Primer for detecting pathogenic microorganisms on basis of RPA-crispr / cas12a and use of primer

Abstract

The present invention relates to the technical field of molecular biological detection, and relates to a primer for detecting pathogenic microorganisms (Neisseria gonorrhoeae and an FC428 ceftriaxone-resistant clone) on the basis of RPA-CRISPR / Cas12a and a use of the primer and a detection method. The pathogenic microorganisms are Neisseria gonorrhoeae or Neisseria gonorrhoeae FC428 ceftriaxone. A special isothermal amplification primer for detecting Neisseria gonorrhoeae is a group of recombinase polymerase amplification (RPA) primers, and base sequences are as shown in SEQ ID NO.1-2 in sequence; and a special isothermal amplification primer for detecting ceftriaxone-resistant Neisseria gonorrhoeae FC428 clone is a group of RPA amplification primers, and base sequences are as shown in SEQ ID NO.3-4 in sequence. Moreover, a micro-fluidic chip platform comprising two parts is designed to achieve on-chip stepwise detection. A sample identification result can be determined by determining whether there are fluorescence signals in cavities, achieving on-site rapid and instant detection, and improving the detection efficiency.

Description

A primer for detecting pathogenic microorganisms based on RPA-CRISPR / Cas12a and its application Technical Field

[0001] The present invention belongs to the field of molecular biology detection technology and relates to a primer for detecting pathogenic microorganisms (Neisseria gonorrhoeae and FC428 ceftriaxone-resistant clones) based on RPA-CRISPR / Cas12a, as well as its application and detection method. Background Art

[0002] Gonorrhea is a common sexually transmitted infection caused by Neisseria gonorrhoeae (NG). The World Health Organization estimated in 2019 that gonorrhea ranks second among bacterial sexually transmitted infections, with 86.9 million new cases annually among adults aged 15 to 49. Currently, ceftriaxone is the recommended first-line treatment for N. gonorrhoeae in most countries. However, ceftriaxone resistance in N. gonorrhoeae is increasing globally. The ceftriaxone-resistant FC428 clone, first discovered in Japan in 2015, has become increasingly prevalent internationally, posing a serious public health threat. The FC428 clone is resistant to multiple antibiotics and faces the risk of becoming untreatable. Therefore, developing timely diagnostic methods for the ceftriaxone-resistant FC428 clone of N. gonorrhoeae to provide clinicians with timely medication guidance is crucial for the clinical treatment, field prevention, and surveillance and early warning of drug-resistant N. gonorrhoeae.

[0003] Currently, common methods for detecting ceftriaxone-resistant clones in Neisseria gonorrhoeae FC428 include antibiotic susceptibility testing (AST), polymerase chain reaction (PCR), and quantitative real-time PCR (qPCR). However, these methods are limited by high costs, long test cycles, and the need for specialized technicians and extensive equipment. Clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) systems, as rapidly developing molecular diagnostic technologies, offer strong specificity and programmability, allowing for easy integration into point-of-care testing devices and demonstrating significant potential in nucleic acid detection. To enhance the sensitivity of CRISPR-based detection methods, their integration with isothermal amplification methods, such as recombinase polymerase amplification (RPA), has enabled single-molecule detection. Therefore, the application of RPA-CRISPR system to detect ceftriaxone-resistant clones of Neisseria gonorrhoeae FC428 provides a rapid (detection time 1 hour), convenient (isothermal reaction at 38°C), and simple (easy to combine with on-site instant detection equipment) method, which is of great significance.

[0004] Microfluidic chip technology can integrate various detection and operation units, connecting the entire system through microchannels. Due to its promising potential in biology, chemistry, medicine, and other fields, microfluidic chip technology has developed into a new, multidisciplinary research field. Equipped with multiple reaction chambers, microfluidic chips enable high-throughput testing, simplifying experimental operations, facilitating on-site prevention and control, and significantly enhancing the ability of real-time on-site screening.

[0005] Summary of the Invention

[0006] The purpose of the present invention is to provide a primer with strong specificity and high sensitivity for detecting pathogenic microorganisms (Neisseria gonorrhoeae and FC428 ceftriaxone-resistant clones) based on RPA-CRISPR / Cas12a, as well as its application and detection method.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] A primer for identifying pathogenic microorganisms, wherein the pathogenic microorganism is Neisseria gonorrhoeae or Neisseria gonorrhoeae FC428 ceftriaxone; wherein,

[0009] The isothermal amplification primers for detecting Neisseria gonorrhoeae are a set of RPA amplification primers, and the base sequences are shown in SEQ ID NO.1-2.

[0010] The isothermal amplification primers specifically used for detecting ceftriaxone-resistant clones of Neisseria gonorrhoeae FC428 are a set of RPA amplification primers, and the base sequences are shown in SEQ ID NO.3-4.

[0011] Application of primers for identifying pathogens, and application of the isothermal amplification primers specifically for detecting Neisseria gonorrhoeae in species identification of Neisseria gonorrhoeae;

[0012] Application of dedicated isothermal amplification primers for detecting ceftriaxone-resistant clones of Neisseria gonorrhoeae FC428.

[0013] A crRNA of Neisseria gonorrhoeae is used for CRISPR reaction to identify the specific crRNA of Neisseria gonorrhoeae, and the base sequence is shown in SEQ ID NO.5.

[0014] A crRNA of a ceftriaxone-resistant clone of Neisseria gonorrhoeae FC428 is used for CRISPR reaction to identify the specific crRNA of the ceftriaxone-resistant clone of Neisseria gonorrhoeae FC428, and the base sequence is shown in SEQ ID NO.6.

[0015] An application of the crRNA, the application of the crRNA in identifying Neisseria gonorrhoeae;

[0016] The application of the crRNA in identifying ceftriaxone-resistant clones of Neisseria gonorrhoeae FC428.

[0017] The above-mentioned identification of Neisseria gonorrhoeae refers to the ability to accurately identify Neisseria gonorrhoeae. RPA amplification primers are designed based on the conserved gene regions that are specific between species and conserved within species of Neisseria gonorrhoeae. At the same time, a specific crRNA is designed based on the CRISPR principle.

[0018] The above-mentioned special primers for identifying ceftriaxone-resistant clones of Neisseria gonorrhoeae FC428 select the gene or region of the penA interspecies variation region as the RPA amplification target and CRISPR / Cas12a detection target, and screen the target sequence containing the protospacer adjacent motif (PAM) site required for CRISPR / Cas12a detection to design crRNA.

[0019] The RPA primers and crRNA specifically designed for identifying Neisseria gonorrhoeae are a set consisting of two primers, SEQ ID NOs. 1-2, one forward primer and one reverse primer, used for RPA amplification reactions; the specific crRNA is a single primer, SEQ ID NO. 5. The RPA primers and crRNA specifically designed for identifying ceftriaxone-resistant clones of Neisseria gonorrhoeae FC428 are a set consisting of two primers, SEQ ID NOs. 3-4, one forward primer and one reverse primer, used for RPA amplification reactions; the specific crRNA is a single primer, SEQ ID NO. 6. Primer sequence information is shown in Table 1.

[0020] Table 1 Primer and crRNA sequence information

[0021] A microfluidic chip assembly detection platform for detecting ceftriaxone-resistant clones of Neisseria gonorrhoeae FC428, comprising RPA primers shown in SEQ ID NOs. 1-4 for detecting Neisseria gonorrhoeae and ceftriaxone-resistant clones of Neisseria gonorrhoeae FC428, and crRNA for detecting Neisseria gonorrhoeae and ceftriaxone-resistant clones of Neisseria gonorrhoeae FC428 shown in SEQ ID NOs. 5-6.

[0022] The detection platform includes a first part 1 and a second part 2, wherein the first part 1 is provided with a first lower cavity 101 and a second lower cavity 102, a slot 105 is provided at the lower part of the second part 2, and a first upper cavity 201, a second upper cavity 202 and an injection port 203 are provided at the upper part of the second part 2, and the injection port 203 is provided between the first upper cavity 201 and the second upper cavity 202, and connecting channels 204 are provided on both sides of the injection port 203, which are respectively connected to the upper cavity on the corresponding side. The first part 1 is inserted into the slot 105 at the lower part of the second part 2, and after the first part 1 is inserted, the first lower cavity 101 and the first upper cavity 201 are correspondingly connected to form a Neisseria gonorrhoeae reaction chamber, and the second lower cavity 102 and the second upper cavity 202 are correspondingly connected to form a Neisseria gonorrhoeae FC428 ceftriaxone-resistant clone reaction chamber.

[0023] The first lower cavity 101 carries the CRISPR system of Neisseria gonorrhoeae, and the second lower cavity 201 carries the CRISPR system of the ceftriaxone-resistant clone of Neisseria gonorrhoeae FC428;

[0024] The first upper cavity 201 carries the RPA reaction system of Neisseria gonorrhoeae, and the second upper cavity 202 carries the RPA reaction system of the ceftriaxone-resistant clone of Neisseria gonorrhoeae FC428.

[0025] 3M is used between the first lower cavity 101 and the first upper cavity 201; and between the second lower cavity 102 and the second upper cavity 202 Brand Tape double-sided tape is used for separation. The lower end of the tape is bonded to cavities 101 and 102. The upper end is not peeled off to protect the hydrophobic paper used to isolate the liquids in 201 and 202.

[0026] The injection port in part 2 is located in the center, and the central injection port is connected to the two RPA reaction chambers on both sides through a capillary channel 204.

[0027] Among them, the construction of the microfluidic chip assembly is as follows: the microfluidic chip is designed by Solidworks2021 software, and the chip contains two parts. The part where the CRISPR reaction chamber is located is 12.4mm long, 11.2mm wide, and 4.5mm high; the CRISPR reaction chamber is a cylinder with a diameter of 3.6mm and a height of 4mm. The part where the RPA reaction chamber is located is 15mm long, 11.2mm wide, and 11.1mm high. The RPA reaction chamber is a cylinder with a diameter of 3mm and a height of 6mm, and the diameter of the central injection port is 1.4mm. After saving the design draft in .stl format, use a 3D printer (Formlabs TM , Form3) printing, using transparent resin (Formlabs TM , FLGPCL04).

[0028] A method for detecting Neisseria gonorrhoeae and ceftriaxone-resistant clones of Neisseria gonorrhoeae FC428 using the detection platform:

[0029] (1) Use a commercial DNA extraction kit to extract nucleic acid from the test sample;

[0030] (2) using the nucleic acid of the sample to be tested as a template, preparing an RPA amplification reaction system under the guidance of the primers described in claim 1, and performing specific amplification in each detection reaction well;

[0031] The RPA system was constructed as follows: The RPA kit was purchased from TwistDx, UK. To the RPA dry powder, 25 μL of buffer, 2 μL of forward primer RPA-porA-F (10 μM), 2 μL of reverse primer RPA-porA-R (10 μM), 2 μL of forward primer RPA-penA-F (10 μM), 2 μL of reverse primer RPA-penA-R (10 μM), and 8 μL of H₂O were added. The mixture was then divided into three equal 13.5 μL aliquots, each containing 1 μL of magnesium acetate and 2 μL of sample (experimental group) / H₂O (control group). One RPA system consisted of 16.5 μL.

[0032] The reaction conditions are as follows: incubation at a constant temperature of 38° C. in a metal bath module for 20 min.

[0033] (3) 2 μL of amplified product was added to the CRISPR / Cas12a system containing specific crRNA respectively; according to the cleavage of the fluorescent reporter probe by the Cas12a protein, the fluorescence of the experimental group changed from zero to positive before and after the reaction, and the presence of ceftriaxone-resistant clones of Neisseria gonorrhoeae and Neisseria gonorrhoeae FC428 in the sample was detected.

[0034] Each CRISPR / Cas12a reaction system was constructed as follows: a reaction system with a final volume of 13.5 μL was prepared by co-preparing a final concentration of 100 nM Cas12a, 100 nM crRNA, 600 nM fluorescent reporter probe, 1x NEB r2.1 Buffer, and nuclease-free water; the fluorescent reporter probe in each CRISPR / Cas12a reaction system was FAM-TTATT-BHQ1.

[0035] The purpose of species identification in the method of the present invention is to classify positive Neisseria gonorrhoeae samples so that they do not cross-react with non-gonococcal Neisseria gonorrhoeae strains.

[0036] The fluorescence change is used to determine that if the sample is positive for Neisseria gonorrhoeae, a fluorescent signal can be observed in the reaction chamber containing the species identification of Neisseria gonorrhoeae. If the sample is positive for the crRNA of the FC428 ceftriaxone-resistant clone of Neisseria gonorrhoeae, a fluorescent signal can be observed in both the species identification and the FC428 ceftriaxone-resistant clone-specific reaction chambers.

[0037] Furthermore, in the CRISPR / Cas12a reaction, due to the sequence specificity of the target sequence, only crRNA with a completely complementary base pair can bind to it, activating the corresponding Cas12a protein. Based on the cleavage of the fluorescent reporter probe by the Cas12a protein, the fluorescence in the experimental group changes from zero to positive before and after the reaction. The CRISPR / Cas12a reaction was performed on the QuantStudio 7 Flex Real-Time PCR System under the following conditions: incubation at 38°C for 30 minutes to complete the reaction, and fluorescence signals were collected every 30 seconds. After the reaction, the fluorescence intensity values ​​of each group were extracted using QuantStudio 6 and 7 Flex Real-Time PCR software v1.0, and the results were plotted using GraphPad Prism 8 and PowerPoint. If the fluorescence intensity is significantly higher than that of the negative control group, it indicates the presence of the detection target in the sample.

[0038] The CRISPR / Cas12a reaction on the microfluidic chip assembly was performed under the following conditions: incubation at 38°C for 20 minutes. When the sample was not N. gonorrhoeae, no fluorescent signal was generated in either chamber. When the sample was N. gonorrhoeae but not the FC428 clone, fluorescent signal was generated only in the chamber containing the N. gonorrhoeae-specific crRNA. When the sample was the ceftriaxone-resistant N. gonorrhoeae FC428 clone, fluorescent signal was generated in both chambers containing both the N. gonorrhoeae-specific crRNA and the FC428 clone-specific crRNA. To maximize system performance, we optimized the RPA and CRISPR reaction conditions.

[0039] Compared with the prior art, the present invention has the following positive effects:

[0040] First, the method of the present invention uses the RPA-CRISPR method to identify Neisseria gonorrhoeae and differentiate the ceftriaxone-resistant clones of Neisseria gonorrhoeae FC428. The reaction is carried out at a constant temperature of 38°C, without complex temperature control requirements. In terms of reaction time, the step-by-step reaction time based on PCR tubes is 20 minutes for RPA and 30 minutes for CRISPR reaction, for a total of 50 minutes, while the total reaction time of the step-by-step method based on the microfluidic chip assembly is 40 minutes, i.e., 20 minutes for RPA reaction and 20 minutes for CRISPR reaction, thereby achieving rapid detection. The RPA-specific primers designed by the method of the present invention can amplify target sequences containing the conserved region of Neisseria gonorrhoeae and the variable region of the ceftriaxone-resistant clone of Neisseria gonorrhoeae FC428 in the same system, and then couple them to the CRISPR / Cas12a reaction system to achieve convenient and specific detection. Furthermore, the method integrates the RPA-CRISPR reaction into the microfluidic chip, and the ceftriaxone-resistant clone of Neisseria gonorrhoeae FC428 can be judged according to the presence or absence of fluorescence in each reaction chamber on the chip. Therefore, this method provides a new technical means for developing detection methods and detection platforms for ceftriaxone-resistant clones of Neisseria gonorrhoeae FC428. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] FIG1 is a part 1 of a microfluidic chip assembly detection platform for identifying Neisseria gonorrhoeae and ceftriaxone-resistant clones of Neisseria gonorrhoeae FC428 provided in an embodiment of the present invention;

[0042] FIG2 is a second part of a microfluidic chip assembly detection platform for identifying Neisseria gonorrhoeae and ceftriaxone-resistant clones of Neisseria gonorrhoeae FC428 provided in an embodiment of the present invention;

[0043] FIG3 is a schematic diagram of the results of identifying ceftriaxone-resistant clones of Neisseria gonorrhoeae and Neisseria gonorrhoeae FC428 based on PCR tube fluorescence monitoring according to an embodiment of the present invention;

[0044] FIG4 is a schematic diagram of a detection method according to an embodiment of the present invention;

[0045] FIG5 is a schematic diagram of the results of identifying ceftriaxone-resistant clones of Neisseria gonorrhoeae and Neisseria gonorrhoeae FC428 based on the microfluidic chip platform provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0046] The specific embodiments of the present invention are further described below with reference to examples. It should be noted that the specific embodiments described here are only for illustrating and explaining the present invention, and are not intended to limit the present invention.

[0047] The present invention designs RPA amplification primers to identify Neisseria gonorrhoeae and ceftriaxone-resistant clones of Neisseria gonorrhoeae FC428, amplifies nucleic acid fragments of the conserved region of Neisseria gonorrhoeae and the variable region of the ceftriaxone-resistant clone of Neisseria gonorrhoeae FC428, and then couples them with a clustered regularly interspaced short palindromic repeats (CRISPR) / CRIPSR-associated (Cas) 12a detection system. By designing two crRNA sequences, they are used to detect the specificity of Neisseria gonorrhoeae and the ceftriaxone-resistant clone of Neisseria gonorrhoeae FC428, respectively. The above detection method is further integrated into a microfluidic chip, and the species identification of Neisseria gonorrhoeae and the identification of ceftriaxone-resistant clones of FC428 can be achieved by interpreting the presence or absence of fluorescent signals in each reaction chamber.

[0048] Example 1: Design of a microfluidic chip assembly detection platform

[0049] As shown in Figures 1-2, the detection platform includes a first part 1 (Figure 1) and a second part 2 (Figure 2), wherein the first part 1 is provided with a first lower cavity 101 and a second lower cavity 102, the lower part of the second part 2 is provided with a slot 105, the upper part of the second part 2 is provided with a first upper cavity 201, a second upper cavity 202 and an injection port 203, and the injection port 203 is provided between the first upper cavity 201 and the second upper cavity 202, and connecting channels 204 are provided on both sides of the injection port 203, which are respectively connected to the upper cavity on the corresponding side. The first part 1 is inserted into the slot 105 at the lower part of the second part 2, and after the first part 1 is inserted, the first lower cavity 101 and the first upper cavity 201 are connected to form a Neisseria gonorrhoeae reaction chamber, and the second lower cavity 102 and the second upper cavity 202 are connected to form a Neisseria gonorrhoeae FC428 ceftriaxone-resistant clone reaction chamber.

[0050] The first lower cavity 101 carries the CRISPR system of Neisseria gonorrhoeae, and the second lower cavity 201 carries the CRISPR system of the ceftriaxone-resistant clone of Neisseria gonorrhoeae FC428;

[0051] The first upper cavity 201 carries the RPA reaction system of Neisseria gonorrhoeae, and the second upper cavity 202 carries the RPA reaction system of the ceftriaxone-resistant clone of Neisseria gonorrhoeae FC428.

[0052] 3M is used between the first lower cavity 101 and the first upper cavity 201 and between the second lower cavity 102 and the second upper cavity 202. Brand Tape double-sided tape is used for separation. The lower end of the tape is bonded to cavities 101 and 102. The upper end is not peeled off to protect the hydrophobic paper used to isolate the liquids in 201 and 202.

[0053] The injection port in part 2 is located in the center, and the central injection port is connected to the two RPA reaction chambers on both sides through a capillary channel 204.

[0054] Among them, the construction of the microfluidic chip assembly is as follows: the microfluidic chip is designed by Solidworks 2021 software, and the chip contains two parts. The part where the CRISPR reaction chamber is located is 12.4mm long, 11.2mm wide, and 4.5mm high; the CRISPR reaction chamber is a cylinder with a diameter of 3.6mm and a height of 4mm. The part where the RPA reaction chamber is located is 15mm long, 11.2mm wide, and 11.1mm high. The RPA reaction chamber is a cylinder with a diameter of 3mm and a height of 6mm, and the diameter of the central injection port is 1.4mm. After saving the design draft in .stl format, use a 3D printer (Formlabs TM , Form3) printing, using transparent resin (Formlabs TM , FLGPCL04).

[0055] First, pre-load the two reaction wells of Part 1 with CRISPR systems targeting Neisseria gonorrhoeae and the ceftriaxone-resistant clone FC428, respectively. Part 1 is fully inserted into Part 2, sealing the lower end of the RPA reaction chamber. The RPA system is then injected into the RPA reaction chamber through the injection port. The device is incubated at 38°C for 20 minutes to allow the RPA reaction to fully react. Then, gently pull Part 1 to connect the RPA and CRISPR reaction wells, mixing the systems. Continue the reaction at 38°C for another 20 minutes, and then interpret the experimental results.

[0056] Example 2: PCR tube-based identification of Neisseria gonorrhoeae species and identification of ceftriaxone-resistant clones of Neisseria gonorrhoeae FC428 were performed on dry samples.

[0057] (1) Sample collection: Collect swabs from patients in sampling tubes containing sample preservation solution and store them in a -80°C ultra-low temperature refrigerator.

[0058] (2) Sample nucleic acid extraction: DNA was extracted from the sample according to the QIAGEN QIAamp DNA Mini Kit instructions. The resulting DNA sample was dissolved in 50 μL of nuclease-free water and set aside.

[0059] (3) Using the nucleic acid of the sample to be tested as a template, an RPA amplification reaction system is prepared under the guidance of the above-mentioned dedicated primer set (primers shown in SEQ ID NOs. 1-4);

[0060] The RPA system was constructed as follows: the RPA kit was purchased from TwistDx, a British company. 25 μL of buffer, 2 μL of forward primer RPA-porA-F (10 μM), 2 μL of reverse primer RPA-porA-R (10 μM), 2 μL of forward primer RPA-penA-F (10 μM), 2 μL of reverse primer RPA-penA-R (10 μM), and 8 μL of H2O were added to the RPA dry powder. The system was divided into three equal parts of 13.5 μL, and 1 μL of magnesium acetate and 2 μL of sample (experimental group) / H2O (control group) were added to each part. One RPA system is 16.5 μL. The sample is DNA obtained after nucleic acid extraction from a swab of a patient carrying the ceftriaxone-resistant clone of Neisseria gonorrhoeae FC428.

[0061] The reaction conditions are as follows: incubation at a constant temperature of 38° C. in a metal bath module for 20 min.

[0062] (4) 1.5 μL of amplified product was added to the CRISPR / Cas12a system containing specific crRNA respectively; according to the cleavage of the fluorescent reporter probe by the Cas12a protein, the fluorescence of the experimental group changed from zero to positive before and after the reaction, and the presence of Neisseria gonorrhoeae in the sample was detected, and the FC428 ceftriaxone-resistant clone was identified.

[0063] Each CRISPR / Cas12a reaction system was constructed as follows: a reaction system with a final volume of 13.5 μL was prepared by co-preparing a final concentration of 100 nM Cas12a, 100 nM crRNA, 600 nM fluorescent reporter probe, 1x NEB r2.1 Buffer, and nuclease-free water; the fluorescent reporter probe in each CRISPR / Cas12a reaction system was FAM-TTATT-BHQ1.

[0064] The reaction conditions were as follows: incubate the reaction at 38°C for 30 minutes in a QuantStudio 7 Flex Real-Time PCR System, and collect the fluorescence signal every 30 seconds.

[0065] (5) Interpretation of results: After the reaction, use QuantStudio 6 and 7 Flex Real-Time PCR software v1.0 to extract the fluorescence intensity values ​​of each group, and use GraphPad Prism 8 and PowerPoint to draw the result graph. If the fluorescence intensity is significantly higher than that of the negative control group, it indicates that the detection target is present in the sample. If the sample contains ceftriaxone-resistant clones of Neisseria gonorrhoeae FC428, both CRISPR / Cas12a reactions should output fluorescent signals.

[0066] (6) The final test result of this method is: porA-crRNA and penA-crRNA produce obvious fluorescent signals, and the target is detected to be the ceftriaxone-resistant clone of Neisseria gonorrhoeae FC428 (see Figure 3).

[0067] Example 3: The microfluidic chip obtained in Example 1 is used to perform microfluidic chip detection on swab samples. The principle is shown in FIG4 .

[0068] (1) Sample collection: Collect swabs from patients in sampling tubes containing sample preservation solution and store them in a -80°C ultra-low temperature refrigerator.

[0069] (2) Sample nucleic acid extraction: DNA was extracted from the sample according to the QIAGEN QIAamp DNA Mini Kit instructions. The resulting DNA sample was dissolved in 50 μL of nuclease-free water and set aside.

[0070] (3) Using the nucleic acid of the sample to be tested as a template, an RPA amplification reaction system is prepared under the guidance of the above-mentioned dedicated primer set (primers shown in SEQ ID NOs. 1-4);

[0071] The RPA system was constructed as follows: The RPA kit was purchased from TwistDx, UK. To the RPA dry powder, 25 μL of buffer, 2 μL of forward primer RPA-porA-F (10 μM), 2 μL of reverse primer RPA-porA-R (10 μM), 2 μL of forward primer RPA-penA-F (10 μM), 2 μL of reverse primer RPA-penA-R (10 μM), 10 μL of H₂O, 3 μL of magnesium acetate, and 4 μL of sample (experimental group) / H₂O (control group) were added. The mixture was then divided equally into two 25 μL systems. One RPA system was 25 μL.

[0072] The sample is DNA obtained after nucleic acid extraction from a swab of a patient carrying the ceftriaxone-resistant clone FC428 of Neisseria gonorrhoeae.

[0073] (4) The prepared 25 μL RPA reaction system was added from the central injection port of the microfluidic chip and flowed into the CRISPR / Cas12a system containing two specific crRNAs. In the CRISPR / Cas12a reaction, due to the sequence specificity of the target sequence, it can only bind to the crRNA with its complete base complementary pairing, activating the corresponding Cas12a protein. According to the cleavage of the fluorescent reporter probe by the Cas12a protein, the change of fluorescence from zero to one before and after the reaction of the experimental group was used to detect the presence of Neisseria gonorrhoeae in the sample and identify the Neisseria gonorrhoeae FC428 clone.

[0074] The CRISPR / Cas12a reaction system was constructed by combining Cas12a at a final concentration of 100 nM, 100 nM crRNA, 600 nM fluorescent reporter probe, 1x NEB r2.1 Buffer, and nuclease-free water in a final volume of 13.5 μL. Two CRISPR / Cas12a systems, each containing crRNAs for identifying Neisseria gonorrhoeae and FC428 ceftriaxone-resistant clones, were pre-placed in the two reaction chambers.

[0075] (4) After incubation at 38°C in the metal bath module for 20 minutes, pull the lower part 1 to connect the RPA and CRISPR reaction holes, thereby mixing the system. Continue the reaction at 38°C for 20 minutes, and then interpret the experimental results.

[0076] (5) Interpretation of test results: If the sample is positive for Neisseria gonorrhoeae, a fluorescent signal can be observed in the reaction chamber on the left containing the porA-crRNA for species identification of Neisseria gonorrhoeae; based on the presence or absence of the fluorescent signal in the reaction chamber of the penA-crRNA specific for the ceftriaxone-resistant clone of Neisseria gonorrhoeae FC428, it is determined whether the sample is infected with the ceftriaxone-resistant clone of Neisseria gonorrhoeae FC428 (see Figure 5).

[0077] (6) The final test results of this method are: based on the presence or absence of fluorescence signals in each reaction chamber, it is determined whether the sample is infected with Neisseria gonorrhoeae species identification and ceftriaxone-resistant clones of Neisseria gonorrhoeae FC428.

Claims

1. A primer for identifying pathogenic microorganisms, characterized in that: The pathogenic microorganism is Neisseria gonorrhoeae or Neisseria gonorrhoeae FC428 ceftriaxone; among them, The isothermal amplification primers for detecting Neisseria gonorrhoeae are a set of RPA amplification primers, and the base sequences are shown in SEQ ID NO.1-2 in sequence; The isothermal amplification primers specifically used for detecting ceftriaxone-resistant clones of Neisseria gonorrhoeae FC428 are a set of RPA amplification primers, and the base sequences are shown in SEQ ID NO.3-4 in sequence.

2. The use of the primer for identifying pathogenic microorganisms according to claim 1, characterized in that: Application of the isothermal amplification primers for detecting Neisseria gonorrhoeae in species identification of Neisseria gonorrhoeae; Application of dedicated isothermal amplification primers for detecting ceftriaxone-resistant clones of Neisseria gonorrhoeae FC428 in detecting ceftriaxone-resistant clones of Neisseria gonorrhoeae FC428.

3. A crRNA of Neisseria gonorrhoeae, characterized in that: The specific crRNA used for CRISPR reaction to identify Neisseria gonorrhoeae, the base sequences are shown in SEQ ID NO.

5.

4. A crRNA of a ceftriaxone-resistant clone of Neisseria gonorrhoeae FC428, characterized in that: The specific crRNA used for CRISPR reaction to identify ceftriaxone-resistant clones of Neisseria gonorrhoeae FC428, the base sequences are shown in SEQ ID NO.

6.

5. An application of the crRNA according to claim 3 or 4, characterized in that: Application of the crRNA in identifying Neisseria gonorrhoeae; The application of the crRNA in identifying ceftriaxone-resistant clones of Neisseria gonorrhoeae FC428.

6. A microfluidic chip assembly detection platform for detecting ceftriaxone-resistant clones of Neisseria gonorrhoeae FC428, characterized in that: Contains RPA primers for detecting ceftriaxone-resistant clones of Neisseria gonorrhoeae and Neisseria gonorrhoeae FC428 as shown in SEQ ID NO.1-4, and crRNA for detecting ceftriaxone-resistant clones of Neisseria gonorrhoeae and Neisseria gonorrhoeae FC428 as shown in SEQ ID NO.5-6.

7. The microfluidic chip assembly detection platform for detecting ceftriaxone-resistant clones of Neisseria gonorrhoeae FC428 according to claim 6, characterized in that: The detection platform includes a first part 1 and a second part 2, wherein the first part 1 is provided with a first lower cavity 101 and a second lower cavity 102, a slot 105 is provided at the lower part of the second part 2, a first upper cavity 201, a second upper cavity 202 and an injection port 203 are provided at the upper part of the second part 2, and the injection port 203 is provided between the first upper cavity 201 and the second upper cavity 202, and connecting channels 204 are provided on both sides of the injection port 203 and are respectively connected with the upper cavities on the corresponding sides, the first part 1 is inserted into the slot 105 at the lower part of the second part 2, and after the first part 1 is inserted, the first lower cavity 101 and the first upper cavity 201 are correspondingly connected to form a Neisseria gonorrhoeae reaction chamber, and the second lower cavity 102 and the second upper cavity 202 are correspondingly connected to form a Neisseria gonorrhoeae FC428 ceftriaxone-resistant clone reaction chamber.

8. The microfluidic chip assembly detection platform for detecting ceftriaxone-resistant clones of Neisseria gonorrhoeae FC428 according to claim 7, characterized in that: The first lower cavity 101 carries the CRISPR system of Neisseria gonorrhoeae, and the second lower cavity 201 carries the CRISPR system of the ceftriaxone-resistant clone of Neisseria gonorrhoeae FC428; The first upper chamber 201 carries the RPA reaction system of Neisseria gonorrhoeae, and the second upper chamber 202 carries Neisseria gonorrhoeae FC428 RPA reaction system for ceftriaxone-resistant clones.

9. A method for detecting Neisseria gonorrhoeae and ceftriaxone-resistant clones of Neisseria gonorrhoeae FC428 using the detection platform of claim 6, characterized in that: (1) Use a commercial DNA extraction kit to extract nucleic acid from the test sample; (2) using the nucleic acid of the sample to be tested as a template, preparing an RPA amplification reaction system under the guidance of the primers described in claim 1, and performing specific amplification in each detection reaction well; (3) 2 μL of amplified product was added to the CRISPR / Cas12a system containing specific crRNA respectively; according to the cleavage of the fluorescent reporter probe by the Cas12a protein, the fluorescence of the experimental group changed from zero to one before and after the reaction, and the presence of ceftriaxone-resistant clones of Neisseria gonorrhoeae and Neisseria gonorrhoeae FC428 in the sample was detected.

10. The detection method according to claim 9, characterized in that: The fluorescence change is used to determine that if the sample is positive for Neisseria gonorrhoeae, a fluorescent signal can be observed in the reaction chamber containing the species identification of Neisseria gonorrhoeae. If the sample is positive for the crRNA of the FC428 ceftriaxone-resistant clone of Neisseria gonorrhoeae, a fluorescent signal can be observed in both the species identification and the FC428 ceftriaxone-resistant clone-specific reaction chambers.

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