A CRISPR / Cas12b-based method for detecting Neisseria gonorrhoeae

Abstract

The present invention provides a LAMP primer and sgRNA combination, a detection kit, and a one-step detection system based on CRISPR / Cas12b, and applications thereof in detecting Neisseria gonorrhoeae.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more specifically to a CRISPR / Cas12b-based rapid detection system and method for Neisseria gonorrhoeae. Background Art

[0002] Infectious diseases, with their high morbidity and mortality rates, pose a serious threat to human life and health. Pathogens with high infectivity and mutation rates are particularly prone to pandemics, posing a significant challenge to global public health systems. Rapid and accurate early diagnosis of infectious diseases is crucial for reducing mortality, improving prognosis, and limiting transmission, and is a crucial component of laboratory medicine research. Nucleic acid testing is a key component of infectious disease diagnosis. Compared to antigen and antibody testing, it offers higher sensitivity and specificity, making it the most accurate and reliable method for early diagnosis.

[0003] Neisseria gonorrhoeae ( Neisseria gonorrhoeae Gonorrhea (NG), also known as Neisseria gonorrhoeae, is a Gram-negative, oxidase-positive, obligate aerobic bacterium that is typically bi-spherical or short rod-shaped, with a diameter of approximately 0.6 μM to 1.5 μM. This bacterium is widespread worldwide and is a common pathogen in sexually transmitted diseases. While some infected individuals may not exhibit any obvious symptoms, becoming asymptomatic carriers, NG can persist in the urogenital tract without symptoms, allowing for efficient transmission to partners through sexual contact. It is estimated that approximately 10%-20% of infected women and 5%-10% of infected men may not develop clinical symptoms, yet these individuals can still be a significant source of disease transmission. Gonorrhea can cause localized infections such as urethritis and cervicitis. If left untreated, it can lead to serious complications such as salpingitis and pelvic inflammatory disease, significantly increasing the risk of infertility in infected women. Furthermore, newborns can also be infected with N. gonorrhoeae during delivery through an infected mother's birth canal, resulting in conjunctivitis, pneumonia, and even sepsis, seriously threatening their health and life.

[0004] Therefore, early identification, rapid diagnosis, and effective treatment of Neisseria gonorrhoeae infection are crucial for controlling disease transmission, alleviating patient suffering, and preventing complications. This requires not only vigilance among clinicians and public health workers, but also continuous strengthening of pathogen monitoring and research to ensure the timeliness and effectiveness of treatment plans.

[0005] In NG pathogen detection, techniques such as PCR and isothermal amplification are typically used to amplify the pathogen's nucleic acid molecules. Subsequently, rapid identification and signal amplification using the CRISPR system enhances detection specificity and sensitivity, resulting in a test result. However, existing detection methods perform pathogen nucleic acid amplification and CRISPR detection as two completely separate steps. Between these steps, either the reaction solution needs to be opened and pipetted, or the CRISPR detection reagent is pre-applied to the tube cap. After amplification is complete, the tube is centrifuged briefly to mix with the amplified product at the bottom of the tube to initiate the CRISPR reaction. This method is known as the one-pot method. The opening of the cap in the former significantly increases the risk of aerosol contamination. While the latter method addresses the issue of aerosol contamination, it is more complex and hinders product standardization and simplification.

[0006] In order to simplify the operation and promote clinical transformation, the present invention has developed a one-step CRISPR detection system for NG pathogens. This technical system completes the reagents for nucleic acid amplification and CRISPR detection in one reaction system at the same time. It does not require partitioning and step-by-step reaction in the tube, and can achieve amplification detection in one step. However, the one-step reaction faces the challenge of incompatibility between LAMP amplification and CRISPR detection. On the one hand, the reagent components are more complex. On the other hand, the cleavage activity of CRISPR / Cas will degrade the products of LAMP amplification, reduce the amplification efficiency, and thus affect the sensitivity of the detection. Therefore, it is necessary to screen a large number of LAMP amplification primers and sgRNA to balance the amplification efficiency and enzyme cleavage efficiency.

[0007] The inventive team screened LAMP primers and sgRNA sequences to ensure that the two reactions were carried out simultaneously under optimal conditions. They finally screened out a combination of sgRNA and primers with good compatibility, and creatively integrated LAMP amplification and CRISPR detection into one reaction system. There is no need to open the lid for pipetting or partition the sample, which simplifies the operation steps and shortens the reaction time. It not only greatly reduces the cost of reagents, consumables and equipment, but also reduces the risk of aerosol contamination, thereby improving the detection efficiency and accuracy, as well as the specificity and reliability of the experimental results, providing a new technical platform for the rapid diagnosis of NG pathogens. Summary of the Invention

[0008] The purpose of the present invention is to provide a rapid detection system and method for Neisseria gonorrhoeae based on CRISPR / Cas12b.

[0009] The present invention provides a LAMP primer and sgRNA combination, characterized in that the combination comprises the following primers and sgRNA:

[0010] outer primers F3 and B3, with nucleotide sequences of 5′-CGGCTCAGTTGGATTTGTCT-3′ and 5′-AAGTGCGCTTGGAAAAATCG-3′, respectively;

[0011] Internal primers FIP and BIP, with nucleotide sequences of 5′-CGGGACTGTATTACCGAAGCGGACCAAAAACAGTACGACCGA-3′ and 5′-GCCCATGGTTTCGACTTTGTCGAATCGACACCGGCGATGA-3′, respectively;

[0012] The nucleotide sequences of the loop primers LF and LB were 5′-GGAAGCAGTGGCGGCAAT-3′ and 5′-CAGAAACGCGAACATACCAGCTA-3′, respectively.

[0013] sgRNA, the nucleotide sequence is shown in SEQ ID NO: 2.

[0014] The present invention also provides a kit, characterized in that the kit comprises:

[0015] (a) Cas12b protein, the amino acid sequence of which is shown in SEQ ID NO: 10;

[0016] (b) the above-mentioned LAMP primer and sgRNA combination, wherein the sgRNA guides the Cas12b protein to specifically bind to the NG nucleic acid molecule;

[0017] (c) A nucleic acid probe having a nucleotide sequence of CCCCCCCC, a fluorescent group FAM labeled at the 5' end, and a quencher group BHQ1 labeled at the 3' end.

[0018] The present invention also provides a method for detecting whether Neisseria gonorrhoeae is present in a sample, which is characterized by comprising the following steps:

[0019] (i) using the above kit to detect the sample to be tested;

[0020] (ii) detecting the cleavage status of the nucleic acid probe in the above kit;

[0021] (iii) making a determination based on the detection result of step (ii): when the nucleic acid probe is cleaved, it indicates that Neisseria gonorrhoeae is present in the sample; when the nucleic acid probe is not cleaved, it indicates that Neisseria gonorrhoeae is not present in the sample.

[0022] In some embodiments, in the above step (ii), the cleavage status of the nucleic acid probe is detected by real-time monitoring of changes in the fluorescence signal; when a fluorescence signal is monitored, indicating that the nucleic acid probe is cleaved, it is determined that the sample contains Neisseria gonorrhoeae with a nucleic acid concentration of not less than 1 pg / μL.

[0023] The present invention also provides the use of the above-mentioned LAMP primer and sgRNA combination, the above-mentioned kit, and the above-mentioned method in detecting Neisseria gonorrhoeae.

[0024] The beneficial effects of the present invention are as follows: after screening a large number of LAMP primers and sgRNAs, a one-step CRISPR detection system was established, integrating LAMP amplification and CRISPR detection into one reaction system, which neither requires opening the lid for pipetting nor partitioning for sample loading, simplifies the operation steps, shortens the reaction time, greatly reduces the cost of reagents, consumables and equipment, and also reduces the risk of aerosol contamination, thereby improving the specificity and reliability of the experimental results. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Other features, objects and advantages of the present application will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings.

[0026] Figure 1 Amplification curves of the LAMP primer set screening experiment. The blue curve is the template of the NG nucleic acid extract, and the purple curve is the no-template control (NTC).

[0027] Figure 2 Screening results of sgRNA, where green represents the porA gene and gray represents NTC.

[0028] Figure 3 Specific identification results.

[0029] Figure 4 Sensitivity test results. DETAILED DESCRIPTION

[0030] The present invention will be further described below with reference to the accompanying drawings. The following examples are only used to illustrate the present invention but do not limit the scope of the present invention.

[0031] The term "guide RNA" or "gRNA" or "sgRNA" refers to an RNA that guides a Cas protein (such as Cas12b protein) to specifically bind to a target DNA sequence.

[0032] The term "CRISPR" refers to clustered regularly interspaced short palindromic repeats, which are the immune system of many prokaryotes.

[0033] The term "Cas protein" refers to a CRISPR-associated protein, which is an associated protein in the CRISPR system.

[0034] The term "Cas12b" refers to a crRNA-dependent endonuclease, which is a type VB enzyme in the CRISPR system classification.

[0035] The term "LAMP" stands for loop-mediated isothermal amplification, which is a constant temperature nucleic acid amplification technology suitable for genetic diagnosis.

[0036] The term "PCR" refers to "polymerase chain reaction," a method used to amplify a target DNA fragment on a large scale.

[0037] CRISPR diagnosis is a rapid molecular detection technology based on the targeted recognition of pathogen nucleic acids by clustered regularly interspaced short palindromic repeats-associated proteins under RNA guidance. Compared with existing molecular diagnostic technologies such as quantitative PCR and second-generation sequencing, CRISPR diagnostic technology has the advantages of sensitivity, specificity, speed, convenience and low cost. It can be widely used in POCT, clinical infection detection, tumor screening, companion diagnosis, food safety and other fields.

[0038] For DNA-based pathogens, CRISPR diagnostics primarily utilize CRISPR / Cas proteins to induce cleavage within each targeted DNA strand, generating double-stranded DNA breaks. In addition to cis-cleavage of the target DNA, target DNA binding also induces trans-cleavage of non-target DNA, specifically cleavage of surrounding single-stranded DNA (ssDNA). This trans-cleavage activity has been utilized in highly sensitive nucleic acid detection. CRISPR / Cas proteins cleave sequence-specific DNA under the guidance of sgRNA (single guide RNA), a complex of CRISPR-derived RNA (crRNA) and trans-activating crRNA (tracRNA). Through base pairing, sgRNA self-folds into a partially double-stranded RNA structure, enabling it to bind to the Cas protein and function.

[0039] The substrate for trans-cleavage is often a molecular probe labeled with a fluorescent group or biotin for colloidal gold detection. In this application, the 5' and 3' ends of the molecular probe for fluorescence detection are respectively connected to the fluorescent group FAM and the quenching group BHQ1, and the probe sequence is FAM-CCCCCCCC-BHQ1. When the genome of the pathogen is present in the detection system, the CRISPR / Cas protein will cut the DNA of the pathogen under the guidance of the sgRNA, resulting in the trans-cleavage activity of the CRISPR / Cas protein being activated. The detection probe is therefore cut and produces fluorescence, which can be detected by the corresponding equipment.

[0040] The present invention will be further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. Experimental procedures in the following examples, where specific conditions are not specified, were generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0041] The porA gene encodes the major outer membrane protein, a key virulence factor of Neisseria gonorrhoeae (NG). The porA gene is highly conserved and shares little homology with other bacteria, making it useful for molecular identification of NG. Detection of the porA gene can assist in confirming whether a bacterium is NG, providing a basis for subsequent diagnosis and treatment.

[0042] To this end, the present invention used the porA gene sequence (SEQ ID NO. 8) as a template and NEB LAMP (https: / / lamp.neb.com / ) to design 10 sets of LAMP primers, Primer 1 to Primer 10, as shown in Table 1. They were synthesized and used for future use.

[0043] Table 1 LAMP primer sequences

[0044]

[0045] 1. Extraction of DNA from NG standard strains

[0046] The NG standard strain with the product number ATCC49226 was purchased and placed in a -80°C refrigerator for use.

[0047] After the strain is taken out and revived, it is inoculated with gonococcal selective culture medium (chocolate medium) on chocolate plates and the plates are placed in a 37°C, 5% CO2 incubator for 24-48 hours.

[0048] The NG strain DNA was extracted using the Tiangen bacterial genomic DNA extraction kit (#DP302).

[0049] 2. Establish LAMP amplification reaction system

[0050] Amplify NG standard strain DNA using different LAMP primers. Monitor the amount of amplified product by adding SybrGreen fluorescent dye to the system. A no-template control (NTC) sample is also included to monitor nonspecific amplification.

[0051] Reaction system: 10× LAMP Buffer (200 mM Tris-HCl, 100 mM (HH4)2SO4, 500 mM KCl, 1% Tween20) 2.5 μL, 10× LAMP Primer (F3 / B3 2 mM, FIP / BIP 16 mM, LF / LB 4 mM) 2.5 μL, 2 M glycine 6 μL, 25 mM dNTP 1 μL, 100 mM MgSO4 2 μL, 8 U / μL Bst 1 μL, template 2.5 μL, 100× SybrGreen 0.25 μL, and enzyme-free sterile water to a total reaction volume of 25 μL.

[0052] Amplification program: 60°C for 1 min (collect FAM fluorescence signal), for 45 cycles.

[0053] 3. LAMP primer screening

[0054] Using the NG standard strain DNA as a template, isothermal amplification tests were carried out using the 10 sets of LAMP primers for the NG porA gene shown in Table 1.

[0055] The results are as follows Figure 1 Among them, Primer 8 had the highest amplification efficiency and no nonspecific amplification occurred in the no-template control (NTC), so Primer 8 was selected as the specific LAMP primer for the porA gene.

[0056] Using the product of the porA gene amplified by primer set Primer 8 (SEQ ID NO. 9) as a template, seven guide RNAs were designed using Snap Gene (6.0.2), namely porA-8-sgRNA1 to porA-8-sgRNA7, as shown in Table 2. sgRNAs were synthesized for future use.

[0057] Table 2 sgRNA sequence list

[0058] serial number Sequence (5'-3') porA-8-sgRNA1 SEQ ID NO. 1 porA-8-sgRNA2 SEQ ID NO. 2 porA-8-sgRNA3 SEQ ID NO. 3 porA-8-sgRNA4 SEQ ID NO. 4 porA-8-sgRNA5 SEQ ID NO. 5 porA-8-sgRNA6 SEQ ID NO. 6 porA-8-sgRNA7 SEQ ID NO. 7

[0059] 1. Establishment of a one-step LAMP-CRISPR detection system for the porA gene

[0060] The LAMP-CRISPR one-step detection reagent system is configured according to the reaction components. While the LAMP system amplifies the target fragment, the CRISPR system in the system performs target detection, breaking the probe through trans-cleavage activity and releasing a fluorescent signal.

[0061] Reaction system: 10× LAMP Buffer (200 mM Tris-HCl, 100 mM (HH4)2SO4, 500 mM KCl, 1% Tween20) 2.5 μL, 10× LAMP Primer (F3 / B3 2 mM, FIP / BIP 16 mM, LF / LB 4 mM) 2.5 μL, 2M glycine 6 μL, 25 mM dNTP 1 μL, 100 mM MgSO4 2 μL, 8 U / μL Bst 1 μL, 10 μM Probe 1.25 μL, 10 μM AapCas12b 0.125 μL, 10 μM gRNA 1 μL, template 2.5 μL, supplemented with enzyme-free sterile water to a total reaction volume of 25 μL.

[0062] Amplification program: 60°C for 1 min (collect FAM fluorescence signal), for 45 cycles.

[0063] 2. sgRNA screening

[0064] Using NG standard strain DNA as a template, the porA gene CRISPR one-step detection was carried out using the gRNA shown in Table 2. The results were analyzed as follows Figure 2 In this system, the sgRNA with the highest fluorescence signal value in the one-step amplification was porA-8-sgRNA2.

[0065] Therefore, porA-8-sgRNA2 was selected as the guide RNA for the one-step detection system of the porA gene.

[0066] The kit for detecting NG was prepared according to the following composition: 10× LAMP Buffer (200 mM Tris-HCl, 100 mM (HH4)2SO4, 500 mM KCl, 1% Tween20), 10× LAMP Primer (F3 / B3 2 mM, FIP / BIP 16 mM, LF / LB 4 mM), 2 M glycine, 100 mM MgSO4, 25 μM dNTPs, 10 μM AapCas12b, 10 μM Probe, 10 μM gRNA and ddH2O.

[0067] The primer is the combination Primer 8 described in Example 1, and the gRNA is the porA-8-gRNA2 described in Example 2.

[0068] The reaction system of this kit can be: 10×LAMP Buffer (200 mM Tris-HCl, 100 mM (HH4)2SO4, 500 mM KCl, 1% Tween20) 2.5 μL, 10×LAMP Primer (F3 / B3 2 mM, FIP / BIP 16mM, LF / LB 4 mM) 2.5 μL, 2 M glycine 6 μL, 25 mM dNTP 1 μL, 100 mM MgSO4 2 μL, 8 U / μLBst 1 μL, 10 μM Probe 1.25 μL, 10 μM AapCas12b 0.125 μL, 10 μM gRNA 1 μL, template 2.5 μL, supplemented with enzyme-free sterile water to a total reaction volume of 25 μL.

[0069] The reaction program of the kit is: 60℃ for 1 min (collect fluorescence signal), for a total of 45 cycles.

[0070] When the kit is used to detect a sample, an amplification curve is obtained through the fluorescence signal detected by the instrument.

[0071] 1. Specificity detection

[0072] The specificity of the porA gene LAMP-CRISPR one-step detection system established in Example 2 was tested using the plasmid NG-Plasmid containing the NG porA gene as a positive control and the plasmid GBS-Plasmid not containing the NG porA gene as a negative control, and genomic DNA of other strains such as NG and GBS as templates.

[0073] The strain templates include Neisseria gonorrhoeae ATCC49226 ( Neisseria gonorrhoeae, NG), Group B Streptococcus ATCC12386 (Group B Streptococcus, GBS), Pseudomonas aeruginosa ATCC27583 ( Pseudomonas aeruginosa , Pa), Enterobacter holmii ATCC700323 ( Enterobacter hormaechei , Eh), Escherichia coli ATCC8739 ( Escherichia coli , E.c1), Escherichia coli ATCC25922 ( Escherichia coli , E.c2), Staphylococcus saprophyticus ATCC BAA750 ( Staphylococcus saprophyticus , Ss), Enterococcus tumefaciens ATCC700327 ( Enterococcus casseliflavus , E.Ca), Staphylococcus aureus ATCC25923 ( Staphylococcus aureus , Sa), Streptococcus pneumoniae ATCC49619 ( Streptococcus pneumoniae , S.Pn) and Staphylococcus epidermidis ATCC12228 ( Staphylococcus epidermidis , Se).

[0074] Test results such as Figure 3 The NG genome and positive control plasmid samples were positive, while other strains, negative control plasmids, and NTC showed no nonspecific detection signals.

[0075] The test results showed that the porA gene LAMP-CRISPR one-step detection system established in Example 2 had good specificity.

[0076] 2. Sensitivity analysis

[0077] Sensitivity refers to the lowest concentration of a sample that can be detected by the amplification reaction, i.e., the minimum detection limit. When using a one-step method to detect NG samples of varying concentrations, if a sample at a certain concentration fails to form an amplification curve, it is considered to have exceeded the minimum detection limit of the system.

[0078] The extracted genomic DNA of the NG strain was measured for concentration using an ultramicro-UV spectrophotometer and adjusted to 10 ng / μL. The concentration was then diluted in a 10-fold gradient to 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, and 100 fg / μL. 2.5 μL of each DNA concentration was used as template and assayed using the one-step assay described in Example 2. The experiment was repeated three times. If no amplification curve was formed for a template at a certain concentration, the concentration was considered to have exceeded the minimum detection limit of the one-step assay.

[0079] The results are as follows Figure 4By diluting the strain genome, the system can successfully detect 1 pg / μL of genome sample, that is, the minimum detection limit of NG is 1 pg / μL.

[0080] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as a preferred embodiment as above, it is not intended to limit the present invention. Any technician familiar with this profession can use the technical content disclosed above to make changes or modifications to equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for detecting Neisseria gonorrhoeae nucleic acid molecules, characterized in that: The following steps are involved: (i) Mix the following reagents with the sample to be tested and perform the test using the one-step detection system: (1) Cas12b protein, the amino acid sequence of which is shown in SEQ ID NO: 10; (2) LAMP primer combination, including: outer primers F3 and B3, with nucleotide sequences of 5′-CGGCTCAGTTGGATTTGTCT-3′ and 5′-AAGTGCGCTTGGAAAAATCG-3′, respectively; Internal primers FIP and BIP, with nucleotide sequences of 5′-CGGGACTGTATTACCGAAGCGGACCAAAAACAGTACGACCGA-3′ and 5′-GCCCATGGTTTCGACTTTGTCGAATCGACACCGGCGATGA-3′, respectively; loop primers LF and LB, with nucleotide sequences of 5′-GGAAGCAGTGGCGGCAAT-3′ and 5′-CAGAAACGCGAACATACCAGCTA-3′, respectively; (3) sgRNA, the nucleotide sequence of which is shown in SEQ ID NO: 2, is used to guide the Cas12b protein to specifically recognize the nucleic acid molecule of Neisseria gonorrhoeae; (4) Nucleic acid probe, nucleotide sequence CCCCCCCC, 5' end labeled with fluorescent group FAM, 3' end labeled with quencher group BHQ1; (ii) detecting the cleavage status of the nucleic acid probe in the system; (iii) determining based on the detection result of step (ii): when the nucleic acid probe is cleaved, it indicates that the Neisseria gonorrhoeae nucleic acid molecule is detected; when the nucleic acid probe is not cleaved, it indicates that the Neisseria gonorrhoeae nucleic acid molecule is not detected; The method is used for diagnosis and treatment of non-diseases.

2. The method according to claim 1, wherein In step (ii), the cleavage state of the nucleic acid probe is detected by real-time monitoring of the change in the fluorescence signal; when the fluorescence signal is monitored, it indicates that the nucleic acid probe is cleaved, that is, the concentration of Neisseria gonorrhoeae nucleic acid molecules is detected to be not less than 1 pg / μL.

3. The method according to claim 1, wherein The one-step detection system is specifically as follows: 10×LAMPBuffer 2.5 μL, 10×LAMP Primer 2.5 μL, 2 M glycine 6 μL, 25 mM dNTP 1 μL, 100 mM MgSO4 2 μL, 8 U / μL Bst 1 μL, 10 μM nucleic acid probe 1.25 μL, 10 μM AapCas12b 0.125 μL, 10 μMgRNA 1 μL, template 2.5 μL, enzyme-free sterile water is added to the total reaction volume of 25 μL; the reaction is incubated at 60°C for 1 min, the fluorescence signal is collected, and a total of 45 cycles are performed.

4. Use of the method according to any one of claims 1 to 3 in detecting Neisseria gonorrhoeae nucleic acid molecules; The applications are for diagnosis and treatment of non-diseases.

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