Development and application of carbapenem drug resistance gene detection test paper based on CRIPSR-dCas9

Abstract

The invention discloses development and application of carbapenem drug-resistant gene detection test paper based on CRIPSR-dCas9. A primer pair, an sgRNA sequence and a recognition probe sequence aiming at an NDM gene are as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4. The invention also discloses a preparation method of the carbapenem drug-resistant gene detection test paper based on CRIPSR-dCas9 and application of the carbapenem drug-resistant gene detection test paper based on CRIPSR-dCas9. According to the primer pair aiming at the KPC gene, an sgRNA sequence and an identification probe sequence are shown as SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8. The invention further discloses a kit for detecting the KPC gene. The method is simple to operate and high in specificity, extra expensive equipment assistance is not needed in the whole detection process, and specific and sensitive detection of common carbapenem drug-resistant genes such as NDM, KPC and OXA-48 in clinical samples can be achieved within 40 min.

Description

[0001] This invention relates to the field of biological detection technology, specifically the development and application of a carbapenem resistance gene detection test strip based on CRIPSR-dCas9. Background Technology

[0002] Due to the long-term, indiscriminate, and extensive use of antibiotics globally, multidrug-resistant bacteria, typically carbapenem-resistant Gram-negative bacteria (CROs), have frequently emerged, posing unprecedented challenges to clinical treatment and being listed by the WHO as one of the top ten global public health threats of the 21st century. Data from the China Antimicrobial Resistance Surveillance Network (CHINET) shows that the situation regarding microbial resistance remains severe, with CROs maintaining a high detection rate for many years, making them one of the most serious drug-resistant bacteria threatening clinical practice. Timely and accurate identification of bacterial resistance in the early stages of clinical infection, helping doctors implement targeted antimicrobial measures, is a crucial step in improving treatment efficiency and curbing the spread of drug-resistant bacteria. However, clinical testing for bacterial resistance mainly involves microbial isolation and culture combined with drug susceptibility testing, which takes several days or even a week. During this period, patients can only receive empirical anti-infective treatment, which may lead to the spread of resistance and worsen the patient's condition.

[0003] To date, most rapid detection technologies for drug-resistant bacteria remain in the laboratory research stage, and there is no universal analytical method for rapid on-site screening of multidrug-resistant bacteria. Immunological methods, such as enzyme-linked immunosorbent assay (ELISA), analyze drug resistance by detecting antibiotic enzymes secreted by drug-resistant bacteria, but their low accuracy limits their clinical application. Real-time fluorescence PCR based on drug-resistant bacterial genotypes has shown high sensitivity and specificity, and has promising applications in the diagnosis of drug-resistant bacteria. However, this method requires large instruments, which cannot meet the needs of rapid clinical screening for multidrug-resistant bacteria. Isothermal amplification technology is promisingly advanced. Enzymatic recombination isothermal amplification (ERA) utilizes recombinases to unwind double strands, achieving high-fold amplification of the target gene at 37-42 °C for 10-30 min. The applicant team previously combined ERA with classic immunochromatographic test strips to develop a test strip detection method that combines the advantages of ERA isothermal amplification and LFS visualization. However, the accuracy of this method mainly depends on the specificity of the amplification primers, and it is easily affected by non-specific amplification when testing actual samples. From a methodological perspective, the main challenges of existing methods lie in their poor accuracy and timeliness. Therefore, achieving accurate detection of key carbapenem resistance genes while ensuring sensor portability and effectively addressing interference from exogenous genes and complex matrices remains a significant challenge.

[0004] In recent years, the emergence of CRISPR / Cas technology has provided a new direction for overcoming these technological bottlenecks. CRISPR / Cas9 consists of two parts: the Cas9 protein and guide RNA (sgRNA). It utilizes the neighboring motif (PAM) in the pre-interstitial region and homologous sequences on the sgRNA to target genes, enabling the identification of mismatches down to the single-base level. The applicant team previously used CRISPR / Cas9 to construct a nucleic acid biosensor, successfully achieving highly sensitive detection of various pathogenic microorganisms. Therefore, if drug resistance gene sequences can be amplified at high magnification using ERA, and conserved fragments of the target gene can be targeted using CRISPR / Cas9, combined with the visualization advantages of test strip technology, it is possible to achieve rapid detection while improving method accuracy, establishing a new method for simultaneously, rapidly, and accurately identifying carbapenem resistance genes, which is of great significance. Summary of the Invention

[0005] The purpose of this invention is to provide a carbapenem resistance gene detection test strip based on CRIPSR-dCas9 and its application, so as to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: The development and application of a carbapenem resistance gene detection strip based on CRIPSR-dCas9 includes the following steps: S1: Rapidly release drug-resistant gene sequences from biological samples using a thermal lysis method to obtain a crude extract containing drug-resistant gene sequences; S2: Using specific primer pairs targeting the drug resistance gene, and with the crude extract as a template, ERA amplification was performed at a constant temperature to obtain terminally biochemical ... S3: Mix an appropriate amount of dsDNA amplification product, dCas9 / sgRNA complex and gold nanoprobe in detection buffer for a certain period of time, then drop it onto the loading area of ​​the test strip. After a certain period of time, the qualitative judgment of the target drug resistance gene fragment in the biological sample to be tested is achieved based on the changes in TL and CL signals in the chromogenic area.

[0007] In a preferred embodiment, the detection system includes primer pairs for ERA amplification of carbapenem resistance genes such as NDM, KPC, and OXA-48, sgRNA sequences for visual detection on test strips, recognition probe sequences, and quality control probe sequences, wherein: The primer pair used for NDM gene ERA amplification is: The sequence of NDM-F is shown in SEQ ID NO.1; The sequence of NDM-R is shown in SEQ ID NO.2; The sgRNA sequence used for NDM gene detection is shown in SEQ ID NO.3; The recognition probe sequence for NDM gene detection is shown in SEQ ID NO.4; The primer pair used for KPC gene ERA amplification is: The sequence of KPC-F is shown in SEQ ID NO.5; The sequence of KPC-R is shown in SEQ ID NO.6; The sgRNA sequence used for KPC gene detection is shown in SEQ ID NO.7; The recognition probe sequence for KPC gene detection is shown in SEQ ID NO.8; The primer pair used for ERA amplification of the OXA-48 gene is: The sequence of OXA-48-F is shown in SEQ ID NO.9; The sequence of OXA-48-R is shown in SEQ ID NO.10; The sgRNA sequence used for OXA-48 gene detection is shown in SEQ ID NO.11; The recognition probe sequence for OXA-48 gene detection is shown in SEQ ID NO.12; The quality control probe sequence is as shown in SEQ ID NO.13.

[0008] In the preferred embodiment, the heating temperature of the thermal pyrolysis method is 80-100℃, and the heating time is 2-5 minutes.

[0009] In a preferred embodiment, the concentration of the specific primers for ERA amplification is 100-1000 nmol / L, and the reaction temperature is 30-40℃. The ERA amplification method employs specific primer pairs to effectively amplify the target drug resistance gene while simultaneously achieving biotinylation labeling of the dsDNA amplification product, which is the key to the design of the test strip detection method of this invention.

[0010] In a preferred embodiment, the gold nanoparticles involved in the gold nanoprobe have a size of 20-40 nm, and the preparation method of the gold nanoprobe includes the following steps: A1: Take 1 mL of gold nanoparticles, centrifuge at 8500 rpm for 10-15 min, and then concentrate to 100 μL; A2: Mix 5-10 μL of a 10-50 μM recognition probe sequence, 2 μL of 1-5 mM TCEP solution, and 1-2 μL of 200-1000 mM acetate buffer at pH 5.0-5.3 in the dark and incubate for 30-60 min. Then inject the mixture into the reaction tube containing 100 μL of concentrated gold nanoparticles described in A1 and incubate at room temperature for 1-2 h. A3: Add an appropriate amount of blocking agent to the reaction tube described in A2. The blocking agent is bovine serum albumin (BSA) solution with a concentration of 5-10% and an addition volume of 2-10 μL.

[0011] A4: Centrifuge the reaction solution described in A3 at 8500 rpm for 10-15 min to obtain a gold nanoparticle probe precipitate. Resuspend the precipitate in 50 μL of Tris-Buffer. The Tris-Buffer contains Tris-HCl, BSA, Tween-20, and sucrose, with a pH of 7.9-8.2. The concentration of Tris-HCl is 1-10 mM, the concentration of BSA is 5-10%, the concentration of Tween-20 is 0.1-1%, and the concentration of sucrose is 5-15%.

[0012] In a preferred embodiment, the test strip includes a sample loading area, a color development area, an absorbent area, and a base plate. The color development area is provided with a detection line (TL) pre-labeled with streptavidin and a control line (CL) for a quality control probe. The sequence of the quality control probe is shown in SEQ ID NO.13.

[0013] In a preferred embodiment, the concentration of streptavidin in the TL region is 0.5-2.5 mg / mL; In a preferred embodiment, the quality control probe in the CL region is bound and immobilized via streptavidin-biotin interaction. The preparation process involves incubating an appropriate amount of streptavidin, 5×PBS solution, and the biotinylated quality control probe at room temperature for a certain period of time, and then spraying them onto an NC membrane using a spray membrane applicator to form the CL region. The streptavidin concentration is 1-5 mg / mL, and the addition volume is 1-3 μL; the addition volume of the 5×PBS buffer is 5-15 μL; and the concentration of the quality control probe is 20-100 μmol / L, with an addition volume of 1-5 μL.

[0014] In a preferred embodiment, during the test strip detection process, the volume of the dsDNA amplification product is 2-5 μL, the final concentration of the dCas9 / sgRNA complex is 100-600 nmol / L, and the detection buffer comprises phosphate buffer, sucrose, BSA, PEG-20000, and MgCl2. The dCas9 / sgRNA complex is prepared by mixing dCas9 protein and sgRNA sequence in a 1:1 ratio; the concentration of the phosphate buffer is 50-200 nmol / L, the pH is 7.4-8.0, the mass concentration of the sucrose is 5-20%, the mass concentration of the BSA is 2-15%, the mass concentration of the PEG-2000 is 0.5-5%, and the concentration of the MgCl2 is 10-500 mmol / L. The incubation time is 2-5 min, and the detection time at the test strip interface is 3-5 min.

[0015] Compared with the prior art, the beneficial effects of the present invention are: 1. Compared with traditional detection methods, this method uses specific primer pairs targeting drug resistance genes for specific amplification, and then uses CRISPR / dCas9 for secondary specific recognition. This can effectively avoid the risks of non-target gene interference and aerosol contamination caused by primer dimers while ensuring sensitivity, and significantly improve the accuracy of detection results. 2. Compared with traditional detection methods, the detection steps involved in this method do not require expensive instruments, making it suitable for rapid on-site detection of carbapenem resistance genes in areas with limited resources. It also features high sensitivity, good specificity, simple operation, and fast result interpretation, showing promising application prospects.

[0016] 3. As a general detection tool, this method can achieve specific, sensitive and rapid detection of any kind of drug resistance gene by simply changing the ERA amplification primer pair, sgRNA sequence and recognition probe sequence. It can also be extended to other gene-dependent detection scenarios, and has excellent universality. Attached Figure Description

[0017] Figure 1 A schematic diagram of the structure and detection application of a carbapenem resistance gene detection test strip based on CRIPSR-dCas9.

[0018] Figure 2 Sensitivity analysis results of rapid detection of NDM gene using the test strip of this invention.

[0019] Figure 3 Sensitivity analysis results of rapid detection of the KPC gene using the test strip of this invention.

[0020] Figure 4 Sensitivity analysis results of rapid detection of the OXA-48 gene using the test strip of this invention. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Please see Figure 1 The present invention provides the following technical solution: a test strip for detecting carbapenem resistance genes based on CRIPSR-dCas9, the steps of which include: S1: Rapidly release drug-resistant gene sequences from biological samples via thermal lysis to obtain a crude extract containing drug-resistant gene sequences; S2: Using specific primer pairs targeting the drug resistance gene, and with the crude extract as a template, ERA amplification was performed at a constant temperature to obtain the terminally biologized dsDNA amplicon sequence. S3: Mix an appropriate amount of dsDNA amplification product, dCas9 / sgRNA complex and gold nanoprobe in detection buffer for a certain period of time, then drop it onto the loading area of ​​the test strip. After a certain period of time, the qualitative judgment of the target drug resistance gene fragment in the biological sample to be tested is achieved based on the changes in TL and CL signals in the chromogenic area.

[0023] The detection system includes primer pairs for ERA amplification of carbapenem resistance genes such as NDM, KPC, and OXA-48, sgRNA sequences for visual detection on test strips, recognition probe sequences, and quality control probe sequences, wherein: The primer pair used for NDM gene ERA amplification is: The sequence of NDM-F is shown in SEQ ID NO.1; The sequence of NDM-R is shown in SEQ ID NO.2; The sgRNA sequence used for NDM gene detection is shown in SEQ ID NO.3; The recognition probe sequence for NDM gene detection is shown in SEQ ID NO.4; The primer pair used for KPC gene ERA amplification is: The sequence of KPC-F is shown in SEQ ID NO.5; The sequence of KPC-R is shown in SEQ ID NO.6; The sgRNA sequence used for KPC gene detection is shown in SEQ ID NO.7; The recognition probe sequence for KPC gene detection is shown in SEQ ID NO.8; The primer pair used for ERA amplification of the OXA-48 gene is: The sequence of OXA-48-F is shown in SEQ ID NO.9; The sequence of OXA-48-R is shown in SEQ ID NO.10; The sgRNA sequence used for OXA-48 gene detection is shown in SEQ ID NO.11; The recognition probe sequence for OXA-48 gene detection is shown in SEQ ID NO.12; The quality control probe sequence is as shown in SEQ ID NO.13.

[0024] The heating temperature for the aforementioned thermal pyrolysis method is 80-10℃, and the heating time is 2-5 min.

[0025] The concentration of the specific primers for ERA amplification is 100-1000 nmol / L, and the reaction temperature is 30-40℃. The ERA amplification uses specific primer pairs to effectively amplify the target drug resistance gene while simultaneously achieving biotinylation labeling of the dsDNA amplification product, which is the key to the design of the test strip detection method of this invention.

[0026] The gold nanoparticles involved in the gold nanoprobe have a size of 20-40 nm, and the preparation method of the gold nanoprobe includes the following steps: A1: Take 1 mL of gold nanoparticles, centrifuge at 8500 rpm for 10-15 min, and then concentrate to 100 μL; A2: Mix 5-10 μL of a 10-50 μM recognition probe sequence, 2 μL of 1-5 mM TCEP solution, and 1-2 μL of 200-1000 mM acetate buffer at pH 5.0-5.3 in the dark and incubate for 30-60 min. Then inject the mixture into the reaction tube containing 100 μL of concentrated gold nanoparticles described in A1 and incubate at room temperature for 1-2 h. A3: Add an appropriate amount of blocking agent to the reaction tube described in A2. The blocking agent is bovine serum albumin (BSA) solution with a concentration of 5-10% and an addition volume of 2-10 μL.

[0027] A4: Centrifuge the reaction solution described in A3 at 8500 rpm for 10-15 min to obtain a gold nanoparticle probe precipitate. Resuspend the precipitate in 50 μL of Tris-Buffer. The Tris-Buffer contains Tris-HCl, BSA, Tween-20, and sucrose, with a pH of 7.9-8.2. The concentration of Tris-HCl is 1-10 mM, the concentration of BSA is 5-10%, the concentration of Tween-20 is 0.1-1%, and the concentration of sucrose is 5-15%.

[0028] The test strip includes a sample loading area, a color development area, an absorbent area, and a base plate. The color development area is provided with a detection line (TL) pre-labeled with streptavidin and a control line (CL) for a quality control probe. The sequence of the quality control probe is shown in SEQ ID NO. 13.

[0029] The concentration of streptavidin in the TL region is 0.5-2.5 mg / mL; The quality control probe in the CL region is bound and immobilized via streptavidin-biotin interaction. The preparation process is as follows: An appropriate amount of streptavidin, 5×PBS solution, and biotinylated quality control probe are incubated at room temperature for a certain period of time, and then sprayed onto an NC membrane using a spray membrane applicator to form the CL region. The concentration of streptavidin is 1-5 mg / mL, and the addition volume is 1-3 μL; the addition volume of the 5×PBS buffer is 5-15 μL; and the concentration of the quality control probe is 20-100 μmol / L, with an addition volume of 1-5 μL.

[0030] During the test strip detection process, the volume of the dsDNA amplification product is 2-5 μL, the final concentration of the dCas9 / sgRNA complex is 100-600 nmol / L, and the detection buffer contains phosphate buffer, sucrose, BSA, PEG-20000, and MgCl2. The dCas9 / sgRNA complex is prepared by mixing dCas9 protein and sgRNA sequence in a 1:1-1:2 ratio. The phosphate buffer concentration is 50-200 nmol / L, pH is 7.4-8.0, the sucrose concentration is 5-20%, the BSA concentration is 2-15%, the PEG-2000 concentration is 0.5-5%, and the MgCl2 concentration is 10-500 mmol / L. The incubation time is 2-5 min, and the detection time at the test strip interface is 3-5 min.

[0031] In summary, the implementation steps of the detection method of this invention are as follows: ① Preparation of gold nanoparticle probes; ② Construction of test strips; ③ Rapid release of drug resistance gene sequences from biological samples; ④ ERA amplification of target drug resistance genes; ⑤ Visual detection using test strips. This invention uses specific primer sets targeting three drug resistance genes: NDM, KPC, and OXA-48, to obtain biolabeled dsDNA amplification products. The dCas9 / sgRNA complex specifically recognizes and unlocks the target sequence in the target dsDNA amplification products, enabling the gold nanoparticle probes to specifically bind to them. The binding of the formed complex on the test strip generates a color signal. The more target drug resistance genes present in the biological sample, the stronger the TL color signal, thus establishing a quantitative relationship between the concentration of target drug resistance genes and the intensity of the TL color signal, achieving rapid target detection. Furthermore, by using different primer pairs, sgRNA sequences, and recognition probe sequences, the detection method of this invention can be used for the detection of different drug resistance genes.

[0032] See the attached table for the sequence list.

[0033] The technical solution of the present invention will be further explained and described below with reference to the accompanying drawings and several preferred embodiments. However, the experimental conditions and setting parameters therein should not be regarded as limitations on the basic technical solution of the present invention. Furthermore, the scope of protection of the present invention is not limited to the following embodiments.

[0034] Example 1 The test strip detection method for the NDM gene in biological samples provided in this embodiment includes the following steps: ① Preparation of gold nanoparticle probes: 1 mL of gold nanoparticles was centrifuged at 8500 rpm for 10 min and concentrated to 100 μL. 10 μL of a 10 μM recognition probe sequence, 2 μL of 1 mM TCEP solution, and 1 μL of 200 mM acetate buffer (pH 5.0) were mixed and incubated in the dark for 30 min. This mixture was then injected into a reaction tube containing 100 μL of concentrated gold nanoparticles and incubated at room temperature for 1 h. 2 μL of blocking agent (10% BSA) was added to the reaction tube. The reaction solution was centrifuged at 8500 rpm for 10 min to obtain a gold nanoparticle probe precipitate. The precipitate was resuspended in 50 μL of Tris-Buffer (1 mM Tris-HCl, 5% BSA, 0.25% Tween-20, 10% sucrose, pH 7.9).

[0035] ② Construction of the test strip: Mix 2.5 μL of quality control probe, 2.5 μL of 5 mg / mL streptavidin, and 15 μL of 1×PBS solution, incubate at room temperature for 30 min, and then spray this mixture onto the colorimetric area of ​​the test strip using a spray film applicator as CL; spray 1 mg / mL of streptavidin onto the colorimetric area of ​​the test strip as TL, and dry at 30℃ for later use. Assemble the sample loading area, colorimetric area, and absorbent area onto the substrate in sequence to obtain the test strip.

[0036] ③ Rapid release of NDM gene sequence from biological samples: Take 1 mL of the biological sample to be tested (including blood, urine and saliva), centrifuge at 5000 rpm for 2 min, aspirate 50 μL (containing precipitate) from the bottom and transfer it to another clean reaction tube, add 50 μL of extraction solution and mix, incubate at 80℃ for 3 min to obtain crude extract containing NDM gene sequence.

[0037] ④ ERA amplification of NDM gene: Dissolve RPA dry powder reagent in 20 μL of solvent, add 2 μL each of 10 μM NDM-F and NDM-R, add water to 46 μL, add 2 μL of crude extract and 2 μL of activator, and immediately react at 40℃ for 20 min.

[0038] ⑤ Visual detection of the test strip: In step ④ of this embodiment, the ERA product is incubated with the dCas9 / sgRNA complex and gold nanoprobe in the detection buffer for 3 min, and then directly loaded onto the sample of the test strip. After standing at room temperature for 3 min, the test strip image is captured using an SLR camera or mobile phone, and the signal intensity of TL is analyzed using ImageJ software.

[0039] For specific experiments in sensitivity analysis, please refer to [link / reference]. Figure 2 The concentration of the NDM gene was diluted to different gradients using saliva, and a blank control group was set up. After the NDM gene of different concentrations was detected by the method of this invention, the corresponding detection signal could be displayed according to the concentration of the NDM gene. This method can detect the NDM gene down to 1 copy / μL, and the entire detection process can be completed within 40 minutes.

[0040] Example 2 The test strip detection method for the KPC gene in biological samples provided in this embodiment includes the following steps: ① Preparation of gold nanoparticle probes: 1 mL of gold nanoparticles was centrifuged at 8500 rpm for 10 min and concentrated to 100 μL. 10 μL of a 10 μM recognition probe sequence, 2 μL of 1 mM TCEP solution, and 1 μL of 200 mM acetate buffer (pH 5.0) were mixed and incubated in the dark for 30 min. This mixture was then injected into a reaction tube containing 100 μL of concentrated gold nanoparticles and incubated at room temperature for 1 h. 2 μL of blocking agent (10% BSA) was added to the reaction tube. The reaction solution was centrifuged at 8500 rpm for 10 min to obtain a gold nanoparticle probe precipitate. The precipitate was resuspended in 50 μL of Tris-Buffer (1 mM Tris-HCl, 5% BSA, 0.25% Tween-20, 10% sucrose, pH 7.9).

[0041] ② Construction of the test strip: Mix 2.5 μL of quality control probe, 2.5 μL of 5 mg / mL streptavidin, and 15 μL of 1×PBS solution, incubate at room temperature for 30 min, and then spray this mixture onto the colorimetric area of ​​the test strip using a spray film applicator as CL; spray 1 mg / mL of streptavidin onto the colorimetric area of ​​the test strip as TL, and dry at 30℃ for later use. Assemble the sample loading area, colorimetric area, and absorbent area onto the substrate in sequence to obtain the test strip.

[0042] ③ Rapid release of KPC gene sequence from biological samples: Take 1 mL of the biological sample to be tested (including blood, urine and saliva), centrifuge at 5000 rpm for 2 min, aspirate 50 μL (containing precipitate) from the bottom and transfer it to another clean reaction tube, add 50 µl of extraction buffer and mix, incubate at 80℃ for 3 min to obtain crude extract containing NDM gene sequence.

[0043] ④ ERA amplification of the KPC gene: Dissolve the RPA dry powder reagent in 20 μL of solvent, add 2 μL each of 10 μM KPC-F and KPC-R, add water to 46 μL, add 2 μL of crude extract and 2 μL of activator, and immediately react at 40℃ for 20 min.

[0044] ⑤ Visual detection of the test strip: In step ④ of this embodiment, the ERA product is incubated with the dCas9 / sgRNA complex and gold nanoprobe in the detection buffer for 3 min, and then directly loaded onto the sample of the test strip. After standing at room temperature for 3 min, the test strip image is captured using an SLR camera or mobile phone, and the signal intensity of TL is analyzed using ImageJ software.

[0045] For specific experiments in sensitivity analysis, please refer to [link / reference]. Figure 3The concentration of KPC gene was diluted to different gradients using serum, and a blank control group was set up. After different concentrations of KPC gene were detected by the method of this invention, the corresponding detection signal could be displayed according to the concentration of KPC gene. This method can detect KPC gene down to 1 copy / μL, and the entire detection process can be completed within 40 minutes.

[0046] Example 3 The test strip detection method for the OXA-48 gene in biological samples provided in this embodiment includes the following steps: ① Preparation of gold nanoparticle probes: 1 mL of gold nanoparticles was centrifuged at 8500 rpm for 10 min and concentrated to 100 μL. 10 μL of a 10 μM recognition probe sequence, 2 μL of 1 mM TCEP solution, and 1 μL of 200 mM acetate buffer (pH 5.0) were mixed and incubated in the dark for 30 min. This mixture was then injected into a reaction tube containing 100 μL of concentrated gold nanoparticles and incubated at room temperature for 1 h. 2 μL of blocking agent (10% BSA) was added to the reaction tube. The reaction solution was centrifuged at 8500 rpm for 10 min to obtain a gold nanoparticle probe precipitate. The precipitate was resuspended in 50 μL of Tris-Buffer (1 mM Tris-HCl, 5% BSA, 0.25% Tween-20, 10% sucrose, pH 7.9).

[0047] ② Construction of the test strip: Mix 2.5 μL of quality control probe, 2.5 μL of 5 mg / mL streptavidin, and 15 μL of 1×PBS solution, incubate at room temperature for 30 min, and then spray this mixture onto the colorimetric area of ​​the test strip using a spray film applicator as CL; spray 1 mg / mL of streptavidin onto the colorimetric area of ​​the test strip as TL, and dry at 30℃ for later use. Assemble the sample loading area, colorimetric area, and absorbent area onto the substrate in sequence to obtain the test strip.

[0048] ③ Rapid release of OXA-48 gene sequence from biological samples: Take 1 mL of the biological sample to be tested (including blood, urine and saliva), centrifuge at 5000 rpm for 2 min, aspirate 50 μL (containing precipitate) from the bottom and transfer it to another clean reaction tube, add 50 µl of extraction buffer and mix, incubate at 80℃ for 3 min to obtain crude extract containing NDM gene sequence.

[0049] ④ ERA amplification of the OXA-48 gene: Dissolve the RPA dry powder reagent in 20 μL of solvent, add 2 μL each of 10 μM OXA-48-F and OXA-48-R, add water to 46 μL, add 2 μL of crude extract and 2 μL of activator, and immediately react at 40℃ for 20 min.

[0050] ⑤ Visual detection of the test strip: In step ④ of this embodiment, the ERA product is incubated with the dCas9 / sgRNA complex and gold nanoprobe in the detection buffer for 3 min, and then directly loaded onto the sample of the test strip. After standing at room temperature for 3 min, the test strip image is captured using an SLR camera or mobile phone, and the signal intensity of TL is analyzed using ImageJ software.

[0051] For specific experiments in sensitivity analysis, please refer to [link / reference]. Figure 3 The concentration of the KPC gene was diluted to different gradients using saliva, and a blank control group was set up. After the KPC gene of different concentrations was detected by the method of this invention, the corresponding detection signal could be displayed according to the concentration of the KPC gene. This method can detect the OXA-48 gene down to 1 copy / μL, and the entire detection process can be completed within 40 minutes.

Claims

1. A CRIPSR-dCas9-based carbapenem resistance gene detection test paper development and application, characterized in that, Comprise: S1: quickly release drug-resistant drug gene sequences in drug-resistant drug gene sequences in biological samples by heating cracking method, obtain the crude extract containing drug-resistant gene sequences; S2: using specific primer pair for target drug-resistant gene, taking crude extract as template, carrying out ERA amplification at constant temperature, obtaining end-biotinylated dsDNA amplification product; S3: mixing a certain amount of dsDNA amplification product, dCas9 / sgRNA complex and gold nanoparticle probe in detection buffer for a certain time, then adding to the loading area of test paper, after a certain time, finally according to the change of TL and CL signal of color developing area, realizing the qualitative judgment of target drug-resistant gene fragment in the biological sample to be measured; The detection system comprises primer pairs for ERA amplification of NDM, KPC and OXA-48 carbapenem-resistant genes, sgRNA sequences, recognition probe sequences and quality control probe sequences for visual detection of test paper, wherein: The primer pair for NDM gene ERA amplification is: The sequence of NDM-F is shown as SEQ ID NO. 1; The sequence of NDM-R is shown as SEQ ID NO. 2; The sgRNA sequence NDM-J1 for NDM gene detection is shown as SEQ ID NO. 3; The recognition probe sequence NDM-J2 for NDM gene detection is shown as SEQ ID NO. 4; The primer pair for KPC gene ERA amplification is: The sequence of KPC-F is shown as SEQ ID NO. 5; The sequence of KPC-R is shown as SEQ ID NO. 6; The sgRNA sequence KPC-J1 for KPC gene detection is shown as SEQ ID NO. 7; The recognition probe sequence KPC-J2 for KPC gene detection is shown as SEQ ID NO. 8; The primer pair for OXA-48 gene ERA amplification is: The sequence of OXA-48-F is shown as SEQ ID NO. 9; The sequence of OXA-48-R is shown as SEQ ID NO. 10; The sgRNA sequence OXA-J1 for OXA-48 gene detection is shown as SEQ ID NO. 11; The recognition probe sequence OXA-J2 for OXA-48 gene detection is shown as SEQ ID NO. 12; The quality control probe sequence ZK-T is shown as SEQ ID NO.

13.

2. The process according to claim 1, characterized in that: The heating temperature is 80-100 DEG C, and the heating time is 2-5 min; the present application realizes the effective amplification of target drug-resistant gene based on the primer of claim 2, and realizes the biotinylated labeling of dsDNA amplification product, which is the key of the test paper detection method design of the present application.

3. The ERA expansion of claim 1, wherein: The specific primer concentration of ERA amplification is 100-1000 nmol / L, and the reaction temperature is 30-40 DEG C.

4. The gold nanoprobe of claim 1, wherein: The size of the gold nanoparticles involved is 20-40 nm, and the preparation method of the gold nanoparticle probe comprises the following steps: A1: centrifuge 1 mL gold nanoparticles at 8500 rpm for 10-15 min, and concentrate to 100 μL; A2: 5-10 μL of the recognition probe sequence with a concentration of 10-50 μM, 2 μL of TCEP solution with a concentration of 1-5 mM, and 1-2 μL of acetic acid buffer with a concentration of 200-1000 mM and a pH of 5.0-5.3 were mixed and incubated in the dark for 30-60 min, and then injected into the reaction tube containing 100 μL of concentrated gold nanoparticles as described in A1, and incubated at room temperature for 1-2 h; A3: An appropriate amount of blocking agent was added to the reaction tube described in A2, the blocking agent was a bovine serum albumin (BSA) solution, the concentration of the BSA solution was 5-10%, and the volume added was 2-10 μL; A4: The reaction solution described in A3 was centrifuged at 8500 rpm for 10-15 min to obtain a gold nanoparticle precipitate, and the obtained precipitate was resuspended with 50 μL of Tris-Buffer. The Tris-Buffer contains Tris-HCl, BSA, Tween-20, and sucrose, etc., with a pH of 7.9-8.2; the concentration of Tris-HCl is 1-10 mM, the concentration of BSA is 5-10%, the concentration of Tween-20 is 0.1-1%, and the concentration of sucrose is 5-15%.

5. The test strip of claim 1, wherein: The detection test paper includes a sample loading area, a color development area, a water absorption area, and a base plate, the color development area is provided with a detection line (TL) pre-labeled with streptavidin and a quality control line (CL) of a quality control probe, and the sequence of the quality control probe is shown in SEQ ID NO.

13.

6. According to claim 5, the concentration of streptavidin in the TL area is 0.5-2.5 mg / mL.

7. According to claim 5, the quality control probe in the CL area is bound and fixed by streptavidin-biotin interaction, and the preparation process is: incubating an appropriate amount of streptavidin, 5×PBS solution, and biotinylated quality control probe at room temperature for a certain period of time, and then using a film sprayer to spray on the NC membrane as the CL area, the concentration of streptavidin is 1-5 mg / mL, the volume added is 1-3 μL, the volume of 5×PBS buffer added is 5-15 μL, and the concentration of the quality control probe is 20-100 μmol / L, and the volume added is 1-5 μL.

8. The test strip assay process of claim 1, wherein: The volume of the dsDNA amplification product is 2-5 μL, the final concentration of the dCas9 / sgRNA complex is 100-600 nmol / L, and the detection buffer comprises a phosphate buffer, sucrose, BSA, PEG-20000 and MgCl2. The dCas9 / sgRNA complex is prepared by mixing dCas9 protein and sgRNA sequence at a ratio of 1:

1. The concentration of the phosphate buffer is 50-200 nmol / L, the pH is 7.4-8.0, the mass concentration of the sucrose is 5-20%, the mass concentration of the BSA is 2-15%, the mass concentration of the PEG-2000 is 0.5-5%, and the concentration of the MgCl2 is 10-500 mmol / L. The incubation time is 2-5 min, and the detection time of the test paper interface is 3-5 min.

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