选到特异靶向tcdB保守基因序列片段且亲和力强的gRNA,开创性的以dCas9-gRNA与DNA特异识别的方式取代了常用的通过抗原抗体结合所开发的胶体金检测,从而避免了因抗体灵敏性和特异性差异所导致的检测结果差异,对找不到抗体的艰难梭菌变种同样有效。采用合理配方对玻璃纤维膜进行预处理保证了dCas9-gRNA胶体金纸片dCas9-gRNA蛋白核酸复合体的稳定性,有效提高产品稳定性及反应灵敏度。Through the above technical scheme, 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.
本公开的其他特征和优点将在随后的具体实施方式部分予以详细说明。Other features and advantages of the present disclosure will be described in detail in the detailed description which follows.
附图是用来提供对本公开的进一步理解,并且构成说明书的一部分,与下面的具体实施方式一起用于解释本公开,但并不构成对本公开的限制。在附图中:The drawings are intended to provide a further understanding of the disclosure, and are in the In the drawing:
图1是胶体金试纸的组装结构示意图。Fig. 1 is a schematic view showing the assembly structure of a colloidal gold test paper.
附图标记说明Description of the reference numerals
1 样品垫 2 被衬底板 3 金标垫1 sample pad 2 by substrate plate 3 gold standard pad
4 T线 5 C线 6 基膜4 T line 5 C line 6 base film
7吸水垫7 absorbent pad
以下结合附图对本公开的具体实施方式进行详细说明。应当理解的是,此处所描述的具体实施方式仅用于说明和解释本公开,并不用于限制本公开。The specific embodiments of the present disclosure will be described in detail below with reference to the accompanying drawings. It is to be understood that the specific embodiments described herein are not to be construed
本公开提供了一种用于检测艰难梭菌的胶体金试纸条,该胶体金试纸条包括依次相连接的吸水垫、基膜、金标垫和样品垫;所述基膜上设置有C线 和T线,其中,所述C线上包被有dCas9抗体,所述T线上包被有tcdB基因片段-牛血清白蛋白偶联物;所述金标垫中含有胶体金标记的dCas9-gRNA蛋白核酸复合物。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.
其中,tcdB基因片段-牛血清白蛋白复合物的制备方法为将合成的tcdB基因片段400-600ng加入8-12wt%牛血清白蛋白中室温孵育30分钟,即得tcdB基因片段-牛血清白蛋白复合物。Wherein, 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.
其中,dCas9-gRNA蛋白核酸复合物的制备方法可以为将dCas9蛋白与体外转录获得的gRNA在含有3mol/L的NaAc且pH为5.2的缓冲液中37℃下孵育60分钟,即得dCas9-gRNA复合体。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.
可选地,其中,所述dCas9-gRNA蛋白核酸复合物中gRNA序列为CATATACAATTGAGACTGGA,如SEQ ID NO.1所示。Optionally, wherein the gRNA sequence in the dCas9-gRNA protein nucleic acid complex is CATATACAATTGAGACTGGA, as shown in SEQ ID NO.
可选地,其中,所述dCas9-gRNA蛋白核酸复合物中dCas9的氨基酸序列为DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD,如SEQ ID NO.2所示。Alternatively, wherein the amino acid sequence of the protein nucleic acid complex in the dCas9 dCas9-gRNA of DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDK AGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD, NO.2 as shown in SEQ ID.
可选地,其中,所述tcdB基因片段-牛血清白蛋白偶联物中,tcdB基因片段序列为GCAGTTGAATATAGTGGTTTAGTTAGAGTTGGTGAAGATGTATATTATTTTGGAGAAACATATACAATTGAGACTGGATGGATATATGATATGGAAAATGAAAGTGATAAATATTATTTCAATCCAGAAACTAAAAAAGCATG,如SEQ ID NO.3所示。Optionally, wherein the tcdB gene fragment-bovine serum albumin conjugate, the tcdB gene fragment sequence is GCAGTTGAATATAGTGGTTTAGTTAGAGTTGGTGAAGATGTATATTATTTTGGAGAAACATATACAATTGAGGGYGGATAGGATATATGATATGGAAAATGAAAGTGATAAATATTATTTCAATCCAGAAACTAAAAAAGCATG, as shown in SEQ ID NO.
可选地,其中,相对于1cm所述T线,tcdB基因片段的DNA量为400-600ng,牛血清白蛋白的量为400-600ng。Alternatively, wherein 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.
可选地,其中,dCas9抗体为购自Abcam的货号为Ab204448的抗体。Alternatively, wherein the dCas9 antibody is an antibody available from Abcam under the trade number Ab204448.
可选地,其中,所述金标垫中所含有的胶体金标记的dCas9-gRNA蛋白核酸复合物是由3-8μg/mL胶体金标记的胶体金标记的dCas9-gRNA蛋白核酸复合物溶液以0.5-5μL/cm的喷涂速度通过喷金点膜机喷涂在金标垫上干燥后得到的;所述胶体金标记的dCas9-gRNA蛋白核酸复合物溶液中,胶体金的含量为3-8μg/mL,dCas9蛋白的含量为10-50ng/mL,gRNA核酸的含量为10-50ng/mL。Optionally, wherein 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 μg/mL colloidal gold. The spraying speed of 0.5-5 μL/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 μg/mL. The content of dCas9 protein is 10-50 ng/mL, and the content of gRNA nucleic acid is 10-50 ng/mL.
可选地,其中,所述T线上包被的tcdB基因片段-牛血清白蛋白复合物是由20-50mg/mL的tcdB基因片段-牛血清白蛋白偶联物溶液以1-10μL/cm的喷涂速度通过喷金点膜机喷涂在T线上干燥后得到的;所述tcdB基因片段-牛血清白蛋白偶联物溶液中,tcdB基因片段的含量为20-50ng/mL,牛血清白蛋白的含量为0.1-0.3w/v%。Optionally, wherein 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 μL/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, and bovine serum white The protein content is from 0.1 to 0.3 w/v%.
可选地,其中,所述C线上包被的dCas9抗体是由0.1-5mg/mL的dCas9抗体溶液以1-10μL/cm的喷涂速度通过喷金点膜机喷涂在C线上干燥后得到的。Optionally, wherein 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 μL/cm through a spray gold film machine on a C line. of.
本公开还提供了一种用于检测艰难梭菌的试剂盒,其中,该试剂盒包括样品提取液以及如上所述的用于检测艰难梭菌的胶体金试纸条。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.
可选地,所述样品提取液的成分可以为2.5-3.5mol/L的NaAc溶液。提取的操作可以包括:在沸水浴中煮沸30分钟。Alternatively, 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.
本发明中,将单克隆抗体进行胶体金标记的方法可以为本领域常规的方法,例如可以包括:将0.01重量%的HAuCl
4溶液加热煮沸后加入1重量%的柠檬酸三钠溶液直至溶液的颜色完全变为透明的红色时,继续回流10min后停止加热,冷却至室温,即得到胶体金;取1mL制取好的胶体金,用1重量%的K
2CO
3调节pH值到8.0,加入15μg的dCas9-gRNA蛋白核酸复合物,混合均匀,室温反应40min;加入5重量%的BSA至终浓度为0.1重量%,静置30min;先用低速(1500×g)离心15分钟,弃去由凝聚的金胶粒形成的沉淀;然后用10000×g离心30分钟;仔细吸出上清,沉淀物用0.1mL含1%BSA的0.1M PBS(pH7.4)复溶,加入5%叠氮钠至终浓度为0.05%,4℃保存。
In the present invention, 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 . 15 μg of dCas9-gRNA protein nucleic acid complex, mix well, react at room temperature for 40 min; add 5 wt% BSA to a final concentration of 0.1 wt%, let stand for 30 min; first centrifuge at low speed (1500 × g) for 15 minutes, discard Precipitate formed by condensed gold colloid; then centrifuged at 10,000 × g for 30 minutes; the supernatant was carefully aspirated, and the precipitate was reconstituted with 0.1 mL of 1% BSA in 0.1 M PBS (pH 7.4), and 5% sodium azide was added. The final concentration was 0.05% and stored at 4 °C.
本发明中,制备免疫胶体金试纸的方法可以为本领域常规的方法,例如可以包括:用喷金点膜机将第一单克隆抗体和羊抗鼠IgG喷于硝酸纤维素膜上,形成相互平行的检测T线和质控C线,然后烘干;将胶体金标记的第二 单克隆抗体用喷金点膜机均匀的喷在金标垫上;然后将样品垫、金标垫、喷有T线和C线的基膜(硝酸纤维素膜)和吸水纸依次连接组装,而后裁切包装备用。In the present invention, 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.
可选地,C线和T线的间距为2.0-7.0mm。Alternatively, the spacing between the C line and the T line is 2.0-7.0 mm.
以下通过实施例进一步详细说明本发明。The invention will now be described in further detail by way of examples.
以下实施例中所用的试剂均为商购得到,例如:dCas9蛋白购自上海近岸科技有限公司,货号为E368-01A;dCas9抗体购自Abcam,货号为Ab204448,tcdB基因片段和gRNA由生工生物工程(上海)股份有限公司合成,RNA酶抑制剂购自上海近岸科技有限公司,gRNA体外转录试剂盒购自北京英茂盛业生物科技有限公司。硝酸纤维素膜、玻璃纤维膜:购自德国赛多利斯公司(SARTORIUS)。The reagents used in the following examples are all commercially available, for example: 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.
实施例1Example 1
1.两步还原法制备胶体金1. Two-step reduction method for preparing colloidal gold
a)氯金酸溶液第一次还原:将6ml 0.0164mol/L的HAuCL
4水溶液加入200ml双蒸水中,煮沸30分钟,缓慢搅拌并加入50ml 0.016mol/L柠檬酸三钠溶液。以30kHZ的频率超声振荡2分钟,冷却至室温,得到15nm粒径的胶体金原核溶液。
a) First reduction of chloroauric acid solution: 6 ml of 0.0164 mol/L aqueous solution of HAuCL 4 was added to 200 ml of double distilled water, boiled for 30 minutes, slowly stirred and 50 ml of 0.016 mol/L trisodium citrate solution was added. The mixture was ultrasonically shaken at a frequency of 30 kHZ for 2 minutes, and cooled to room temperature to obtain a colloidal gold pronucleus solution having a particle diameter of 15 nm.
b)氯金酸溶液第二次还原:取第一次还原后得到的胶体金原核溶液26ml在4℃条件下,加入4℃预冷后的0.035mol/L的HAuCL
4溶液,缓慢搅拌并以每秒1-2滴的速度滴加4℃预冷后的0.018mol/L的抗坏血酸与0.138g/L PVP混合液,反应1小时至溶液呈现透明酒红色,即得到40nm粒径的胶体金溶液。
b) The second reduction of the chloroauric acid solution: 26 ml of the colloidal gold pronuclear solution obtained after the first reduction is added to the 0.035 mol/L HAuCL 4 solution after pre-cooling at 4 ° C at 4 ° C, and slowly stirred and At a rate of 1-2 drops per second, a mixture of 0.018 mol/L ascorbic acid and 0.138 g/L PVP after pre-cooling at 4 ° C was added dropwise, and the reaction was carried out for 1 hour until the solution exhibited a transparent wine-red color, thereby obtaining a colloidal gold solution having a particle diameter of 40 nm. .
2.制备胶体金包被的dCas9-gRNA2. Preparation of colloidal gold-coated dCas9-gRNA
a)gRNA体外转录a) gRNA in vitro transcription
合成体外转录所需引物,序列如下:5’-TTAATACGACTCACTATAGGG
CATATACAATTGAGACTGGAGTTTTAGAGCTAGAAATAG-3’(SEQ ID NO.4),按照gRNA体外转录试剂盒中的操作步骤对gRNA进行体外转录。
The primers required for in vitro transcription were synthesized, and the sequence was as follows: 5'-TTAATACGACTCACTATAGGG CATATACAATTGAGACTGGA GTTTTAGAGCTAGAAATAG-3' (SEQ ID NO. 4), gRNA was in vitro transcribed according to the procedure in the gRNA in vitro transcription kit.
b)制备dCas9-gRNA复合体b) Preparation of dCas9-gRNA complex
将dCas9蛋白与体外转录获得的gRNA在3M NaAc pH5.2缓冲液中37℃孵育60分钟,即得dCas9-gRNA复合体,孵育过程中务必采用无RNA酶的耗材进行实验,注意防止RNA酶污染。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. .
3.dCas9-gRNA胶体金预处理3.dCas9-gRNA colloidal gold pretreatment
a)将获得的dCas9-gRNA复合体用0.1M pH7.8的磷酸盐缓冲液稀释至浓度1mg/ml。a) The obtained dCas9-gRNA complex was diluted to a concentration of 1 mg/ml with 0.1 M phosphate buffer of pH 7.8.
b)将1000ml胶体金溶液与含有500u/ml RNA酶抑制剂的100ml0.1M pH7.8的磷酸盐缓冲液混合,快速搅拌3分钟。之后,以每秒1-2滴的速度滴加稀释后的dCas9-gRNA复合体溶液8ml,缓慢搅拌室温反应5分钟。b) 1000 ml colloidal gold solution was mixed with 100 ml of 0.1 M phosphate buffer pH 7.8 containing 500 u/ml RNase inhibitor and stirred rapidly for 3 minutes. Thereafter, 8 ml of the diluted dCas9-gRNA complex solution was added dropwise at a rate of 1-2 drops per second, and the mixture was slowly stirred at room temperature for 5 minutes.
c)在上述反应液中快速加入20ml 10wt%的牛血清白蛋白溶液,缓慢搅拌室温反应5分钟。c) Quickly add 20 ml of a 10 wt% bovine serum albumin solution to the above reaction solution, and slowly react at room temperature for 5 minutes.
d)将获得的溶液8000rpm/min离心20分钟,取沉淀,并收集上清,将上清12500rpm/min离心30分钟,取沉淀。将两次沉淀合并用含有0.1wt%BSA的硼酸缓冲液复溶到OD540值为14。d) The obtained solution was centrifuged at 8000 rpm/min for 20 minutes, a precipitate was taken, and the supernatant was collected, and the supernatant was centrifuged at 12,500 rpm/min for 30 minutes to take a precipitate. The two precipitates were combined and reconstituted with borate buffer containing 0.1 wt% BSA to an OD540 value of 14.
4.dCas9-gRNA胶体金纸制备4.dCas9-gRNA colloidal gold paper preparation
a)喷金缓冲液制备:在800ml双蒸水中加入100ml 1.0M Tris溶液,调pH至8.5。在溶液中加入3g聚乙二醇20000、2g牛血清白蛋白,2g脱脂牛奶,3g酪蛋白和0.5g叠氮钠,充分溶解,至总体积1000ml。a) Preparation of gold spray buffer: 100 ml of 1.0 M Tris solution was added to 800 ml of double distilled water to adjust the pH to 8.5. 3 g of polyethylene glycol 20,000, 2 g of bovine serum albumin, 2 g of skim milk, 3 g of casein and 0.5 g of sodium azide were added to the solution, and dissolved sufficiently to a total volume of 1000 ml.
b)用喷金缓冲液稀释dCas9-gRNA胶体金,至溶液OD540值为2。b) Dilute dCas9-gRNA colloidal gold with a gold spray buffer until the solution has an OD540 value of 2.
c)取7ml Tween-20,160g蔗糖,用双蒸水定容至1L,配置成玻璃纤维 膜预处理液。用每30ml预处理液浸泡玻璃纤维膜261mm*220mm 30分钟后,至37℃干燥;再用OD540值为2的dCas9-gRNA胶体金溶液喷涂玻璃纤维膜,每261mm*220mm的玻璃纤维膜上喷涂20ml,干燥,制得dCas9-gRNA胶体金纸。c) Take 7ml of Tween-20, 160g of sucrose, and dilute to 1L with double distilled water to prepare a glass fiber membrane pretreatment solution. Soak the glass fiber membrane with 261mm*220mm per 30ml pretreatment solution for 30 minutes, then dry at 37 °C; spray the glass fiber membrane with dCas9-gRNA colloidal gold solution with OD540 value of 2, spray on each 261mm*220mm glass fiber membrane. 20 ml, dried, to obtain dCas9-gRNA colloidal gold paper.
5.含检测线及质控线硝酸纤维素膜的制备5. Preparation of nitrocellulose membrane containing test line and quality control line
将dCAS9抗体用磷酸盐缓冲液稀释成0.5mg/ml,制得质控线(C线)溶液。The dCAS9 antibody was diluted to 0.5 mg/ml with a phosphate buffer to prepare a quality control line (C line) solution.
tcdB基因片段-牛血清白蛋白偶联物的制备方法为将合成的tcdB基因片段400-600ng加入10wt%牛血清白蛋白中室温孵育30分钟,即得tcdB基因片段-牛血清白蛋白复合物;将tcdB基因片段-牛血清白蛋白偶联物用磷酸盐缓冲液稀释成0.5mg/ml,tcdB基因片段DNA含量为500ng/ml,制得检测线(T线)溶液。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.
a)用点膜机喷点C、T线溶液,每1m长硝酸纤维素膜分别包被有1ml的C线和T线溶液,C线和T线间距6mm。a) Spray the C and T line solutions with a spotting machine. Each 1m long nitrocellulose membrane is coated with 1ml of C line and T line solution, and the C line and T line are 6mm apart.
将滤样纸、dCas9-gRNA胶体金纸片、硝酸纤维素膜、吸水纸依次黏贴在胶板上,切割成宽度为4mm的试剂条。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.
实施例2Example 2
按照实施例1的方法制备胶体金试纸条,不同的是,gRNA序列更改为TATAGTGGTTTAGTTAGAGT(SEQ ID NO.5)。A colloidal gold test strip was prepared as in Example 1, except that the gRNA sequence was changed to TATAGTGGTTTAGTTAGAGT (SEQ ID NO. 5).
测试实施例1Test Example 1
(1)胶体金试纸条特异性实验:(1) Colloidal gold test strip specific experiment:
交叉实验检测样品为分别含有下列常见肠道细菌DNA浓度为200ng/ml的磷酸盐溶液:布氏瘤胃球菌、双歧杆菌、黄化瘤胃球菌、史密斯甲烷短杆 菌、沙门氏菌、志贺氏菌感染、弯曲杆菌。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.
检测方法:将检测试剂条置于待测交叉反应实验的样品中吸取样品约150ul,5-8分钟后判读结果。每种菌检测3次。Detection method: The 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.
表1特异性实验结果Table 1 specific experimental results
菌种Strain | 实施例1制备的试纸条Test strip prepared in Example 1 | 实施例2制备的试纸条Test strip prepared in Example 2 |
布氏瘤胃球菌Ruminococcus | 阴性negative | 阴性negative |
双歧杆菌Bifidobacterium | 阴性negative | 阴性negative |
黄化瘤胃球菌Rhizobium | 阴性negative | 阴性negative |
史密斯甲烷短杆菌Brevibacterium methane | 阴性negative | 阴性/阳性Negative/positive |
沙门氏菌salmonella | 阴性negative | 阴性negative |
志贺氏菌感染Shigella infection | 阴性negative | 阴性negative |
弯曲杆菌Campylobacter | 阴性negative | 阴性/阳性Negative/positive |
实验结果如表1所示,采用实施例1方法制备的试纸条在上述肠道细菌DNA浓度等于或小于200ng/ml时不会被检出,采用实施例1方法制备的试纸条的特异性显著大于采用实施例2方法制备的试纸条。The experimental results are shown in Table 1. The test strip prepared by the method of Example 1 was 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.
(2)试剂盒灵敏度检测:(2) Kit sensitivity detection:
检测样品:配制含有艰难梭菌基因组DNA浓度为50ng/ml、100ng/ml、120ng/ml、150ng/ml、180ng/ml、200ng/ml样品。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.
检测方法:将检测试剂条置于待测样品中吸取样品约150ul,5-8分钟后判读结果,每个浓度检测3次。Detection method: The 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.
表2灵敏度检测结果Table 2 sensitivity test results
实验结果如表2所示,采用实施例1方法制备的试纸条的检出灵敏度为 100ng/ml显著大于采用实施例2方法制备的试纸条的检出灵敏度150ng/ml。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.
(3)试剂盒批间差异检测(3) Detection of differences between kits
检测样品:将含有艰难梭菌基因组DNA浓度为200ng/ml、500ng/ml、1000ng/ml的样品分别标号为1,2,3;将不含艰难梭菌基因组DNA的溶液4份分别标号为4,5,6,7。Samples were tested: samples containing C. difficile genomic DNA at 200 ng/ml, 500 ng/ml, 1000 ng/ml were labeled 1, 2, 3; 4 samples containing C. difficile genomic DNA were labeled 4 respectively. , 5, 6, 7
检测方法:将不同标号的待测样品,用三个不同批次的检测试剂盒进行检测,每个样品检测3次,比较待测样品总体阳性率检测及重复性。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.
表3table 3
实验结果如表3所示,采用实施例1方法制备的试纸条批间差异优于采用实施例2方法制备的试纸条。The experimental results are shown in Table 3. The difference between the batches of the test strips prepared by the method of Example 1 was better than that of the test strips prepared by the method of Example 2.
以上结合附图详细描述了本公开的优选实施方式,但是,本公开并不限于上述实施方式中的具体细节,在本公开的技术构思范围内,可以对本公开的技术方案进行多种简单变型,这些简单变型均属于本公开的保护范围。The preferred embodiments of the present disclosure have been described in detail above with reference to the accompanying drawings. However, the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solutions of the present disclosure within the scope of the technical idea of the present disclosure. These simple variations are all within the scope of the disclosure.
另外需要说明的是,在上述具体实施方式中所描述的各个具体技术特征,在不矛盾的情况下,可以通过任何合适的方式进行组合,为了避免不必要的重复,本公开对各种可能的组合方式不再另行说明。It should be further noted that the specific technical features described in the above specific embodiments may be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present disclosure is applicable to various possibilities. The combination method will not be described separately.
此外,本公开的各种不同的实施方式之间也可以进行任意组合,只要其不违背本公开的思想,其同样应当视为本公开所公开的内容。In addition, any combination of various embodiments of the present disclosure may be made as long as it does not deviate from the idea of the present disclosure, and should also be regarded as the disclosure of the present disclosure.
Claims (10)
- 一种用于检测艰难梭菌的胶体金试纸条,该胶体金试纸条包括依次相连接的吸水垫、基膜、金标垫和样品垫;所述基膜上设置有C线和T线,其特征在于,所述C线上包被有dCas9抗体,所述T线上包被有tcdB基因片段-牛血清白蛋白复合物;所述金标垫中含有胶体金标记的dCas9-gRNA蛋白核酸复合物。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 a C line and a T a line characterized in that 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 colloidal gold-labeled dCas9-gRNA Protein nucleic acid complex.
- 根据权利要求1所述的胶体金试纸条,其中,所述dCas9-gRNA蛋白核酸复合物中gRNA序列为CATATACAATTGAGACTGGA。The colloidal gold test strip according to claim 1, wherein the gRNA sequence in the dCas9-gRNA protein nucleic acid complex is CATATACAATTGAGACTGGA.
- 根据权利要求1所述的胶体金试纸条,其中,所述dCas9-gRNA蛋白核酸复合物中dCas9的氨基酸序列为SEQ ID NO.2。The colloidal gold test strip according to claim 1, wherein the amino acid sequence of dCas9 in the dCas9-gRNA protein nucleic acid complex is SEQ ID NO.
- 根据权利要求1所述的胶体金试纸条,其中,所述tcdB基因片段-牛血清白蛋白复合物中,tcdB基因片段序列为GCAGTTGAATATAGTGGTTTAGTTAGAGTTGGTGAAGATGTATATTATTTTGGAGAAACATATACAATTGAGACTGGATGGATATATGATATGGAAAATGAAAGTGATAAATATTATTTCAATCCAGAAACTAAAAAAGCATG。The colloidal gold test strip according to claim 1, wherein the tcdB gene fragment sequence is GCAGTTGAATATAGTGGTTTAGTTAGAGTTGGTGAAGATGTATATTATTTTGGAGAAACATATACAATTGAGGGATGGATATATGATATGGAAAATGAAAGTGATAAATATTATTTCAATCCAGAAACTAAAAAAGCATG.
- 根据权利要求1所述的胶体金试纸条,其中,相对于1cm所述T线,tcdB基因片段的DNA量为400-600ng,牛血清白蛋白的量为0.1-0.3w/v%。The colloidal gold test strip according to claim 1, wherein the amount of DNA of the tcdB gene fragment is 400-600 ng and the amount of bovine serum albumin is 0.1-0.3 w/v% with respect to 1 cm of the T line.
- 根据权利要求1所述的胶体金试纸条,其中,dCas9抗体为购自Abcam的货号为Ab204448的抗体。The colloidal gold test strip of claim 1 wherein the dCas9 antibody is an antibody of Ab204448 available from Abcam.
- 根据权利要求1所述的胶体金试纸条,其中,所述金标垫中所含有 的胶体金标记的dCas9-gRNA蛋白核酸复合物是由胶体金标记的dCas9-gRNA蛋白核酸复合物溶液以0.5-5μL/cm的喷涂速度通过喷金点膜机喷涂在金标垫上干燥后得到的;所述胶体金标记的dCas9-gRNA蛋白核酸复合物溶液中,胶体金的含量为3-8μg/mL,dCas9蛋白的含量为10-50ng/mL,gRNA核酸的含量为10-50ng/mL。The colloidal gold test strip according to claim 1, wherein 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. The spraying speed of 0.5-5 μL/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 μg/mL. The content of dCas9 protein is 10-50 ng/mL, and the content of gRNA nucleic acid is 10-50 ng/mL.
- 根据权利要求7所述的胶体金试纸条,其中,所述T线上包被的tcdB基因片段-牛血清白蛋白复合物是由tcdB基因片段-牛血清白蛋白偶联物溶液以1-10μL/cm喷涂速度通过喷金点膜机喷涂在T线上干燥后得到的;所述tcdB基因片段-牛血清白蛋白偶联物溶液中,tcdB基因片段的含量为20-50ng/mL,牛血清白蛋白的含量为0.1-0.3w/v%。The colloidal gold test strip according to claim 7, wherein the TcdB gene fragment-bovine serum albumin complex coated on the T line is composed of a tcdB gene fragment-bovine serum albumin conjugate solution The spray rate of 10 μL/cm was sprayed on a T-line by a spray gold film machine; the content of the tcdB gene fragment in the tcdB gene fragment-bovine serum albumin conjugate solution was 20-50 ng/mL, and the bovine The serum albumin content is from 0.1 to 0.3 w/v%.
- 根据权利要求8所述的胶体金试纸条,其中,所述C线上包被的dCas9抗体是由0.1-5mg/mL的dCas9抗体溶液以1-10μL/cm的喷涂速度通过喷金点膜机喷涂在C线上干燥后得到的。The colloidal gold test strip according to claim 8, wherein the C-line coated dCas9 antibody is passed through a gold spray film at a spraying speed of 1-10 μL/cm from a solution of 0.1-5 mg/mL of dCas9 antibody. Machine sprayed on the C line after drying.
- 一种用于检测艰难梭菌的试剂盒,其特征在于,该试剂盒包括样品提取液以及权利要求1-9中任意一项所述的用于检测艰难梭菌的胶体金试纸条。A kit for detecting C. difficile, characterized in that the kit comprises a sample extract and a colloidal gold test strip for detecting C. difficile according to any one of claims 1-9.
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