CN110596384A

CN110596384A - Human papilloma virus 6 type and 11 type immunoassay kit prepared based on Cas protein and gRNA compound

Info

Publication number: CN110596384A
Application number: CN201910965480.8A
Authority: CN
Inventors: 刘光明; 张树文; 黄翔; 孙娈燕
Original assignee: LIMING BIO-PRODUCTS Co Ltd
Current assignee: LIMING BIO-PRODUCTS Co Ltd
Priority date: 2019-10-12
Filing date: 2019-10-12
Publication date: 2019-12-20
Anticipated expiration: 2039-10-12
Also published as: CN110596384B

Abstract

The invention relates to the technical field of microbial detection, in particular to human papilloma virus 6-type and 11-type immunodetection kits prepared based on a Cas protein and a gRNA compound, which comprise a GNP1 compound and a GNP2 compound; the GNP1 compound is compounded by Cas protein and gRNA1 of HPV-6 type, and the gene sequence of gRNA1 consists of a primer sequence 1 and a gene E1 replication protein MK 313768.12399-2421; the GNP2 complex is composed of Cas protein and gRNA2 of HPV-11 type, and the gene sequence of gRNA2 is composed of primer sequence 2 and gene E2 regulatory protein MK 313768.13414-3436. The immunoassay kit has higher sensitivity, specificity and accuracy.

Description

Human papilloma virus 6 type and 11 type immunoassay kit prepared based on Cas protein and gRNA compound

Technical Field

The invention relates to the technical field of microbial detection, in particular to human papilloma virus 6-type and 11-type immunodetection kits prepared based on a Cas protein and a gRNA compound.

Background

Condyloma acuminata is a sexually transmitted disease mainly manifested by proliferative lesions at anogenital parts caused by Human Papilloma Virus (HPV) infection, and mostly occurs in young and middle-aged people of 18-50 years old. Onset occurs after a latency period of about half a month to 8 months, averaging 3 months, which is common and is primarily transmitted by sexual contact. There are different subtypes of HPV, with types 6 and 11 being the most common HPV causing condyloma acuminata.

Currently, there are several methods for clinically diagnosing condyloma acuminatum:

1. acetic acid white test: the local external application or wet dressing of 3-5% acetic acid solution for 5-10 minutes can whiten the HPV infected area, namely the so-called acetic acid whitening phenomenon. However, the specificity is not high, and false positive can occur in some chronic inflammations, such as candidal vulvitis, genital trauma and nonspecific inflammations.

2. And (3) cytological examination: when the vaginal or cervical wart tissue smear is used and is subjected to Papanicolaou staining, two cells, namely vacuolated cells and keratinocytes, can be seen to exist simultaneously, and the diagnostic value for condyloma acuminatum is realized.

3. And (3) histopathological examination: the presence of vacuolated cells, e.g., above the spinous layer and in the granular layer, is important evidence for diagnosis of HPV infection.

4. Immunological tests: the method for detecting the HPV antigen in the lesion tissue by using the antibody of the HPV protein has low sensitivity and the detection rate of only about 50 percent.

5. Nucleic acid hybridization assay: important means for detecting HPV infection include dot blot, tissue in situ hybridization, and nucleic acid blotting. These methods are highly specific and sensitive and are sensitive and reliable methods for diagnosing HPV infection. But the technical operation is complicated and is not generally developed clinically.

6. Polymerase Chain Reaction (PCR): is the most sensitive method for detecting HPV infection at present, can be used for type specificity analysis, and has the characteristics of high sensitivity and simple, convenient and rapid method.

Among the six detection methods, PCR detection is the most widely used method in third-class hospitals, but PCR use requires specially trained personnel and places qualified in acceptance, and equipment is expensive, and the PCR detection cannot be carried out in second-class hospitals and the following hospitals.

In contrast, immunological detection utilizes specific binding of antibody antigen, has high accuracy and lower cost than PCR detection, and thus has great significance for detecting HPV antigen. However, the premise for realizing immunological detection is to find an antibody against HPV, since HPV cannot be cultured in vitro, natural HPV virus cannot be extracted and used for immunizing animals to obtain the antibody against HPV, the HPV polypeptide synthesized in vitro does not have a natural conformation, and the antibody obtained by immunization cannot react with the natural HPV virus, so that an HPV immunodetection kit cannot be developed by using the existing method.

In order to solve the problem that a corresponding immunoassay kit cannot be developed when an HPV antibody cannot be prepared or the activity of the antibody is weak in the prior art, the inventor of the application develops an artificially synthesized GNP complex (guide RNA-Protein) as a substitute antibody for developing the HPV immunoassay kit.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a human papillomavirus type 6 and type 11 immunodetection kit prepared based on a Cas protein and a gRNA compound, which connects the gRNA specific to HPV with the Cas protein to form a specific GNP compound aiming at HPV to replace an HPV antibody, so that the developed HPV immunodetection kit has higher sensitivity, specificity and accuracy.

In order to achieve the purpose, the invention provides the following technical scheme:

a human papillomavirus type 6 and type 11 immunodetection kit prepared based on a Cas protein and a gRNA compound comprises a GNP1 compound and a GNP2 compound;

the GNP1 compound is formed by compounding a Cas protein and a gRNA1 of an HPV-6 type, and the gene sequence of the gRNA1 consists of a primer sequence 1 shown as SEQ ID NO:1 and a gene E1 replication protein MK 313768.12399-2421; the GNP2 compound is compounded by a Cas protein and a gRNA2 of HPV-11 type, and the gene sequence of the gRNA2 consists of a primer sequence 2 shown as SEQ ID NO. 2 and a gene E2 regulatory protein MK 313768.13414-3436; 1 is: 5'-ATGATGCCACACAACCATGTTGG-3' the flow of the air in the air conditioner,

SEQ ID NO 2 is: 5'-ACGGCGTGTCGGCGCCGCCTAGG-3' are provided.

Further, the Cas protein is one of a Cas9 protein and a Cas13 protein.

Further, the immunodetection kit is one of an HPV enzyme-linked immunosorbent kit, an HPV immunochromatographic kit and an HPV chemiluminescent immunoassay kit.

Further, the preparation method of the HPV ELISA kit comprises the following steps:

designing and synthesizing gRNA1 and gRNA2 specific to HPV in vitro by using computer software;

secondly, mixing the Cas protein with gRNA1 and gRNA2 respectively to form a GNP1 compound and a GNP2 compound correspondingly;

thirdly, coating the GNP1 compound on an enzyme label plate;

marking the GNP2 complex by HRP as an HRP marker;

preparing substrate color developing solution A, substrate color developing solution B, cleaning solution and stop solution;

sixthly, packing the semi-finished products obtained in the third step, the fourth step and the fifth step separately and then boxing the semi-finished products to obtain the final enzyme linked immunosorbent assay kit;

or

Firstly, designing gRNA1 and gRNA2 aiming at HPV specificity by using computer software;

thirdly, coating the GNP2 compound on an enzyme label plate;

marking the GNP1 complex by HRP as an HRP marker;

sixthly, packing the semi-finished products obtained in the third step, the fourth step and the fifth step separately and then boxing the semi-finished products to obtain the final enzyme linked immunosorbent assay kit.

Further, in step (c), 1mL of 1mg/mL of Cas protein is mixed with 1OD of gRNA1 or gRNA2, and reacted at 37 ℃ for 30min, corresponding to the formation of GNP1 complex and GNP2 complex.

Further, in step (c), the GNP1 complex or GNP2 complex was diluted to 2 μ g/mL when coated on a microplate in an amount of 100 μ L/well, and after 6 hours of coating, blocked with 15 wt% FCS at 4 ℃ overnight.

Further, in the step (iv), the HRP labeling the GNP1 complex or GNP2 complex, comprising the following steps: 4-1, weighing 5mg of HRP, and dissolving the HRP in 1mL of distilled water to obtain an HRP solution;

4-2, adding 0.2mL of newly prepared 0.1M NaIO into the HRP solution₄Stirring the solution at room temperature in the dark for 20min to obtain HRP-NaIO₄A solution;

4-3, mixing HRP-NaIO₄Loading the solution into a dialysis bag, dialyzing the solution against 1mM sodium acetate buffer solution having a pH of 4.4 at 4 ℃ overnight to obtain a hydroformylation RP;

4-4, adding 20. mu.L of 0.2M carbonate buffer solution with pH 9.5 to raise the pH of the hydroformylation RP to 9.0-9.5, immediately adding 10mg of GNP1 complex or GNP2 complex, and gently stirring at room temperature in the absence of light for 2 h;

4-5, adding 0.1mL of newly-formulated 4mg/mL NaBH₄Uniformly mixing the solution, and placing the mixture at the temperature of 4 ℃ for 2 hours to obtain HRP mixed solution;

4-6, putting the HRP mixed solution into a dialysis bag, dialyzing with PBS (0.15M pH 7.4) and standing at 4 ℃ overnight;

4-7, dropwise adding saturated ammonium sulfate with the same volume under stirring, placing at 4 ℃ for 1h, and transferring to a centrifuge tube to obtain a centrifugate;

4-8, placing the centrifugate in a centrifuge, centrifuging at 4 ℃ and 3000rpm for 30min, removing the supernatant, collecting the precipitate, washing the precipitate twice with half-saturated ammonium sulfate, and finally dissolving the precipitate in a small amount of PBS (0.15M) with pH value of 7.4 to obtain an HRP (horse radish peroxidase) labeling solution;

4-9, filling the HRP labeling solution into a dialysis bag, dialyzing with PBS (0.15M pH 7.4), removing ammonium ions, centrifuging at 4 ℃ and 10,000rpm for 30min, collecting supernatant to obtain the HRP-labeled GNP1 complex or the HRP-labeled GNP2 complex, subpackaging, and freezing at-20 ℃.

Further, in the fifth step, the substrate color developing A solution is prepared according to the following mixture ratio: adding 13.6g of sodium acetate, 1.6g of citric acid and 0.3mL of 30 wt% hydrogen peroxide into 500mL of distilled water,

the substrate developing B liquid is prepared from the following components in parts by weight: adding 0.2g of disodium ethylene diamine tetraacetate, 0.95g of citric acid, 50mL of glycerol and 3mL of DMSO solution dissolved with 0.15g of TMB into 500mL of distilled water;

the cleaning solution is 0.01M PBST;

the stop solution is 2N H₂SO₄。

Further, the HPV ELISA kit using method comprises the following steps:

A. reagent balance: taking the prepared ELISA plate, the sample diluent and the quality control product/product to be detected to balance to room temperature;

B. diluting: diluting a quality control product/a product to be detected to a specific concentration by using a sample diluent;

C. sample adding: adding the quality control product/product to be detected into the corresponding enzyme label plate by 100 mu L/hole;

D. and (3) incubation: placing the ELISA plate at 37 ℃ for incubation for 60min, and washing the plate for 5 times by using a washing liquid for an automatic plate washer;

E. adding an enzyme: adding an HRP marker into a corresponding enzyme label plate at a rate of 100 mu L/hole;

F. and (3) incubation: placing the ELISA plate at 37 ℃ for incubation for 60min, and washing the plate for 5 times by using a washing solution for an automatic plate washing machine;

G. color development: mixing the color-developing agent A solution and the color-developing agent B solution in equal volume, adding 100 μ L/hole into the enzyme label plate, and incubating at 37 deg.C for 30min in an electrothermal constant-temperature incubator;

H. and (4) terminating: adding stop solution into 50 mu L/hole to stop reaction;

I. reading a plate: absorbance was measured at a wavelength of 450nm using a microplate reader, and the reference wavelength was 620nm or 630 nm.

Further, the preparation method of the HPV immunochromatographic kit comprises the following steps:

(1) blue latex particle labeled biotin

Diluting the blue latex particles to a final concentration of 1 wt% with 0.01M PBS (pH 7.4), adding biotin to give a final concentration of 0.1mg/mL, and reacting at 4 ℃ overnight; adding BSA with final concentration of 1 wt% for blocking for 1h, centrifuging at 4 deg.C and 10,000rpm for 20min, removing supernatant, re-dissolving the precipitate with 0.01M PBS (pH 7.4) containing 0.1 wt% BSA to obtain biotin emulsion, and standing at 4 deg.C;

(2) red latex particle marker GNP1

The red latex particles were diluted to a final concentration of 1 wt% with 0.01M PBS pH 7.4, GNP1 was added to give a final concentration of 0.1mg/mL, and the reaction was allowed to proceed overnight at 4 ℃; blocking with 1 wt% BSA at 4 deg.C for 1h, centrifuging at 10,000rpm for 20min, removing supernatant, re-dissolving the precipitate with 0.01M PBS (pH 7.4) containing 0.1 wt% BSA to obtain GNP1 emulsion, and standing at 4 deg.C;

(3) spot film

Respectively diluting the avidin antibody and the GNP2 to proper concentrations by using PBS (0.01M) with the pH value of 7.4, respectively spotting the avidin antibody diluent and the GNP2 diluent on a quality control area and a test area on a nitrocellulose membrane by using a membrane spotting machine, drying for 2 hours at 37 ℃, sealing and then placing at room temperature for later use;

(4) spot emulsion

Diluting the biotin latex solution and GNP1 latex solution marked in steps (1) and (2) to proper concentration with 0.01M PBS (pH 7.4) containing 0.1 wt% BSA, spraying on polyester film by machine, drying at 37 deg.C for 2 hr, sealing, and standing at room temperature;

(5) assembly

Sticking the dotted nitrocellulose membrane and a polyester membrane on a PVC sheet of the test strip, sticking the polyester membrane with a glass fiber feeding area, sticking two ends of the nitrocellulose membrane with the polyester membrane and a water absorption area respectively, cutting into corresponding widths, and loading into a plastic card to obtain a final immunochromatographic kit;

or

(1) Blue latex particle labeled biotin

(2) red latex particle marker GNP2

The red latex particles were diluted to a final concentration of 1 wt% with 0.01M PBS pH 7.4, GNP2 was added to give a final concentration of 0.1mg/mL, and the reaction was allowed to proceed overnight at 4 ℃; blocking with 1 wt% BSA at 4 deg.C for 1h, centrifuging at 10,000rpm for 20min, removing supernatant, re-dissolving the precipitate with 0.01M PBS (pH 7.4) containing 0.1 wt% BSA to obtain GNP2 emulsion, and standing at 4 deg.C;

(3) spot film

Respectively diluting the avidin antibody and the GNP1 to proper concentrations by using PBS (0.01M) with the pH value of 7.4, respectively spotting the avidin antibody diluent and the GNP1 diluent on a quality control area and a test area on a nitrocellulose membrane by using a membrane spotting machine, drying for 2 hours at 37 ℃, sealing and then placing at room temperature for later use;

(4) spot emulsion

Diluting the biotin latex solution and GNP2 latex solution marked in steps (1) and (2) to proper concentration with 0.01M PBS (pH 7.4) containing 0.1 wt% BSA, spraying on polyester film by machine, drying at 37 deg.C for 2 hr, sealing, and standing at room temperature;

(5) assembly

And adhering the dotted nitrocellulose membrane and a polyester membrane to a PVC sheet of the test strip, adhering the polyester membrane to a glass fiber feeding area, adhering two ends of the nitrocellulose membrane to the polyester membrane and a water absorption area respectively, cutting into corresponding widths, and filling into a plastic card to obtain the final immunochromatographic kit.

Further, the method for using the HPV immunochromatographic kit comprises the following steps:

a. reagent balance: taking the prepared immunochromatography reagent, the sample diluent and the quality control product/product to be tested to balance to room temperature;

c. sample adding: adding the quality control product/to-be-detected product into the feeding area of the test strip at a rate of 100 mu L/hole;

d. and (4) interpretation of results: and judging the result 15min after sample addition, wherein a red line appearing in the detection line is positive, and the detection line is negative otherwise.

In conclusion, the invention has the following beneficial effects:

1. according to the HPV immunodetection kit developed by the application, the HPV GNP compound designed and synthesized by the application replaces an HPV antibody, can be combined with natural HPV viruses and a carrier of the immunoassay kit, and has high sensitivity, specificity and accuracy;

2. compared with a PCR detection method, the immunoassay method can effectively reduce the detection cost of HPV and is suitable for the basic level.

Drawings

FIG. 1 is a test strip of an HPV immunochromatographic kit;

FIG. 2 is a diagram showing the test result of HPV ELISA clinical specimen.

In the figure, 1, a PVC sheet; 2. a feeding zone; 3. a polyester film; 4. a nitrocellulose membrane; 5. a test zone; 6. a quality control region; 7. a water absorption area.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings.

1. Materials and reagents

1.1 Cas protein

It should be noted that, in the GNP1 complex and GNP2 complex of the present application, any protein in the Cas protein family can be used, wherein, the GNP complex prepared by using the Cas9 protein and the Cas13 protein has higher detection sensitivity, specificity and accuracy. The application is specifically illustrated with Cas9 protein (from sigma).

1.2, gRNA1 of HPV-6 type and gRNA2 of HPV-11 type

The application designs HPV specific gRNA1 and gRNA2 gene sequences by using a computer, and then synthesizes the genes in vitro by using a PCR technology, wherein the synthesis method is a conventional means in the field and is not described in detail.

1.3 sample dilution

The application uses a phosphate Tween buffer solution, and the specific ratio is 0.01M PBS + 0.5% Tween 20.

In addition, other materials and reagents in the application are all commercial products, and the reagents are all high-grade pure.

2. Examples of the embodiments

2.1, example 1

A human papilloma virus 6 type and 11 type immunodetection kit prepared based on a Cas protein and a gRNA compound comprises a GNP1 compound and a GNP2 compound.

The GNP1 compound is compounded by Cas protein and gRNA1 of HPV-6 type, and the gene sequence of gRNA1 is composed of a primer sequence 1 shown in SEQ ID NO:1 and a gene E1 replication protein MK 313768.12399-2421.

The GNP2 compound is compounded by Cas protein and gRNA2 of HPV-11 type, the gene sequence of gRNA2 is composed of a primer sequence 2 shown as SEQ ID NO. 2 and a gene E2 regulatory protein MK 313768.13414-3436;

wherein SEQ ID NO 1 is: 5'-ATGATGCCACACAACCATGTTGG-3', SEQ ID NO:2 is: 5'-ACGGCGTGTCGGCGCCGCCTAGG-3' are provided.

The immunoassay kit is an HPV enzyme linked immunosorbent assay kit, and the preparation method comprises the following steps:

first, a gRNA1 and a gRNA2 specific to HPV were designed and synthesized in vitro using computer software.

② 1mL of 1mg/mL Cas9 protein is mixed with 1OD gRNA1 and gRNA2 respectively, and reacted for 30min at 37 ℃ to form a GNP1 complex and a GNP2 complex correspondingly.

③ coating the GNP1 complex on a microplate, diluting the GNP1 complex to 2 μ g/mL, coating the diluted GNP1 complex to 100 μ L/well, and sealing the coated GNP1 complex with 15 wt% FCS at 4 ℃ overnight after 6 h.

And (iv) labeling the GNP2 complex with HRP (horseradish peroxidase) as an HRP label, wherein the labeling step comprises the following steps: 4-1, weighing 5mg of HRP, and dissolving the HRP in 1mL of distilled water to obtain an HRP solution;

the substrate color developing A solution is prepared according to the following mixture ratio: adding 13.6g of sodium acetate, 1.6g of citric acid and 0.3mL of 30 wt% hydrogen peroxide into 500mL of distilled water;

the substrate developing B liquid is prepared according to the following mixture ratio: adding 0.2g of disodium ethylene diamine tetraacetate, 0.95g of citric acid, 50mL of glycerol and 3mL of DMSO solution dissolved with 0.15g of TMB into 500mL of distilled water;

the cleaning solution was 0.01M PBST;

the stop solution is 2N H₂SO₄。

The HPV ELISA kit takes a quality control product as a sample to verify the detection effect of the kit, the type and the concentration of the quality control product are shown in the following table I, and the using method comprises the following steps:

A. reagent balance: balancing the prepared enzyme label plate, the sample diluent and the quality control product to room temperature;

B. diluting: diluting a quality control product to a specific concentration by using a sample diluent;

C. sample adding: adding the quality control product into the corresponding enzyme label plate at a rate of 100 mu L/hole;

2.2 example 2

Example 2 based on the method of example 1, the GNP1 complex was coated on a microplate and the GNP1 complex was labeled with HRP. The corresponding detection results are consistent with example 1, therefore the order of the GNP1 complex and GNP2 complex can be interchanged, and the effect on the sensitivity, specificity and accuracy of the immunoassay kit is negligible.

2.3, example 3

The immunodetection kit of example 3 is an HPV immunochromatographic kit using the GNP1 complex and GNP2 complex of example 1, and the preparation method comprises the following steps:

(1) blue latex particle labeled biotin

Blue latex particles were diluted to a final concentration of 1 wt% with 0.01M PBS (phosphate buffered saline) at pH 7.4, biotin was added to a final concentration of 0.1mg/mL, and the reaction was carried out overnight at 4 ℃; adding BSA (bovine serum albumin) with a final concentration of 1 wt% for blocking for 1h, centrifuging at 4 deg.C and 10,000rpm for 20min, removing supernatant, redissolving the precipitate with 0.01M PBS (pH 7.4) containing 0.1 wt% BSA to obtain biotin emulsion, and standing at 4 deg.C;

(2) red latex particle marker GNP1

(3) spot film

(4) spot emulsion

(5) assembly

And (3) adhering the dotted nitrocellulose membrane and the polyester membrane to a PVC sheet of the test strip, adhering the polyester membrane to the glass fiber feeding area, adhering two ends of the nitrocellulose membrane to the polyester membrane and the water absorption area respectively, cutting into corresponding widths to prepare the test strip shown in the figure 1, and filling the test strip into a plastic card to obtain the final immunochromatographic kit.

The using method of the HPV immunochromatographic kit takes the quality control product as a sample to verify the detection effect of the kit, the type and the concentration of the quality control product refer to the following table I, and the using method comprises the following steps:

a. reagent balance: balancing the prepared immunochromatography reagent, the sample diluent and the quality control product to room temperature;

c. sample adding: adding the quality control substance into the feeding area of the test strip at a rate of 100 mu L/hole;

2.4, example 4

Example 4 based on the procedure of example 3, red latex particles were labeled with GNP 2. The corresponding detection results are consistent with example 3, therefore the order of the GNP1 complex and GNP2 complex can be interchanged, and the effect on the sensitivity, specificity and accuracy of the immunoassay kit is negligible.

The principle of the chemiluminescence immunoassay kit is similar to that of an enzyme linked immunosorbent assay kit, so that the GNP1 compound and the GNP2 compound are simultaneously suitable for the chemiluminescence immunoassay kit. However, the ELISA kit has excellent chemiluminescence immunity, so the ELISA kit is preferably selected as an example for development.

3. Performance detection

The immunodetection kits of example 1 and example 3 were used to detect HPV type 6 and HPV type 11 with sensitivity and accuracy, respectively, and the results are shown in tables one and two below.

3.1 sensitivity

TABLE sensitivity test results of example 1 and example 3

Detecting items	concentration/copies/mL	Enzyme linked immunosorbent assay result	Results of immunochromatography
				HPV type 6	1*10⁸	Positive for	Positive for
HPV type 6	1*10⁷	Positive for	Positive for
				HPV type 6	1*10⁶	Positive for	Positive for
HPV type 6	1*10⁵	Positive for	Negative of
				HPV type 6	1*10⁴	Negative of	Negative of
HPV type 11	1*10⁸	Positive for	Positive for
				HPV type 11	1*10⁷	Positive for	Positive for
HPV type 11	1*10⁶	Positive for	Positive for
				HPV type 11	1*10⁵	Positive for	Negative of
HPV type 11	1*10⁴	Negative of	Negative of

As can be seen from the results in Table I, the HPV ELISA kit has 1 x 10 detection sensitivity on HPV types 6 and 11⁵copies/ml; the detection sensitivity of the immunochromatography kit on HPV 6 type and HPV 11 type is 1 x 10⁶copies/ml, all have higher sensitivity.

3.2, specificity

TABLE II results of specificity detection in example 1 and example 3

Detecting items	concentration/copies/mL	Enzyme linked immunosorbent assay result	Results of immunochromatography
				HPV type 16	1*10⁸	Negative of	Negative of
HPV type 18	1*10⁸	Negative of	Negative of
				Chlamydia trachomatis	1*10⁸	Negative of	Negative of
Neisseria gonorrhoeae	1*10⁸	Negative of	Negative of
				Ureaplasma urealyticum	1*10⁸	Negative of	Negative of
Mycoplasma hominis	1*10⁸	Negative of	Negative of
				Staphylococcus aureus	1*10⁸	Negative of	Negative of
Escherichia coli	1*10⁸	Negative of	Negative of

From the results in Table two, it can be seen that the HPV ELISA kit and the immunochromatography kit can treat the above microorganisms at a concentration of 1 x 10⁸No cross reaction occurs at copies/mL, and the specificity is excellent.

3.3 detection of clinical specimens of HPV immunodetection kits

Taking 48 suspected condyloma acuminatum patient samples, using PCR detection as a reference, and simultaneously using PCR detection, HPV enzyme linked immunosorbent assay kit (embodiment 1) and HPV immunochromatography kit (embodiment 3) for detection, wherein the clinical sample test results of the HPV enzyme linked immunosorbent assay kit are shown in figure 2, and the test results are shown in the third table below.

Detection results of clinical samples by using epi-PCR method, enzyme-linked immunosorbent assay and immunochromatography method

	PCR method	Enzyme linked immunosorbent assay	Immunochromatography
				Positive for	17	15	14
Negative of	31	33	34
				Sensitivity/%)	100.00	88.24	82.35
Specificity/%	100.00	100.00	100.00
				Accuracy/%	100.00	95.83	93.75

The results of the table III show that the specificity of the HPV ELISA kit and the HPV immunochromatographic kit can reach 100%, wherein the sensitivity of the HPV ELISA kit and the HPV immunochromatographic kit is greater than 82%, the accuracy of the HPV ELISA kit and the HPV immunochromatographic kit is greater than 93%, the requirements of basic HPV detection can be met, the detection cost of HPV can be effectively reduced compared with PCR detection, and the HPV detection kit and the HPV immunochromatographic kit are convenient to popularize and use. Among them, the HPV ELISA kit has a better detection result, so that the embodiment 1 is taken as a preferred embodiment.

The present embodiment is only for explaining the present invention, and it is not limited to the present invention, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present invention.

Claims

1. A human papilloma virus 6 type and 11 type immunodetection kit prepared based on a Cas protein and a gRNA compound is characterized by comprising a GNP1 compound and a GNP2 compound;

the GNP1 compound is formed by compounding a Cas protein and a gRNA1 of an HPV-6 type, and the gene sequence of the gRNA1 consists of a primer sequence 1 shown as SEQ ID NO:1 and a gene E1 replication protein MK 313768.12399-2421;

the GNP2 compound is compounded by a Cas protein and a gRNA2 of HPV-11 type, and the gene sequence of the gRNA2 consists of a primer sequence 2 shown as SEQ ID NO. 2 and a gene E2 regulatory protein MK 313768.13414-3436;

1 is: 5'-ATGATGCCACACAACCATGTTGG-3' the flow of the air in the air conditioner,

SEQ ID NO 2 is: 5'-ACGGCGTGTCGGCGCCGCCTAGG-3' are provided.

2. The human papillomavirus type 6 and type 11 immunodetection kits prepared based on a CAS protein and a GRNA complex of claim 1, wherein the CAS protein is one of a CAS9 protein and a CAS13 protein.

3. The human papillomavirus type 6 and type 11 immunodetection kits prepared based on CAS proteins and GRNA complexes according to claim 1, wherein the immunodetection kits are one of HPV enzyme linked immunosorbent kits, HPV immunochromatographic kits, and HPV chemiluminescent immunoassay kits.

4. The human papillomavirus type 6 and type 11 immunodetection kits prepared based on CAS protein and GRNA complexes of claim 3, wherein the HPV ELISA kit preparation method comprises the following steps:

thirdly, coating the GNP1 compound on an enzyme label plate;

marking the GNP2 complex by HRP as an HRP marker;

or

thirdly, coating the GNP2 compound on an enzyme label plate;

marking the GNP1 complex by HRP as an HRP marker;

5. The human papillomavirus type 6 and type 11 immunodetection kits prepared based on CAS protein and GRNA complexes of claim 4, wherein in step (ii), 1mL of 1mg/mL of the Cas protein is mixed with 1OD gRNA1 or gRNA2, and reacted at 37 ℃ for 30min, corresponding to the formation of GNP1 complex and GNP2 complex; in step (c), the GNP1 complex or GNP2 complex is diluted to 2 μ g/mL when coated on the microplate, the coating amount is 100 μ L/well, and after 6h of coating, the coated plate is blocked with 15 wt% FCS at 4 ℃ overnight.

6. The human papillomavirus type 6 and type 11 immunodetection kits prepared based on CAS protein and GRNA complexes of claim 4 wherein in step (iv), the HRP labels GNP1 complex or GNP2 complex, comprising the steps of:

4-1, weighing 5mg of HRP, and dissolving the HRP in 1mL of distilled water to obtain an HRP solution;

7. The human papillomavirus type 6 and type 11 immunoassay kit prepared based on the CAS protein and the GRNA complex according to claim 4, wherein in the fifth step, the substrate chromogenic A solution is prepared according to the following mixture ratio: adding 13.6g of sodium acetate, 1.6g of citric acid and 0.3mL of 30 wt% hydrogen peroxide into 500mL of distilled water,

the cleaning solution is 0.01M PBST;

the stop solution is 2N H₂SO₄。

8. The human papillomavirus type 6 and type 11 immunodetection kits prepared based on CAS protein and GRNA complexes of claim 4, wherein the HPV ELISA kit usage method comprises the following steps:

9. The human papillomavirus type 6 and type 11 immunodetection kits prepared based on CAS proteins and GRNA complexes of claim 3, wherein the HPV immunochromatographic kit preparation method comprises the following steps:

(1) blue latex particle labeled biotin

(2) red latex particle marker GNP1

(3) spot film

(4) spot emulsion

(5) assembly

or

(1) Blue latex particle labeled biotin

(2) red latex particle marker GNP2

(3) spot film

(4) spot emulsion

(5) assembly

10. The human papillomavirus type 6 and type 11 immunodetection kits prepared based on CAS proteins and GRNA complexes according to claim 9, wherein the HPV immunochromatography kit is used in a method comprising the steps of: