High-risk HPV16 rapid detection system based on RPA-CRISPR / Cas12a and application

Abstract

The invention belongs to the technical field of molecular diagnosis, and particularly discloses a high-risk HPV16 rapid detection system based on RPA-CRISPR / Cas12a, which is prepared by the following steps: (1) mixing a nucleic acid lysis solution containing Tris (pH = 8.0), NaHCO3, polyvinylpyrrolidone and Tween-20 with Proteinase K to treat a sample, and performing pyrolysis at room temperature for 5 minutes to obtain a nucleic acid template; (2) an RPA reaction system contains an RNA inhibitor, a reverse transcriptase and an RPA reaction essential enzyme, and after the template and the starting agent in the step (1) are added, RPA amplification is carried out at 37 DEG C; (3) dropping the reaction liquid on a colloidal gold test strip subjected to position exchange and transformation of a sample pad and a water absorption pad, and carrying out result interpretation; and (4) adding the RPA product into a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) reaction system containing the LbCas12a protein, the specific gRNA and the probe. According to the scheme, a rapid, high-sensitivity and cross-reaction-free detection system of the HPV16 is realized by optimizing a nucleic acid lysis solution formula, designing a specific primer and gRNA and modifying a test strip for detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular diagnostic technology, and in particular to a rapid, sensitive and specific detection method and system for high-risk human papillomavirus type 16 (HPV16) based on the combination of recombinase polymerase amplification (RPA) and CRISPR / Cas12a system. BACKGROUND

[0002] Persistent infection with high-risk human papillomavirus (hrHPV) is the main cause of the occurrence and development of cervical cancer, and about 95% of cervical cancer cases are associated with hrHPV infection, especially HPV types 16 and 18. Cervical cancer is the fourth most common cancer among women worldwide, with an estimated 660,000 new cases and 350,000 deaths worldwide in 2022. Low- and middle-income countries have the highest prevalence and mortality rates, reflecting problems such as inadequate vaccination and screening coverage. Persistent infection with high-risk HPV can cause abnormal cervical epithelial cells, leading to cervical intraepithelial neoplasia (CIN) and cervical cancer. In addition, clinical data show that persistent infection with HPV type 16 significantly increases the risk of cervical cancer, and its detection rate in patients with cervical cancer is as high as about 70%. Therefore, it is of great significance to establish a rapid, sensitive and specific HPV16 detection method for early screening and prevention of cervical cancer.

[0003] The 'three-step' approach, namely cervical cytology, HPV testing, colposcopy and cervical biopsy, is the main method for early cervical cancer screening. The pathological examination of cervical biopsy is the 'gold standard' for clinical diagnosis. With the development of molecular techniques, PCR and nucleic acid hybridization have also been developed for HPV detection. Although they have high sensitivity and specificity, they are complex, time-consuming, require expensive equipment and professional technicians, and are difficult to promote in primary medical institutions and resource-limited areas. In recent years, nucleic acid detection techniques based on recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeat (CRISPR / Cas) systems have shown great potential in pathogen detection due to their rapid, sensitive, specific, and simple operation. RPA is an isothermal nucleic acid amplification technique that can rapidly amplify target DNA at 37-42℃ without the need for complex temperature cycling equipment. The CRISPR / Cas system is a prokaryotic adaptive immune system. Cas12a protein, under the guidance of guide CRISPR RNAs (gRNA), can not only recognize target DNA with high specificity, but also has non-specific single-stranded DNA (ssDNA) cleavage activity after activation, which can be used for signal amplification. The combination of RPA and CRISPR / Cas12a system can achieve rapid amplification and high-sensitivity detection of target DNA.

[0004] HPV16 E6 and E7 oncoproteins are considered to be the main participants in the transformation of infected cervical cells to cervical cancer. Studies have found that E6 and E7 can exacerbate cell proliferation through a glutamine-dependent manner. The oncogenic mechanism of HPV mainly depends on the active expression of two oncogenes E6 and E7, and E6 / E7 gene expression directly participates in the destruction of tumor suppressor proteins (such as p53 and Rb) of host cells, promoting the process of cell carcinogenesis. In another clinical study, it was found that HPV16 E6 and E7 mRNA can reflect the degree of E6 / E7 transcriptional activity, and can reflect whether the virus is truly active and integrated in the cell, and distinguish between persistent infection and transient infection. A Meta analysis showed that HPV E6 / E7 mRNA detection had comparable sensitivity to HPV DNA in screening for high-grade cervical lesions, but its specificity was significantly higher, which was beneficial to improve the accuracy of screening. In addition, for patients with HPV-positive and abnormal cervical cytology, E6 / E7 mRNA detection can help risk stratification management and guide subsequent diagnosis and treatment decisions, especially for the monitoring of infection with high-risk HPV16 and early screening for cervical cancer.

[0005] The patent aims to establish an RPA-CRISPR / Cas12a detection technology based on HPV16 E6 and E7 mRNA. The technology combines the rapid amplification capability of RPA with the high sensitivity detection capability of CRISPR / Cas12a, and at the same time, the existing nucleic acid lysis solution is optimized to obtain the best nucleic acid formula, which is expected to provide a rapid, sensitive and specific detection method for HPV16 infection, and provide a new technical means for early screening and prevention of cervical cancer. SUMMARY

[0006] In order to overcome the defects of the prior art, a HPV16 detection method based on RPA-CRISPR / Cas12a is provided, which realizes the rapid, high sensitivity and non-cross reaction detection system of HPV16 by optimizing the nucleic acid lysis solution formula, designing specific primers and gRNA, and modifying the test paper strip.

[0007] The present application provides a nucleic acid lysis solution formula, which comprises Tris (pH=8.0), NaHCO3, 2% polyvinylpyrrolidone (PVP) and Tween-20. The lysis solution combines with Proteinase K to process the sample, and the complete RNA / DNA template is quickly obtained.

[0008] Specific primers F1, R1, F2 and R2 designed for HPV16 E6 / E7 gene are used, and the sequences are SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, respectively. The RPA isothermal amplification reaction at 40 DEG C is used, and the amplification time is controlled at 30 minutes. The reaction system is 50 μL, and the details are shown in Table 1: the band is present in the amplification results of the primer pair of SEQ ID NO:1 and SEQ ID NO:2, the primer pair of SEQ ID NO:3 and SEQ ID NO:4, and no band is present in the negative amplification, indicating that the selected primers are suitable for RPA detection. And the band in the nucleic acid protein gel imager (Bio-Rad) of the amplification product of the primer pair of SEQ ID NO:1 and SEQ ID NO:2 is brighter. Preferably, the primer pair of SEQ ID NO:1 and SEQ ID NO:2 is selected for subsequent experiments.

[0009] Table 1 RPA reaction system

[0010] Reagent Volume (μl) RPA reaction unit 1 tube SEQ ID NO: 1 / 3 (10 μM) 2 SEQ ID NO: 2 / 4 (10 μM) 2 Template 5 Magnesium acetate 2.5 Ultra-pure water 38.5

[0011] Under the premise of setting the reaction time (30 min) unchanged, the isothermal amplification was carried out in a constant temperature water bath at 30, 33, 37, 40 and 43℃ to change the incubation temperature, and the optimal reaction temperature of the scheme was determined to be 37℃; under the premise of setting the reaction temperature (37℃) unchanged, the isothermal amplification was carried out in a constant temperature water bath to change the incubation time of 5, 10, 15, 20, 30 and 35 minutes, and the optimal reaction time of the scheme was determined to be 30 minutes.

[0012] The structure of the colloidal gold test strip was modified, and the sample pad and the water absorption pad were designed to be reversed, so that the sample liquid first contacted with the detection line, which significantly improved the readability of the CRISPR / Cas12a system detection result, and the color of the positive reaction detection line and the quality control line was deepened, and the color of the negative reaction detection line was not changed. Among them, the combination pad contains rabbit anti-FITC antibody colloidal gold latex microspheres and mouse anti-FAM antibody colloidal gold latex microspheres, the detection line contains goat anti-mouse secondary antibody, and the quality control line contains colloidal gold labeled streptavidin which can bind to biotin.

[0013] On the basis of the RPA amplification product, a CRISPR / Cas12a detection system was constructed, and the system composition included LbCas12a (cpf1) protein, paired gRNA (SEQ ID NO: 5), and probe (SEQ ID NO: 6). After optimization, the final concentration of the probe was 0.04μM, the reaction system was 20μl, and the details were shown in Table 2 below. The reaction temperature was 37℃, and the cutting time was 20 minutes.

[0014] Table 2 CRISPR / Cas12a reaction system

[0015] Reagent Volume (μl) 10*Cas12a Buffer 2 LbCas12a (cpf1) protein 1.5 gRNA (5 μM) 1.2 RPA amplification product 0.5 Probe 2 Ultra-pure water 12.8

[0016] An application of a high-risk HPV16 rapid detection system based on RPA-CRISPR / Cas12a, the application comprising the following steps:

[0017] (1) A recombinant plasmid containing a target gene was constructed, and RPA template was obtained by lysis for 5 minutes using the lysis scheme in claim 1. RPA amplification was performed using the RPA amplification primer pair, and the reaction was carried out at 37℃ for 30 minutes to obtain RPA amplification product;

[0018] (2) The CRISPR / Cas12a reaction solution was prepared, which included the probe, gRNA and LbCas12a protein, and the RPA amplification product prepared in step (1);

[0019] (3) using the CRISPR / Cas12a reaction solution prepared in step (2) to perform a CRISPR / Cas12a reaction for 20 minutes, and then using the modified colloidal gold lateral flow chromatographic test strip to perform detection.

[0020] (4) the positive result is that the color of the test line and the quality control line is deepened, and the negative result is that only the color of the quality control line is changed.

[0021] Further detection of clinical samples, the steps are as follows:

[0022] (1) the clinical samples collected in the clinic are lysed for 5 minutes using the lysis solution in claim 1 to obtain RPA templates, and the RPA amplification primer pair is used for RPA amplification, and the reaction is performed at 37 DEG C for 30 minutes to obtain the RPA amplification product;

[0023] (2) the CRISPR / Cas12a reaction solution is prepared, and the CRISPR / Cas12a reaction solution includes the probe, gRNA and LbCas12a protein, and the RPA amplification product prepared in step (1);

[0024] (3) using the CRISPR / Cas12a reaction solution prepared in step (2) to perform a CRISPR / Cas12a reaction for 20 minutes, and then using the modified colloidal gold lateral flow chromatographic test strip to perform detection.

[0025] (4) the positive result is that the color of the test line and the quality control line is deepened, and the negative result is that only the color of the quality control line is changed.

[0026] The experimental data show that the detection sensitivity of the method to HPV16 reaches 10 1 copies / muL, the sensitivity is high, and the specificity is strong.

[0027] The total time of the whole nucleic acid extraction to result interpretation is controlled within 58 minutes, which is suitable for the rapid detection needs of primary medical institutions and on-site.

[0028] The above is only a preferred embodiment of the present application, and does not limit the present application. Any simple modification, change and equivalent change made according to the technical essence of the application to the above embodiment are still within the protection scope of the technical solution of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 Agarose gel electrophoresis result after lysing pUC57 E6 / E7 recombinant plasmid with nucleic acid lysis solution.

[0030] Lane M is 250bp DNA ladder; Lane 1, 2 and 3 represent the results of agarose gel electrophoresis after cracking pUC57 E6 / E7 recombinant plasmid by cracking scheme ①; Lane 4, 5 and 6 represent the results of agarose gel electrophoresis after cracking pUC57 E6 / E7 recombinant plasmid by cracking scheme ②; Lane 7, 8 and 9 represent the results of agarose gel electrophoresis after cracking pUC57 E6 / E7 recombinant plasmid by cracking scheme ③.

[0031] Figure 2 RPA basic primer screening results.

[0032] Lane M is 2000bp Marker; Lane 1 is the amplification results of the primer pair of SEQ ID NO: 1 and SEQ ID NO: 2; Lane 2 is the amplification results of the primer pair of SEQ ID NO: 3 and SEQ ID NO: 4; Lane 3 is the negative control with water as the template.

[0033] Figure 3 RPA reaction temperature optimization results.

[0034] Test strips 1, 2, 3, 4 and 5 are the results of RPA reaction at temperatures of 30, 33, 37, 40 and 43℃, respectively.

[0035] Figure 4 RPA reaction time optimization results.

[0036] Test strips 1, 2, 3, 4, 5 and 6 are the results of RPA reaction at times of 5, 10, 15, 20, 30 and 35 minutes, respectively.

[0037] Figure 5 RPA-CRISPR / Cas12a specific test strip schematic diagram.

[0038] A is a schematic diagram of the modification of a nucleic acid detection test strip for CRISPR / Cas12a system; B is a schematic diagram of result interpretation.

[0039] Figure 6 Test strip verification.

[0040] In A, test strips 1, 2 and 3 represent the results of conventional test strips after RPA-CRISPR / Cas12a reaction, with templates of HPV16, HPV52 and water, respectively; In B, test strips 1, 2 and 3 represent the results of the modified test strips in this study after RPA-CRISPR / Cas12a reaction, with templates of HPV16, HPV52 and water, respectively.

[0041] Figure 7 RPA-CRISPR / Cas12a system probe concentration and reaction time optimization results.

[0042] A The test strips 1, 2, 3, 4, 5 and 6 in A represent the reaction results when the probe concentration is 0, 0.02, 0.04, 0.08, 0.1 and 0.2 μM, respectively; B The test strips 1, 2, 3, 4 and 5 in B represent the reaction results when the reaction time is 5, 10, 15, 20 and 30 minutes, respectively.

[0043] Figure 8 The results of RPA-CRISPR / Cas12a sensitivity detection.

[0044] The test strips 1, 2, 3, 4, 5, 6, 7 and 8 represent the RPA-CRISPR / Cas12a reaction results when the template concentration is 106, 105, 104, 103, 102, 101, 100 copies / μL and the template is water, respectively.

[0045] Figure 9 The results of RPA-CRISPR / Cas12a specificity detection.

[0046] The test strips 1, 2, 3, 4, 5, 6, 7, 8 and 9 represent the RPA-CRISPR / Cas12a reaction results when the template is HPV16, HPV18, HPV26, HPV35, HPV52, HPV68, HPV11, HPV61 and water, respectively.

[0047] Figure 10 The results of RPA-CRISPR / Cas12a technology clinical sample detection. DETAILED DESCRIPTION

[0048] The 776 bp sequence containing E6 and E7 genes in Genebank: MG850604.1 was selected as the target gene (SEQ ID NO: 7), and was constructed on the pUC57 vector and verified by sequencing to generate the pUC57-E6 / E7 recombinant plasmid. The nucleic acid extracted from the recombinant plasmid was used as the template in RPA. Figures 1-10 As shown in the following table:

[0049] Example 1: Optimization of nucleic acid lysis solution

[0050] ① Tris (pH = 8.0), NaHCO3, 2% PVP, Tween-20; ② Guanidine hydrochloride, Tris (pH = 8.0), Triton-X100, Tween-20; ③ Guanidine hydrochloride, Tris (pH = 8.0), NaHCO3, EDTA, Tween-20 lysis solution was used to treat the cell sample, and Proteinase K enzyme was used for digestion, and the reaction was carried out at room temperature for 5 minutes, followed by centrifugation at 10000 r for 3 minutes at 4°C. Agarose gel electrophoresis confirmed that complete HPV16 RNA / DNA Figure 1 ) was obtained by scheme ①.

[0051] Example 2: RPA basic primer screening

[0052] According to the design of HPV16 E6 E7 primers, the nucleic acid in the lysate of the synthesized pUC57 recombinant plasmid was used as a template, water was used as a negative control, and RPA amplification was performed (40°C, 30 minutes). The results were detected by 2% agarose gel electrophoresis. The results are shown in Figure 2. As shown in Figure 2, there were bands in the amplification results of the primer pairs of SEQ ID NO: 1 and SEQ ID NO: 2, and SEQ ID NO: 3 and SEQ ID NO: 4 (SEQ ID NO: 8 and SEQ ID NO: 9), and there was no band in the negative amplification, indicating that the selected primers were suitable for RPA detection. Moreover, the band of the amplification product of the primer pair of SEQ ID NO: 1 and SEQ ID NO: 2 was brighter in the nucleic acid protein gel imager (Bio-Rad). Preferably, the primer pair of SEQ ID NO: 1 and SEQ ID NO: 2 was selected for subsequent experiments. Figure 2 Figure 2

[0053] Example 3: RPA reaction condition optimization

[0054] Using the sample nucleic acid obtained from the above lysate as a template, adding the primer pair of SEQ ID NO: 1 and SEQ ID NO: 2, the reaction time (30 minutes) was unchanged, and the incubation temperature was changed to 30, 33, 37, 40 and 43°C for isothermal amplification in a constant temperature water bath; the reaction temperature (37°C) was unchanged, and the incubation time was changed to 5, 10, 15, 20, 30 and 35 minutes for isothermal amplification in a constant temperature water bath. It was confirmed that 37°C was the best reaction temperature for RPA ( Figure 3 ), and 30 minutes was the best time for RPA ( Figure 4 ).

[0055] Example 4: Test strip modification

[0056] The structure of the conventional CRISPR system colloidal gold test strip was adjusted, and the positions of the sample pad and the water absorption pad were exchanged, so that the sample liquid first flowed through the detection line, thereby improving the color development probability and interpretation of the positive signal. The effect was obviously improved through clinical sample verification. Among them, the combination pad contains rabbit anti-FITC antibody colloidal gold emulsion microspheres and mouse anti-FAM antibody colloidal gold emulsion microspheres, the detection line contains goat anti-mouse secondary antibody, and the quality control line contains colloidal gold-labeled streptavidin that can bind to Biotin. The color of the positive reaction detection line and the quality control line is darker, and the color of the negative reaction detection line is darker, and the detection line does not change color. Figure 5

[0057] ​​​Example 5: Select the best parameters by comparing different probe concentrations and reaction times of the CRISPR / Cas12a detection system

[0058] The detection system containing LbCas12a protein, specific gRNA and probe was constructed, and the reaction was carried out at 37°C for 30 minutes. The probe concentrations were 0, 0.02, 0.04, 0.08, 0.1 and 0.2 μM, respectively, and the best probe concentration was finally determined to be 0.04 μM, which was sensitive and robust. Under the condition of 0.04 μM probe concentration, the reaction conditions were changed to 5, 10, 15, 20 and 30 minutes, and positive results appeared when the reaction time was 20 minutes, indicating that the best cutting time of the CRISPR / Cas12a system was 20 minutes. Figure 6 ).

[0059] Example 6: Sensitivity and specificity test

[0060] The detection was carried out on the pUC57 recombinant plasmid nucleic acid diluted with different gradients (10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 copies / μL), and the minimum detection limit reached 10 1 copies / μL Figure 7 ); 7 non-target subtypes including HPV18, HPV26, HPV35, HPV52, HPV68, HPV11 and HPV61 showed no cross reaction Figure 8 ).

[0061] Example 7: Clinical sample detection

[0062] As shown in Figure 10 , 20 cases of clinically diagnosed HPV16, 18 cases of HPV52 and 5 cases of healthy samples were detected by the detection system of the application. The results are shown in Figure 9 , all of the 20 clinically tested HPV16 positive samples were detected as positive, with a coincidence rate of 100%.

[0063] The above is only an embodiment of the present application, and the well-known specific structure and characteristics in the scheme are not described in detail. It should be noted that for those skilled in the art, without departing from the structure of the present application, some modifications and improvements can be made, which should also be regarded as the protection scope of the present application, which will not affect the effect and practicality of the patent. The protection scope claimed in this application should be subject to the content of its claims, and the specific embodiments and the like in the description can be used to explain the content of the claims.

Claims

1. A rapid detection system for high-risk HPV16 based on RPA-CRISPR / Cas12a, characterized in that, Includes the following steps: (1) Samples were treated with a nucleic acid lysis buffer containing Tris (pH=8.0), NaHCO3, polyvinylpyrrolidone and Tween-20 and Proteinase K, and the samples were lysed at room temperature for 5 minutes to obtain nucleic acid templates. (2) The RPA reaction system contains RNA inhibitors, reverse transcriptase and RPA reaction essential enzymes. Preferably, specific primers SEQ ID NO:1 and SEQ ID NO:2 are used. After adding the template and initiator in (1), RPA amplification is carried out at 37°C for 30 minutes. (3) Drop the reaction solution onto the colloidal gold test strip that has been modified by changing the positions of the sample pad and the absorbent pad, and interpret the results. If the test line and the control line become darker, it is a positive reaction; if only the control line becomes darker and there is no color change at the test line, it is a negative reaction. (4) Add the RPA product to a CRISPR reaction system containing LbCas12a protein, specific gRNA and probe, and react at 37°C for 20 minutes.

2. A rapid detection system for high-risk HPV16 based on RPA-CRISPR / Cas12a, characterized in that the optimal reaction temperature of the RPA reaction is 37°C and the optimal reaction time is 30 minutes.

3. A rapid detection system for high-risk HPV16 based on RPA-CRISPR / Cas12a, characterized in that the mass fraction of polyvinylpyrrolidone in the lysis buffer formulation is 2%, and the concentration of Proteinase K is 40 μg / ml.

4. A rapid detection system for high-risk HPV16 based on RPA-CRISPR / Cas12a, characterized in that the Cas12a protein can be any one of FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a, or Lb4Cas12a.

5. A rapid detection system for high-risk HPV16 based on RPA-CRISPR / Cas12a, characterized in that the gRNA sequence in the CRISPR / Cas12a system is SEQ ID NO:

5.

6. A rapid detection system for high-risk HPV16 based on RPA-CRISPR / Cas12a, characterized in that the probe sequence in the CRISPR / Cas12a system is SEQ ID NO:6, and the optimal reaction concentration is 0.04 μM.

7. A rapid detection system for high-risk HPV16 based on RPA-CRISPR / Cas12a, comprising a nucleic acid lysis device, an RPA amplification device, a CRISPR / Cas12a detection module, and a modified colloidal gold test strip, wherein the method described in any one of claims 1-6 is implemented.

8. An application of a rapid detection system for high-risk HPV16 based on RPA-CRISPR / Cas12a, characterized in that, The application includes the following steps: (1) Construct a recombinant plasmid containing the target gene, lyse it for 5 minutes using the lysis protocol in claim 1 to obtain an RPA template, perform RPA amplification using the RPA amplification primer pair, react at 37°C for 30 minutes to obtain the RPA amplification product. (2) Prepare CRISPR / Cas12a reaction solution, wherein the CRISPR / Cas12a reaction solution includes the probe, gRNA and LbCas12a protein, and the RPA amplification product prepared in step (1); (3) The CRISPR / Cas12a reaction was carried out for 20 minutes using the CRISPR / Cas12a reaction solution prepared in step (2), and then the modified colloidal gold side-flow chromatography test strip was used for detection. (4) A positive result is that both the test line and the control line become darker, while a negative result is that only the control line changes color. Further testing of clinical samples follows these steps: (1) Clinical samples collected clinically were lysed for 5 minutes using the lysis protocol in claim 1 to obtain an RPA template. The RPA amplification primers described above were used for RPA amplification, and the reaction was carried out at 37°C for 30 minutes to obtain the RPA amplification product. (2) Prepare CRISPR / Cas12a reaction solution, wherein the CRISPR / Cas12a reaction solution includes the probe, gRNA and LbCas12a protein, and the RPA amplification product prepared in step (1); (3) The CRISPR / Cas12a reaction was carried out for 20 minutes using the CRISPR / Cas12a reaction solution prepared in step (2), and then the modified colloidal gold side-flow chromatography test strip was used for detection. (4) A positive result is that both the test line and the control line become darker, while a negative result is that only the control line changes color.

9. The method according to any one of claims 1 to 7, characterized in that the detection sensitivity is 10. 1 It has a per-copy / μL content and no cross-reactivity with HPV18, HPV26, HPV35, HPV52, HPV68, HPV11 and HPV61.

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