CRISPR / Cas12b-based neisseria gonorrhoeae detection method

Abstract

The invention provides an LAMP primer and sgRNA combination, a detection kit, a one-step detection system based on CRISPR / Cas12b and application of the one-step detection system in detection of 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 pose a serious threat to human life and health due to their high morbidity and mortality rates. In particular, pathogens with high infectivity and mutation rates are prone to pandemics, which is a huge test for the global public health system. Rapid and accurate early diagnosis of infectious diseases is a key measure to reduce mortality, improve prognosis and limit transmission, and is also an important part of laboratory medicine research. Nucleic acid testing is the focus of infectious disease diagnosis. Compared with antigen and antibody testing, it has higher sensitivity and specificity, and is the most accurate and reliable for early diagnosis.

[0003] Neisseria gonorrhoeae ( Neisseria gonorrhoeae NG, also known as Neisseria gonorrhoeae, is a Gram-negative, oxidase-positive obligate aerobic bacterium. It is mostly bi-spherical or short rod-shaped, with a diameter of about 0.6 μM~1.5 μM. This bacterium is widely spread around the world and is one of the common pathogens of sexually transmitted diseases. Although some infected people may not show any obvious symptoms and become asymptomatic carriers, NG can persist in the urogenital system in an asymptomatic state and then be efficiently transmitted to partners through sexual contact. It is estimated that about 10%-20% of female infected people and 5%-10% of male infected people may not show clinical symptoms, but even so, they can still become an important source of disease transmission. Gonorrhea can cause local infections such as urethritis and cervicitis. If not treated in time, it may further lead to serious complications such as salpingitis and pelvic inflammatory disease, and the risk of infertility in female infected people is significantly increased. In addition, newborns may also be infected with Neisseria gonorrhoeae during delivery through the infected maternal birth canal, causing eye inflammation, pneumonia and even sepsis, which seriously threatens the health and life of newborns.

[0004] Therefore, early identification, rapid diagnosis and effective treatment of Neisseria gonorrhoeae infection are crucial to controlling the spread of the disease, alleviating the suffering of patients and preventing complications. This requires not only clinicians and public health workers to remain vigilant, but also to continuously strengthen the monitoring and research of pathogens to ensure the timeliness and effectiveness of treatment plans.

[0005] In NG pathogen detection, PCR, isothermal amplification and other technologies are usually used to amplify the nucleic acid molecules of pathogens; then, through the rapid identification and signal amplification of the CRISPR system, the detection specificity and sensitivity are improved to obtain the detection results. However, in the existing detection methods, the amplification of pathogen nucleic acid molecules and CRISPR detection are two completely independent steps. Between the two steps, either the reaction solution needs to be opened and pipetted, or the CRISPR detection reagent is pre-added to the tube cap. After the amplification is completed, it is mixed with the amplification product at the bottom of the tube through instantaneous centrifugation to start the CRISPR reaction. This method is called the one-pot method. The opening operation of the former greatly increases the risk of aerosol contamination; although the latter solves the problem of aerosol contamination, the operation is more complicated, which is not conducive to the standardization and simplification of the product.

[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, without the need for partitioned sample addition 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, and on the other hand, the cleavage activity of CRISPR / Cas will degrade the product 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 invention 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 loading, 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 detection efficiency and accuracy, as well as the specificity and reliability of 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: The outer primers F3 and B3 had nucleotide sequences of 5′-CGGCTCAGTTGGATTTGTCT-3′ and 5′-AAGTGCGCTTGGAAAAATCG-3′, respectively; The internal primers FIP and BIP had nucleotide sequences of 5′-CGGGACTGTATTACCGAAGCGGACCAAAAACAGTACGACCGA-3′ and 5′-GCCCATGGTTTCGACTTTGTCGAATCGACACCGGCGATGA-3′, respectively; The nucleotide sequences of loop primers LF and LB were 5′-GGAAGCAGTGGCGGCAAT-3′ and 5′-CAGAAACGCGAACATACCAGCTA-3′, respectively.

[0010] sgRNA, the nucleotide sequence is shown in SEQ ID NO:2.

[0011] The present invention also provides a kit, characterized in that the kit comprises: (a) Cas12b protein, the amino acid sequence of which is shown in SEQ ID NO: 10; (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; (c) A nucleic acid probe, wherein the nucleotide sequence of the probe is CCCCCCCC, the 5' end is labeled with a fluorescent group FAM, and the 3' end is labeled with a quenching group BHQ1.

[0012] The present invention also provides a method for detecting whether Neisseria gonorrhoeae exists in a sample, characterized in that it comprises the following steps: (i) using the above kit to detect the sample to be tested; (ii) detecting the cleavage status of the nucleic acid probe in the above kit; (iii) making a determination based on the detection result of step (ii): when the nucleic acid probe is cut, it indicates that Neisseria gonorrhoeae is present in the sample; when the nucleic acid probe is not cut, it indicates that Neisseria gonorrhoeae is not present in the sample.

[0013] In some embodiments, in the above 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, 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.

[0014] 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.

[0015] 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 is established, and LAMP amplification and CRISPR detection are integrated into one reaction system, which does not require opening the lid for pipetting or partitioning for sample loading, simplifies the operation steps, shortens the reaction time, and not only greatly reduces the cost of reagents, consumables and equipment, but also reduces the risk of aerosol contamination, thereby improving the specificity and reliability of the experimental results. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] 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.

[0017] Figure 1 Amplification curves of the LAMP primer set screening experiment, where the template of the blue curve is NG nucleic acid extract and the purple curve is the no-template control (NTC).

[0018] Figure 2 Screening results of sgRNA, where green represents the porA gene and gray represents NTC.

[0019] Figure 3 Specific identification results.

[0020] Figure 4 Sensitivity test results. DETAILED DESCRIPTION

[0021] The present invention will be further described below in conjunction with the accompanying drawings. The following examples are only used to illustrate the present invention but do not limit the scope of the present invention.

[0022] 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.

[0023] The term "CRISPR" refers to clustered regularly interspaced short palindromic repeats, which are the immune system of many prokaryotes.

[0024] The term "Cas protein" refers to CRISPR-associated protein, which is the associated protein in the CRISPR system.

[0025] The term "Cas12b" refers to a crRNA-dependent endonuclease, which is a type VB enzyme in the CRISPR system classification.

[0026] The term "LAMP" stands for loop-mediated isothermal amplification, which is a constant temperature nucleic acid amplification technology suitable for genetic diagnosis.

[0027] The term "PCR" refers to "polymerase chain reaction", a method used to amplify a target DNA fragment in large quantities.

[0028] CRISPR diagnosis is a rapid molecular detection technology based on the targeted recognition of pathogen nucleic acids by clustered regularly interspaced short palindromic repeat-associated proteins under the guidance of RNA. Compared with existing molecular diagnostic technologies such as quantitative PCR and second-generation sequencing, CRISPR diagnostic technology has the advantages of sensitivity, specificity, rapidity, convenience and low cost. It can be widely used in POCT, clinical infection detection, tumor screening, companion diagnosis, food safety and other fields.

[0029] For pathogens whose detection targets are DNA, CRISPR diagnosis mainly uses CRISPR / Cas proteins to induce cutting in each targeted DNA strand, resulting in double-stranded DNA breaks. In addition to inducing cis-cutting of target DNA, target DNA binding also induces trans-cutting of non-target DNA, that is, cutting of surrounding single-stranded DNA (ssDNA), and its trans-cutting activity has been applied to high-sensitivity nucleic acid detection. CRISPR / Cas proteins cut specific sequence DNA under the guidance of sgRNA. sgRNA (singleguide RNA, sgRNA) is an integration of crRNA (CRISPR-derived RNA, crRNA) and tracRNA (trans-activatingcrRNA, tracRNA). Through base pairing, it self-folds into a partially double-stranded RNA structure and binds to Cas proteins to exert its function.

[0030] 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' end and 3' end 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 exists in the detection system, the CRISPR / Cas protein will cut the DNA of the pathogen under the guidance of sgRNA, resulting in the activation of the trans-cleavage activity of the CRISPR / Cas protein, and the detection probe is therefore cut and produces fluorescence, which can be detected by the corresponding equipment.

[0031] The present invention will be further described below in conjunction with 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. The experimental methods in the following examples without specifying specific conditions are usually performed under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer.

[0032] The main outer membrane protein encoded by the porA gene is an important virulence factor of Neisseria gonorrhoeae (NG). The porA gene is highly conserved and has low homology with other bacteria, so porA can be used for molecular identification of NG. By detecting the porA gene, it can assist in confirming whether the bacteria is NG, thereby providing a basis for subsequent disease diagnosis and treatment.

[0033] To this end, the present invention uses 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 Primer1 to Primer 10, as shown in Table 1. They are synthesized and used for later use.

[0034] Table 1 LAMP primer sequence list

[0035] 1. Extraction of DNA from NG standard strains The NG standard strain with the product number ATCC49226 was purchased and placed in a -80°C refrigerator for future use.

[0036] After the strain was taken out and revived, it was inoculated with gonococcal selective culture medium (chocolate culture medium) on chocolate plates, and the plates were placed in a 37°C, 5% CO2 incubator for 24 to 48 hours.

[0037] The NG strain DNA was extracted using the Tiangen bacterial genomic DNA extraction kit (#DP302).

[0038] 2. Establish LAMP amplification reaction system Different LAMP primers were used to amplify the NG standard strain DNA. SybrGreen fluorescent dye was added to the system to monitor the amount of amplified products in the amplification system. At the same time, a no-template sample was set as a control group (NTC) to monitor the non-specific amplification of the primers.

[0039] 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, add enzyme-free sterile water to a total reaction volume of 25 μL.

[0040] Amplification program: 60°C for 1 min (collect FAM fluorescence signal), for 45 cycles.

[0041] 3. LAMP primer screening Using NG standard strain DNA as template, isothermal amplification tests were carried out with 10 sets of LAMP primers of NG porA gene shown in Table 1 .

[0042] The results are as follows Figure 1 As shown in the figure, 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.

[0043] Using the product of the porA gene amplified by primer set Primer 8 (SEQ ID NO. 9) as a template, 7 guide RNAs were designed using Snap Gene (6.0.2), namely porA-8-sgRNA1 to porA-8-sgRNA7, as shown in Table 2. Synthesize sgRNA for later use.

[0044] Table 2 sgRNA sequence list 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 1. Establishment of a one-step LAMP-CRISPR detection system for the porA gene 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, breaks the probe through trans-cleavage activity, and releases a fluorescent signal.

[0045] 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, supplement with enzyme-free sterile water to a total reaction volume of 25 μL.

[0046] Amplification program: 60°C for 1 min (collect FAM fluorescence signal), for 45 cycles.

[0047] 2. Screening of sgRNA 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 are analyzed as follows Figure 2 In this system, the sgRNA with the highest fluorescence signal value in the one-step amplification is porA-8-sgRNA2.

[0048] Therefore, porA-8-sgRNA2 was selected as the guide RNA for the one-step detection system of the porA gene.

[0049] 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 16mM, LF / LB 4 mM), 2 M glycine, 100 mM MgSO4, 25 μM dNTPs, 10 μM AapCas12b, 10 μMProbe, 10 μM gRNA and ddH2O.

[0050] The primer is the combination Primer 8 described in Example 1, and the gRNA is the porA-8-gRNA2 described in Example 2.

[0051] The reaction system of the 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 1μL, 100 mM MgSO4 2 μL, 8 U / μLBst 1μL, 10 μM Probe 1.25 μL, 10 μM AapCas12b 0.125 μL, 10 μM gRNA 1 μL, template 2.5 μL, and enzyme-free sterile water is added to a total reaction volume of 25 μL.

[0052] The reaction program of the kit is: 60℃ for 1 min (collecting fluorescence signals), for a total of 45 cycles.

[0053] When the kit is used to detect a sample, an amplification curve is obtained through the fluorescent signal detected by the instrument.

[0054] 1. Specificity detection The plasmid NG-Plasmid containing the NG porA gene was used as a positive control, the plasmid GBS-Plasmid not containing the NG porA gene was used as a negative control, and the genomic DNA of other strains such as NG and GBS was used as a template to test the specificity of the porA gene LAMP-CRISPR one-step detection system established in Example 2.

[0055] The strain templates include Neisseria gonorrhoeae ATCC49226 ( Neisseria gonorrhoeae , NG), Group B Streptococcus ATCC12386 (Group B Streptococcus, GBS), Pseudomonas aeruginosa ATCC27583 ( Pseudomonas aeruginosa , Pa), E. coli 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 ferulatus 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).

[0056] Test results such as Figure 3 As shown in the figure, the test results of NG genome and positive control plasmid samples were positive, and there were no non-specific detection signals for other strains, negative control plasmids and NTC.

[0057] The test results show that the porA gene LAMP-CRISPR one-step detection system established in Example 2 has good specificity. 2. Sensitivity analysis Sensitivity refers to the lowest concentration of a sample that can be detected by the amplification reaction, that is, the lowest detection limit. When using the one-step method to detect NG samples of different concentrations, if a sample of a certain concentration cannot form an amplification curve, it is considered that the concentration exceeds the lowest detection limit of the system.

[0058] The extracted genomic DNA was measured by ultra-micro UV spectrophotometer, and the concentration of NG strain genomic DNA was adjusted to 10 ng / μL, and diluted to 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL and 100 fg / μL according to a 10-fold gradient. 2.5 μL of each concentration of DNA was taken as a template, and the test was performed according to the one-step system of Example 2. The test was repeated 3 times. When a certain concentration of template cannot form an amplification curve, it is considered that the concentration exceeds the minimum detection limit of the one-step system.

[0059] The results are as follows Figure 4 By diluting the strain genome, the system can successfully detect 1 pg / μL of genome samples, that is, the minimum detection limit of NG is 1 pg / μL.

[0060] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used 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 of equivalent changes 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 LAMP primer and sgRNA combination for detecting Neisseria gonorrhoeae nucleic acid molecules, characterized in that: The combination includes the following primers and sgRNA: The outer primers F3 and B3 had nucleotide sequences of 5′-CGGCTCAGTTGGATTTGTCT-3′ and 5′-AAGTGCGCTTGGAAAAATCG-3′, respectively; The internal primers FIP and BIP had 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; sgRNA, the nucleotide sequence is shown in SEQ ID NO:

2.

2. A kit, characterized in that: The kit comprises: (a) Cas12b protein, the amino acid sequence of which is shown in SEQ ID NO: 10; (b) The LAMP primer and sgRNA combination of claim 1, wherein the sgRNA can guide the Cas12b protein to specifically bind to the Neisseria gonorrhoeae nucleic acid molecule; (c) A nucleic acid probe, wherein the nucleotide sequence of the probe is CCCCCCCC, the 5' end is labeled with a fluorescent group FAM, and the 3' end is labeled with a quenching group BHQ1.

3. A method for detecting Neisseria gonorrhoeae nucleic acid molecules, characterized in that: The following steps are involved: (i) using the kit according to claim 2 for detection; (ii) detecting the cleavage status of the nucleic acid probe in the kit; (iii) making a determination based on the detection result of step (ii): when the nucleic acid probe is cut, it indicates that the Neisseria gonorrhoeae nucleic acid molecule is detected; when the nucleic acid probe is not cut, it indicates that the Neisseria gonorrhoeae nucleic acid molecule is not detected; The method is used for non-disease diagnosis and treatment.

4. The method according to claim 3, characterized in that In the 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 not less than 1 pg / μL is detected.

5. Use of the LAMP primer and sgRNA combination according to claim 1, the kit according to claim 2, and the method according to any one of claims 3 to 4 in detecting Neisseria gonorrhoeae nucleic acid molecules; The applications are for non-disease diagnosis and treatment.

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