WO2019179174A1

WO2019179174A1 - Colloidal gold test strip and kit for detecting clostridium difficile

Info

Publication number: WO2019179174A1
Application number: PCT/CN2018/119838
Authority: WO
Inventors: 李程; 丁秋蓉
Original assignee: 杭州观梓健康科技有限公司
Priority date: 2018-03-21
Filing date: 2018-12-07
Publication date: 2019-09-26
Also published as: CN108445212A; CN108445212B

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

Colloidal gold test strip and kit for detecting C. difficile

Technical field

The present disclosure relates to the field of biomedicine, and in particular to a colloidal gold test strip and kit for detecting C. difficile.

Background technique

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.

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.

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.

Summary of the invention

It is an object of the present disclosure to provide a C. difficile assay kit that is highly sensitive, specific, and inexpensive.

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.

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.

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.

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.

Other features and advantages of the present disclosure will be described in detail in the detailed description which follows.

DRAWINGS

The drawings are intended to provide a further understanding of the disclosure, and are in the In the drawing:

Fig. 1 is a schematic view showing the assembly structure of a colloidal gold test paper.

Description of the reference numerals

1 sample pad 2 by substrate plate 3 gold standard pad

4 T line 5 C line 6 base film

7 absorbent pad

detailed description

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

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.

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.

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.

Optionally, wherein the gRNA sequence in the dCas9-gRNA protein nucleic acid complex is CATATACAATTGAGACTGGA, as shown in SEQ ID NO.

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.

Optionally, wherein the tcdB gene fragment-bovine serum albumin conjugate, the tcdB gene fragment sequence is GCAGTTGAATATAGTGGTTTAGTTAGAGTTGGTGAAGATGTATATTATTTTGGAGAAACATATACAATTGAGGGYGGATAGGATATATGATATGGAAAATGAAAGTGATAAATATTATTTCAATCCAGAAACTAAAAAAGCATG, as shown in SEQ ID NO.

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.

Alternatively, wherein the dCas9 antibody is an antibody available from Abcam under the trade number Ab204448.

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.

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%.

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.

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.

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.

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.

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.

Alternatively, the spacing between the C line and the T line is 2.0-7.0 mm.

The invention will now be described in further detail by way of examples.

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.

Example 1

1. Two-step reduction method for preparing colloidal gold

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) 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. Preparation of colloidal gold-coated dCas9-gRNA

a) gRNA in vitro transcription

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) Preparation of dCas9-gRNA complex

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 colloidal gold pretreatment

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) 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) 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) 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 colloidal gold paper preparation

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) Dilute dCas9-gRNA colloidal gold with a gold spray buffer until the solution has an OD540 value of 2.

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. Preparation of nitrocellulose membrane containing test line and quality control line

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.

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.

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.

Example 2

A colloidal gold test strip was prepared as in Example 1, except that the gRNA sequence was changed to TATAGTGGTTTAGTTAGAGT (SEQ ID NO. 5).

Test Example 1

(1) Colloidal gold test strip specific experiment:

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.

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.

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

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) Kit sensitivity detection:

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.

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.

Table 2 sensitivity test results

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) Detection of differences between kits

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

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.

table 3

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.

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.

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.

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

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.
The colloidal gold test strip according to claim 1, wherein the gRNA sequence in the dCas9-gRNA protein nucleic acid complex is CATATACAATTGAGACTGGA.
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.
The colloidal gold test strip according to claim 1, wherein the tcdB gene fragment sequence is GCAGTTGAATATAGTGGTTTAGTTAGAGTTGGTGAAGATGTATATTATTTTGGAGAAACATATACAATTGAGGGATGGATATATGATATGGAAAATGAAAGTGATAAATATTATTTCAATCCAGAAACTAAAAAAGCATG.
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.
The colloidal gold test strip of claim 1 wherein the dCas9 antibody is an antibody of Ab204448 available from Abcam.
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.
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%.
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.
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.