Cas12a-crRNA, crisper-cas system for detecting gardnerella vaginalis and application thereof

Abstract

This invention relates to the field of medical testing technology, and provides a Cas12a-crRNA for detecting Gardnerella vaginalis, the base sequence of which is shown in SEQ ID NO.1. This invention also provides the application of the above-mentioned Cas12a-crRNA in constructing a Gardnerella vaginalis detection system. Furthermore, this invention provides a CRISPR-Cas system, a method for rapid detection of Gardnerella vaginalis based on CRISPR, and the application of the above-mentioned CRISPR-Cas system in the preparation of a Gardnerella vaginalis detection kit. The advantages of this invention are: based on CRISPR technology, it can be used for rapid early detection of specimens from patients suspected of having BV infection due to excessive Gardnerella vaginalis proliferation, and the detection cost is low and the procedure is simple, which is of great significance for clinical diagnosis.

Description

Technical Field

[0001] This invention relates to the field of medical testing technology, and in particular to a Cas12a-crRNA, CRISPR-Cas system and its application for detecting Gardnerella vaginalis. Background Technology

[0002] The vaginal microecological system of a healthy woman is a highly dynamic and balanced complex system, dominated by hydrogen peroxide-producing lactobacilli (such as *Lactobacillus curvaturei* and *Lactobacillus gasseri*). These beneficial bacteria maintain the acidic environment of the vagina (pH 3.8–4.5) by producing lactic acid and secrete antimicrobial peptides and bacteriocins to form a biological barrier. When this precisely regulated microecological balance is disrupted, the BV-associated microbiota, centered on *Gardnerella vaginalis*, including strictly anaerobic bacteria such as *Mobiluncus spp.*, *Atopobium vaginae*, *Prevotella spp.*, and *Mycoplasma hominis*, forms a highly virulent biofilm community. Gardnerella vaginalis, the most specific biomarker of vaginal vaginitis (BV), is a facultative anaerobic bacterium with unique Gram staining variability (it can appear Gram-positive or Gram-negative). The adhesins expressed on its surface (such as sialidase and vaginolysin) can disrupt the vaginal epithelial barrier function. Clinical studies have confirmed that its overgrowth not only directly leads to typical BV symptoms (such as homogeneous thin discharge, elevated pH >4.5, clue cells >20%, and a fishy odor, according to Amsel criteria), but also closely relates to various serious gynecological complications by inducing local inflammatory responses. Epidemiological survey data show that the prevalence of BV among women of reproductive age worldwide exhibits significant regional differences, resulting in approximately $4 billion in direct medical expenditure annually. It has been listed by the WHO as one of the key diseases for prevention and control in the field of reproductive health.

[0003] The current gold standard for diagnosing vaginal vaginitis (BV) primarily relies on microscopic examination techniques (Nugent scoring) and clinical criteria (Amsel criteria). While both methods are widely used, they both have significant limitations. The Nugent scoring system, currently the most authoritative laboratory diagnostic method, uses a semi-quantitative scoring (0-10 points) of specific bacterial morphologies (such as Lactobacillus, Gardnerella vaginalis, and Molecularcurvature) in Gram-stained smears of vaginal secretions, achieving a diagnostic accuracy of 89-97%. However, this method requires experienced laboratory personnel to observe at least five fields of view under 1000x oil immersion, with a single sample testing time exceeding 30 minutes, and significant subjective variability in result interpretation. As for the Amsel clinical criteria, although simple to operate (requiring only pH test strips, a microscope, and KOH solution), its specificity is only 60-77%, and the sensitivity of clue cell interpretation is significantly affected by the sampling site and the experience of the examiner.

[0004] The development of molecular biology techniques has brought breakthroughs to the diagnosis of vaginal vaginitis (BV). Multiple studies have confirmed that real-time quantitative PCR (qPCR) can accurately quantify Gardnerella vaginalis (detection limit 10). 2 ~10 3 It can detect BV-related pathogens such as *Prevotella* and *Sneathia*, and can also detect emerging pathogens such as *Atopobium vaginae*, which are difficult to identify using traditional methods. In particular, the multiplex qPCR technology can simultaneously detect 8–15 BV-related bacteria, with a diagnostic sensitivity of 95.3% and a specificity of 91.7%. In recent years, some BV multiplex qPCR diagnostic kits have also appeared on the market, such as… (LabCorp, Burlington, NC), real-time PCR assay (Quest diagnostics, Secaucus, NJ), BD MAXTM real-time PCR assay (Becton, Dickinson and company, Franklin Lakes, NJ), BVmultiplex assay (Medical Diagnostic Laboratories, Hamilton Township, NJ), assay (Gryphus Diagnostics, LLC, Knoxville, TN), card(Litmus Concepts,Santa Clara,CA)、 Immunoassay (Siemens Healthcare, Erlangen, Germany) and QuickLine immunoassay (Milenia Biotec, Giebren, Germany), among others, are used. However, these technologies generally face three major application bottlenecks: first, high equipment costs; second, complex testing procedures (requiring nucleic acid extraction, system preparation, and 45–60 thermal cycles); and third, a total processing time of 2–4 hours. These factors severely restrict the widespread application of molecular diagnostic technologies in BV screening.

[0005] Therefore, developing novel molecular diagnostic technologies is crucial for point-of-care testing (POCT) of vaginal Gardnerella vaginalis overgrowth leading to BV, especially in resource-constrained environments. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a Cas12a-crRNA and CRISPR-Cas system and its application for detecting Gardnerella vaginalis. Based on CRISPR technology, it can be used for rapid (30-40 minutes) early detection of BV infection in patients suspected of having Gardnerella vaginalis overgrowth. The detection cost is low and the procedure is simple, which is of great significance for clinical diagnosis.

[0007] The present invention solves the above-mentioned technical problems by adopting the following technical solutions:

[0008] A Cas12a-crRNA for detecting Gardnerella vaginalis has the base sequence shown in SEQ ID NO.1.

[0009] As one of the preferred embodiments of the present invention, the Cas12a-crRNA is used to guide the Cas12a protein to recognize the PAM sequence containing TTTN, and form a Cas12a / crRNA / target DNA ternary complex with the RAA-amplified target DNA, thereby activating its side branch cleavage activity and cutting any single-stranded DNA molecule in the reaction system.

[0010] An application of the above-mentioned Cas12a-crRNA in constructing a vaginal Gardnerella vaginalis detection system.

[0011] A CRISPR-Cas system for detecting Gardnerella vaginalis includes the aforementioned Cas12a-crRNA for detecting Gardnerella vaginalis.

[0012] As one of the preferred embodiments of the present invention, it specifically includes Cas12a protein, Cas12a-crRNA, FAM-ssDNA-BHQ1 probe, and RAA amplification product; wherein, the amplification primers of the RAA amplification product are RAA-F and RAA-R, and the base sequences are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively; the sequence of the FAM-ssDNA-BHQ1 probe is FAM-TTATT-BHQ1.

[0013] As one of the preferred embodiments of the present invention, ...

[0014] A method for rapid detection of Gardnerella vaginalis based on CRISPR is proposed, which utilizes the aforementioned CRISPR-Cas system to detect the sample to be tested.

[0015] As one of the preferred embodiments of the present invention, the following specific steps are included:

[0016] S1. DNA extraction from swab samples:

[0017] Total DNA was extracted from swab samples;

[0018] S2 and RAA primer design and amplification:

[0019] Design nucleotide sequence RAA primers for conserved fragments of Gardnerella vaginalis; the RAA primers are RAA-F and RAA-R, with base sequences as shown in SEQ ID NO.2 and SEQ ID NO.3, respectively;

[0020] The RAA reaction system consisted of: 2 μL of 10 μM primer RAA-F, 2 μL of 10 μM primer RAA-R, 25 μL of buffer A, 5 μL of DNA template, 2.5 μL of buffer B, and 13.5 μL of ddH2O.

[0021] RAA amplification conditions: 39℃, incubation for 30 min;

[0022] Identification of S3 and RAA amplification products:

[0023] The amplified RAA product is placed in a gel imaging system for imaging and photographs are taken; if a band is present, the RAA amplification is complete.

[0024] S4. The amplified fragment is recognized and cleaved by the Cas12a protein, which is then used to cleave the "FAM-ssDNA-BHQ1 probe".

[0025] Constructing the CRISPR-Cas system lysis reaction mixture: 1 μL of 100 nM Cas12a protein, 2 μL of 250 nM Cas12a-crRNA, 1 μL of 1 μM FAM-ssDNA-BHQ1 probe, 2 μL of 10×HOLMES Buffer, 12 μL of ddH2O, and 2 μL of LRAA amplification product;

[0026] After centrifugation and mixing, the reaction system was placed in a real-time PCR instrument and incubated at 37°C for 20 min. The changes in the fluorescence value of the FAM channel of the cleavage product after the lysis reaction were observed. When the fluorescence value of the FAM channel increased, the result was considered positive; when the fluorescence value of the FAM channel did not increase, the result was considered negative.

[0027] Application of the above-mentioned CRISPR-Cas system in the preparation of a vaginal Gardnerella vaginalis detection kit.

[0028] The advantages of this invention compared to the prior art are:

[0029] (1) This invention uses recombinase-assisted isothermal amplification technology to amplify Gardnerella vaginalis DNA. It does not require a complicated temperature cycle process. Only a metal bath is needed to control the temperature at 39°C and incubate at a constant temperature for 20 minutes to complete the process. This method can be implemented in areas with scarce medical resources. It does not rely on large instruments and equipment such as PCR instruments. It is low in cost, short in time (30-40 minutes), highly sensitive, and has low technical requirements for operators. It can be applied in various places.

[0030] (2) The Cas12a-crRNA of the present invention can guide the Cas12a protein to bind to the target sequence to recognize and cleave the target sequence, and at the same time, cleave the single-stranded DNA reporter molecule in the reaction system; when the single-stranded DNA reporter molecule is the FAM-ssDNA-BHQ1 probe, it can be used with a fluorescence detection instrument to directly read the detection results; accordingly, the present invention can be used as a method for large-scale clinical testing of the population, and compared with traditional diagnostic testing methods, the detection method of the present invention greatly shortens the testing time and does not require repeated training of operators;

[0031] (3) The new detection platform established by combining RAA amplification with CRISPR-Cas12a technology is innovative and can be further promoted for the detection of various pathogens and genes, with great market potential. Attached Figure Description

[0032] Figure 1 This is a diagram showing the RAA amplification primers and Cas12a-crRNA sequence and position information designed in this invention;

[0033] Figure 2This is a diagram illustrating the verification process of the CRISPR-based rapid detection method for Gardnerella vaginalis in this invention.

[0034] Figure 3 This is a graph showing the fluorescence values ​​of the trans-cleavage of the Cas12a protein after the addition of the fluorescently modified probe in this invention (in the graph, NTC is the template-free negative control, GV is the Gardnerella vaginalis test sample; "****" indicates a P value < 0.0001);

[0035] Furthermore, it should be noted that SEQ ID NO.1 in the sequence listing of this invention is an RNA sequence, in which "T" = "U". Detailed Implementation

[0036] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments. Furthermore, unless otherwise specified, the reagents, kits, and other materials used in the following embodiments are all commercially available products conventional in the art; the experimental methods used, unless otherwise specified, are all conventional methods in the art and will not be described further.

[0037] Example 1

[0038] This embodiment describes a Cas12a-crRNA for detecting Gardnerella vaginalis in the vagina.

[0039] In this embodiment, Cas12a-crRNA was designed targeting a conserved region of the Gardnerella vaginalis genome. The base sequence of Cas12a-crRNA is shown in SEQ ID NO.1, and its positional information can be found in [link to SEQ ID NO.1]. Figure 1 .

[0040] The Cas12a-crRNA is the core of CRISPR, used to guide the Cas12a protein to recognize the PAM sequence containing TTTN, and form a Cas12a / crRNA / target DNA ternary complex with the RAA-amplified target DNA, activating its side branch cleavage activity to cut any single-stranded DNA molecule in the reaction system.

[0041] Example 2

[0042] This embodiment presents a CRISPR-Cas system for detecting Gardnerella vaginalis, comprising Cas12a protein, Cas12a-crRNA (Example 1), FAM-ssDNA-BHQ1 probe (FAM-TTATT-BHQ1), RAA amplification products (amplification primers RAA-F, RAA-R), and other reagents required for RAA amplification reaction and CRISPR-Cas12a system lysis reaction.

[0043] The RAA amplification reaction system (50 μL) consisted of: 2 μL of 10 μM primer RAA-F (SEQ ID NO. 2), 2 μL of 10 μM primer RAA-R (SEQ ID NO. 3), 25 μL of buffer A (RAA nucleic acid amplification kit), 5 μL of DNA template, 2.5 μL of buffer B (RAA nucleic acid amplification kit), and 13.5 μL of ddH2O. The RAA amplification conditions were: 39℃, incubation for 30 min.

[0044] The CRISPR-Cas12a lysis reaction system (20 μL) consisted of: 1 μL of 100 nM Cas12a protein, 2 μL of 250 nM Cas12a-crRNA, 1 μL of 1 μM FAM-ssDNA-BHQ1 probe, 2 μL of 10×HOLMES Buffer, 12 μL of ddH2O, and 2 μL of RAA amplification product.

[0045] Example 3

[0046] This embodiment presents a method for rapid detection of Gardnerella vaginalis based on CRISPR. The method utilizes the CRISPR-Cas system described in Example 2 to detect the sample. The steps are as follows:

[0047] S1. DNA extraction from vaginal swab samples:

[0048] DNA was extracted from vaginal swab specimens (using a DNA extraction kit and following the corresponding instructions).

[0049] S2 and RAA primer design and amplification:

[0050] (1) RAA primers for the nucleotide sequence of the target gene fragment were designed using Primer Explorer V5 and sent to a company for synthesis. The RAA primers were RAA-F and RAA-R, with base sequences shown in SEQ ID NO.2 and SEQ ID NO.3, respectively (position information is available in [link to SEQ ID NO.3]). Figure 1 ).

[0051] (2) Construction of the RAA reaction system (50 μL): 2 μL of 10 μM primer RAA-F (SEQ ID NO.2), 2 μL of 10 μM primer RAA-R (SEQ ID NO.3), 25 μL of A buffer (RAA nucleic acid amplification kit), 5 μL of DNA template, 2.5 μL of B buffer (RAA nucleic acid amplification kit), and 13.5 μL of ddH2O. The sample addition process was performed on ice.

[0052] (3) Perform RAA amplification; RAA amplification conditions are: 39℃, incubation for 30 min.

[0053] Identification of S3 and RAA amplification products:

[0054] The amplified RAA product is placed in a gel imaging system for imaging and photographs are taken; if a band is present, the RAA amplification is complete.

[0055] S4. The amplified fragment is recognized and cleaved by the Cas12a protein: "fluorescein-quencher dual-labeled ssDNA probe (FAM-ssDNA-BHQ1 probe)":

[0056] (1) Construction of the CRISPR-Cas12a system lysis reaction system (20 μL): 1 μL of 100 nM Cas12a protein, 2 μL of 250 nM Cas12a-crRNA, 1 μL of 1 μM FAM-ssDNA-BHQ1 probe, 2 μL of 10×HOLMES Buffer, 12 μL of ddH2O, and 2 μL of RAA amplification product. The sample loading process was performed on ice.

[0057] (2) After centrifugation and mixing, the reaction system was placed in a real-time PCR instrument and incubated at 37°C for 20 min. The changes in the fluorescence value of the FAM channel of the cleavage product after the lysis reaction were observed. When the fluorescence value of the FAM channel increased, the result was considered positive; when the fluorescence value of the channel did not increase, the result was considered negative.

[0058] Verification Example

[0059] This validation example is used to verify the feasibility of the present invention's CRISPR-based method for rapid detection of Gardnerella vaginalis, referring to... Figure 2 The steps are as follows:

[0060] I. DNA extraction from vaginal swab samples:

[0061] DNA was extracted from vaginal swab specimens (using a DNA extraction kit and following the corresponding instructions).

[0062] II. RAA Amplification and Identification of Amplification Products:

[0063] The RAA reaction system (50 μL) was constructed as follows: 2 μL of 10 μM primer RAA-F (SEQ ID NO. 2), 2 μL of 10 μM primer RAA-R (SEQ ID NO. 3), 25 μL of buffer A (RAA nucleic acid amplification kit), 5 μL of DNA template, 2.5 μL of buffer B (RAA nucleic acid amplification kit), and 13.5 μL of ddH2O. The sample addition was performed on ice. RAA amplification was then carried out under the following conditions: 39 °C for 30 min.

[0064] The amplified RAA product was placed in a gel imaging system for imaging and photographed; the presence of bands indicates that the RAA amplification was complete.

[0065] III. CRISPR reaction:

[0066] The CRISPR-Cas12a lysis reaction system was constructed using a 20 μL mixture: 1 μL of 100 nM Cas12a protein, 2 μL of 250 nM Cas12a-crRNA, 1 μL of 1 μM FAM-ssDNA-BHQ1 probe, 2 μL of 10×HOLMES Buffer, 12 μL of ddH2O, and 2 μL of RAA amplification product. The sample loading was performed on ice.

[0067] After centrifugation and mixing, the reaction system was placed in a real-time PCR instrument and incubated at 37°C for 20 min for CRISPR reaction. The changes in the FAM channel fluorescence value of the cleavage product after the lysis reaction were observed.

[0068] IV. Results Reading

[0069] Fluorescence value results as follows Figure 3 As shown in the figure (NTC is a template-free negative control; GV is a vaginal Gardnerella vaginalis test sample). According to... Figure 3 As a result, the fluorescence value of the FAM channel increased, and the result was positive.

[0070] Accordingly, the Gardnerella vaginalis detection method based on the CRISPR-Cas system of this invention can accurately and rapidly detect Gardnerella vaginalis, has high feasibility, and can be prepared into a Gardnerella vaginalis detection kit for widespread application. It is suitable for various places and has strong practicality.

[0071] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A Cas12a-crRNA for detecting Gardnerella vaginalis, characterized in that, Its base sequence is shown in SEQ ID NO.1; the Cas12a-crRNA is used to guide the Cas12a protein to recognize the PAM sequence containing TTTN, and form a Cas12a / crRNA / target DNA ternary complex with the RAA-amplified target DNA, thereby activating its side branch cleavage activity and cutting any single-stranded DNA molecule in the reaction system.

2. The application of the Cas12a-crRNA as described in claim 1 in constructing a vaginal Gardnerella vaginalis detection system.

3. A CRISPR-Cas system for detecting Gardnerella vaginalis, characterized in that, Includes Cas12a-crRNA for detecting Gardnerella vaginalis as described in claim 1.

4. The CRISPR-Cas system according to claim 3, characterized in that, Specifically, it includes Cas12a protein, Cas12a-crRNA, FAM-ssDNA-BHQ1 probe, and RAA amplification product; wherein, the amplification primers for the RAA amplification product are RAA-F and RAA-R, and the base sequences are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively; the sequence of the FAM-ssDNA-BHQ1 probe is FAM-TTATT-BHQ1.

5. The use of the CRISPR-Cas system as described in claim 3 or 4 in the preparation of a vaginal Gardnerella vaginalis detection kit.

Images