Kit for detecting drug resistance gene of mycoplasma pneumoniae

Abstract

The invention provides a kit for detecting a mycoplasma pneumoniae drug resistance gene based on a CRISPR / Cas12 technology, and belongs to the field of biomedical detection. The kit comprises an LbCas12a protein, a buffer agent of the LbCas12a protein, crRNA of a targeted mycoplasma pneumoniae mutation site A2063G / A2064G, a reporter probe and an RNase inhibitor. The kit is simple to operate, the detection only needs 25 minutes, and the detection result is accurate and reliable; meanwhile, the kit can also be combined with an isothermal amplification technology to improve the detection sensitivity.

Description

[0001] This application claims priority to the earlier Chinese application, application number: 2025100243641, filed on January 7, 2025;

[0002] All of its contents are part of this invention. Technical Field

[0003] This invention relates to the field of biomedical testing, specifically to a kit for detecting drug resistance genes in Mycoplasma pneumoniae. Technical Background

[0004] Mycoplasma pneumoniae (MP) is the second leading cause of community-acquired pneumonia (CAP) in children, accounting for 20%–40% of CAP cases, and up to 70% in closed populations. Since 2000, the proportion of infections with macrolide-resistant Mycoplasma pneumoniae (MRMPI) has risen rapidly worldwide, especially in East Asia, such as China, where MRMPI strains account for 69%–95% of cases. This is a significant cause of severe and / or critical cases, complications, and sequelae.

[0005] Mycoplasma pneumoniae lacks a cell wall, thus exhibiting inherent resistance to antibiotics that act on the cell wall, such as penicillin and cephalosporins, meaning these antibiotics cannot be used to treat it. While Mycoplasma pneumoniae is sensitive to macrolide antibiotics that inhibit microbial protein synthesis, in recent years, some strains of Mycoplasma pneumoniae have emerged that are insensitive to macrolide antibiotics. This resistance is not limited to azithromycin but extends to the entire macrolide class, including clarithromycin, erythromycin, josamycin, and roxithromycin.

[0006] Macrolide antibiotics bind to the 23S rRNA domain of Mycoplasma pneumoniae. Variations in regions II and V of the 23S rRNA domain reduce the affinity between the antibiotic and the ribosome, leading to resistance in Mycoplasma pneumoniae. Identified variant sites include 2063, 2064, 2067, and 2617. A2063G positivity indicates resistance to 14-cyclic macrolides, A2064G positivity indicates resistance to both 14- and 16-cyclic macrolides, C2617G positivity indicates resistance to both 14- and 15-cyclic macrolides, and A2067G positivity indicates resistance to josamycin.

[0007] Currently, the main methods for detecting drug resistance genes in Mycoplasma pneumoniae are gene sequencing and quantitative real-time PCR (qPCR). Gene sequencing is one of the most commonly used methods for detecting drug resistance genes. It involves analyzing the 23S ribosomal RNA gene of Mycoplasma pneumoniae using sequencing technology to identify known drug resistance gene mutation sites. This method has the advantages of high sensitivity and high specificity, but it is relatively slow and often expensive. qPCR is widely used for the rapid detection of drug resistance genes. It involves designing specific primers and probes to specifically amplify and detect drug resistance gene fragments. Compared to sequencing methods, qPCR detection takes only a few hours, significantly improving speed. It also has lower reagent costs and higher sensitivity and specificity, making it suitable for clinical laboratories. However, qPCR typically requires laboratories with high-performance instruments and complete equipment, and the equipment maintenance costs are high. The machine setup and operation are complex and require specialized personnel, making it difficult to popularize in primary healthcare units.

[0008] In summary, there is currently a lack of a sensitive, specific, rapid, and simple method for detecting drug-resistant Mycoplasma pneumoniae. Summary of the Invention

[0009] To address the aforementioned problems, this invention provides a kit for detecting Mycoplasma pneumoniae drug resistance genes based on CRISPR / Cas12 technology, belonging to the field of biomedical testing. The kit contains LbCas12a protein and its buffer, crRNA targeting the Mycoplasma pneumoniae mutation sites A2063G / A2064G, a reporter probe, and an RNase inhibitor. The kit is simple to operate, requiring only 25 minutes for detection, and provides accurate and reliable results. Furthermore, it can be coupled with isothermal amplification technology to improve detection sensitivity.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0011] In a first aspect, the present invention provides a reagent for detecting drug-resistant mutation sites on the 23S rRNA of Mycoplasma pneumoniae, wherein the mutation sites are A2063G and / or A2064G; the reagent comprises crRNA and / or an amplification primer pair, both of which are suitable for recombinase-based isothermal amplification and / or conventional PCR techniques. The crRNA comprises an anchor sequence and a guide sequence, wherein the anchor sequence binds to the Cas12 protein, and the guide sequence matches the sequence amplified by the primer pair. Therefore, it is understood that the specific sequences of the anchor sequence and the guide sequence are determined by the type of Cas12 protein selected and the mutation site to be detected.

[0012] Furthermore, the Cas12 protein is selected from at least one of Cas12a, Cas12b, Cas12f, and Cas12j;

[0013] Preferably, the Cas12 protein is selected from at least one of FnCas12a, LbCas12a, AsCas12a, Lb5Cas12a, Mb2Cas12a, Mb3Cas12a, SuCas12a2, MbCas12a, PcCas12a, PbCas12a, ErCas12a, EeCas12a, AacCas12b, AapCas12b, Un1Cas12f1, and Cas12j.

[0014] In some embodiments, the Cas12 protein is Lb5Cas12a with an anchoring sequence of 5'-UAAUUUCUACUAAGUGUAGAU-3'; in other embodiments, the Cas protein is AsCas12a with an anchoring sequence of 5'-UAAUUUCUACUCUUGUAGAU-3'.

[0015] This invention uses different Cas12 proteins and their corresponding anchoring sequences to detect relevant sites of drug-resistant Mycoplasma pneumoniae, and found that Lb5Cas12a has higher detection sensitivity and accuracy compared with AsCas12a protein.

[0016] Furthermore, the guide sequence is either 5'-ACGGGGUCUUCCCGUCCCGU-3' or 5'-ACGGGGUCUCUCCGUCCCGU-3'.

[0017] Specifically, when detecting the A2063G mutation site, the guide sequence used is 5'-ACGGGGUCUUCCCGUCCCGU-3', and the crRNA sequence is the same as shown in SEQ ID NO.11 of the sequence listing; when detecting the A2064G mutation site, the guide sequence used is 5'-ACGGGGUCUCUCCGUCCCGU-3', and the crRNA sequence is the same as shown in SEQ ID NO.16 of the sequence listing.

[0018] This invention optimizes the guide sequence length for detecting different mutation sites, and finds that the detection efficiency is highest when the guide sequence length is 20 bases. This may be because the RNA fragment provides sufficient binding energy (ΔG) to form a stable RNA / DNA hybrid nucleic acid while maintaining high specificity (reducing off-target effects); and avoids off-target effects and steric hindrance, maximizing cleavage efficiency.

[0019] Furthermore, the amplification primer pair is used to amplify the full length or a fragment of the 23S rRNA, preferably, to amplify any fragment shown in SEQ ID NO. 21 to 23 in the sequence listing.

[0020] Further, the primer pair includes an upstream primer and a downstream primer. The upstream primer is a specially designed modified primer and consists of a template hybridization region and a template extension region. Both the template hybridization region and the template extension region are template complementary sequences. The template hybridization region is a base sequence used for amplification with a length of 25-35 bases. The template extension region is a base sequence extending along the 3' end with a length of 5-20 bases. Its 3' end is blocked by any one of C3Spacer, NH2, phosphorylation, BHQ, dideoxy-terminal bases, and trans bases. In some specific embodiments, C3Spacer blocking is selected. An Abasic-type base site is spaced between the template hybridization region and the template extension region. The Abasic-type base (such as apurinic / apyrimidinic depurinic or depyrimidine bases) can be efficiently and specifically cleaved by Exonuclease III, endonuclease IV, or APE1.

[0021] Preferably, the template hybridization region and the template extension region are separated by one Abasic-type base site; further, the Abasic-type base site is tetrahydrofuran (THF); preferably, the enzyme that cleaves the Abasic-type base site is Exonuclease III; more specifically, the upstream primer is selected from SEQ ID NO. 1 to 4 in the sequence listing.

[0022] Furthermore, the downstream primer is a normal RPA primer; more specifically, the downstream primer is selected from SEQ ID NO. 5 to 8 in the sequence listing.

[0023] Furthermore, the reagent also contains Cas12 protein, preferably LbCas12a protein.

[0024] Furthermore, the reagent also includes a reporter probe.

[0025] It is important to understand that by introducing the reporter probe, the trans-cleavage capability of the Cas12 amplifies the detection signal in the system, thereby improving detection sensitivity. The reporter probe contains a nucleic acid sequence, preferably 5'-TTTTT-3'; the probe can be used for both fluorescence detection and test strip detection.

[0026] Specifically, when the reporter probe is used for fluorescence detection, both ends of the probe are modified with fluorescent groups or quenching groups, respectively. The fluorescent dye is one of SYTO-13, SYTO-82, FAM, FITC, SYBR Green I, SYTO-13, SYTO-82, VIC, HEX, JOE, TAMRA, TET, Cy3, ROX, TEXAS-Red, or Cy5; the quenching group is one of BHQ-0, BHQ-1, BHQ-2, or BHQ-3. In some specific embodiments, the fluorescent reporter probe is 5'-FAM-TTTTT-BHQ1-3' (SEQ ID NO.19).

[0027] Specifically, when the report probe is used for test strip detection, both ends of the probe are modified with fluorescent groups or biotin, and the fluorescent dye is one of SYTO-13, SYTO-82, FAM, FITC, SYBR Green I, SYTO-13, SYTO-82, VIC, HEX, JOE, TAMRA, TET, Cy3, ROX, TEXAS-Red, or Cy5. In some specific embodiments, the test strip report probe is 5'-FAM-TTTTT-Biotin-3' (SEQ ID NO.20).

[0028] The reagent also includes at least one of the following: an isothermal amplification system containing recombinase, Exonuclease III, endonuclease IV, APE1, RNase inhibitor, and dNTPs.

[0029] Preferably, the recombinase-based isothermal amplification system is selected from at least one of enzyme-mediated isothermal amplification (ERA), recombinase polymerase amplification (RPA), recombinase-mediated isothermal amplification (RAA), and multi-enzyme isothermal rapid amplification (MIRA); further, it is an RPA technology amplification system, and the specific amplification system is shown in Table 3.

[0030] It is important to understand that when using the above reagents to detect drug-resistant Mycoplasma pneumoniae, only one mutation site can be detected at a time, and multiple mutation sites require multiple tests. In addition, when detecting different mutation sites, apart from the different crRNAs used, the other reagent components and concentrations are the same.

[0031] In a second aspect, the present invention provides a method for detecting drug resistance genes in Mycoplasma pneumoniae, the method using the reagents described above.

[0032] Furthermore, the method includes the following steps:

[0033] (1) Sample pretreatment: nucleic acid extraction or sample lysis;

[0034] Specifically, a sample release agent is used to release nucleic acid from the sample to be tested, or to extract nucleic acid from the sample to be tested.

[0035] (2) Use the above-mentioned reagents to prepare the detection system;

[0036] Specifically, the detection system comprises the nucleic acid, Cas protein, crRNA, reporter probe, and RNase inhibitor described in step (1). Preferably, the detection system can be used in conjunction with RPA isothermal amplification technology, i.e., the nucleic acid sample is amplified first, and then detected. The isothermal amplification system is a full-component amplification microsphere system, comprising the amplification primer pair and DNA reverse transcriptase. It should be understood that the addition of DNA reverse transcriptase expands the application range of this detection technology, i.e., the detection target can be either DNA or RNA, and also significantly improves the detection sensitivity. Further, the isothermal amplification conditions are 37–45°C, and the reaction time is 10–15 minutes.

[0037] (3) Conduct the detection reaction, obtain and interpret the detection results.

[0038] Specifically, the detection reaction refers to incubation at 37–45°C for 5–10 minutes; the detection results are displayed on a fluorescence thermostat, qPCR instrument, or test strip.

[0039] More specifically, when the fluorescence isothermal detector or qPCR instrument generates a curve that first rises and then enters a plateau phase, the test result is positive; conversely, when it generates a straight line without a clear upward trend, the test result is negative.

[0040] More specifically, the test result is positive when two bands appear on the test strip; conversely, the test result is negative when only one band appears.

[0041] The beneficial effects of this invention include:

[0042] 1. The method for detecting drug resistance gene mutation sites of Mycoplasma pneumoniae based on CRISPR / Cas12 provided by this invention overcomes the shortcomings of molecular detection technologies such as PCR or DNA sequencing, which are cumbersome to operate, expensive to use, have high requirements for experimental sites and personnel, and are difficult to apply to large-scale on-site screening. The amplification and detection steps are both carried out at a constant temperature, so the entire detection process can be completed with only a portable constant temperature fluorescence signal detector, which is very suitable for use in grassroots units and battlefields with limited environmental conditions.

[0043] 2. The detection method provided by this invention integrates the RPA amplification step and the CRISPR / Cas12 detection step into the same reaction tube, and the detection can be completed in only 25 minutes, which greatly shortens the detection time compared with traditional PCR;

[0044] 3. The detection method provided by this invention has outstanding sensitivity. Specifically, it can detect at least 10 copies of the drug-resistant A2063G mutant gene in each test, and the detection limit of the drug-resistant A2064G mutant gene is 5 copies / test.

[0045] 4. The detection results of the method provided by this invention have high accuracy. Specifically, this method has been validated on nucleic acid samples with known drug resistance mutation sites, and the results are consistent with expectations.

[0046] 5. This invention provides a simple, highly sensitive, specific, and rapid method for detecting drug-resistant mutation sites in Mycoplasma pneumoniae, which can be widely used in the future for the rapid detection and screening of drug-resistant mutation sites in patients with Mycoplasma pneumoniae infection in respiratory departments and fever clinics. Attached Figure Description

[0047] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0048] Figure 1 The effect of A2063G crRNA detection on negative templates (wild-type plasmids) is shown in the figure, where the x-axis represents the cycle number and the y-axis represents the detected fluorescence value.

[0049] Figure 2 The effect of A2063G crRNA detection on positive templates (plasmids containing the A2063G mutation site), where the horizontal axis represents the cycle number and the vertical axis represents the detected fluorescence value;

[0050] Figure 3 The effect of A2064G crRNA detection on negative templates (wild-type plasmids) is shown in the figure, where the x-axis represents the cycle number and the y-axis represents the detected fluorescence value.

[0051] Figure 4 The effect of A2064G crRNA detection on positive templates (plasmids containing the A2064G mutation site), where the horizontal axis represents the cycle number and the vertical axis represents the detected fluorescence value;

[0052] Figure 5 The effect of different RPA primers on detecting the drug resistance mutation site A2063G of Mycoplasma pneumoniae is shown in the figure, where the horizontal axis represents the cycle number and the vertical axis represents the detected fluorescence value.

[0053] Figure 6The effect of different RPA primers on detecting the drug resistance mutation site A2064G of Mycoplasma pneumoniae is shown in the figure, where the x-axis represents the cycle number and the y-axis represents the detected fluorescence value.

[0054] Figure 7 Sensitivity test results of RPA-CRISPR / Cas12 method for detecting the drug resistance mutation site A2063G of Mycoplasma pneumoniae. The horizontal axis represents the cycle number and the vertical axis represents the detected fluorescence value.

[0055] Figure 8 The results of RPA-CRISPR / Cas12 assay for the specificity of the drug resistance mutation site A2063G in Mycoplasma pneumoniae are shown in the figure. The x-axis represents the cycle number and the y-axis represents the detected fluorescence value.

[0056] Figure 9 Sensitivity test results of RPA-CRISPR / Cas12 method for detecting the drug resistance mutation site A2064G of Mycoplasma pneumoniae. The horizontal axis represents the cycle number and the vertical axis represents the detected fluorescence value.

[0057] Figure 10 The results of RPA-CRISPR / Cas12 assay for the specificity of the drug resistance mutation site A2064G in Mycoplasma pneumoniae are shown in the figure. The x-axis represents the cycle number and the y-axis represents the detected fluorescence value.

[0058] Figure 11 Results of RPA-CRISPR / Cas12 fluorescence assay for detecting different Mycoplasma pneumoniae nucleic acid samples (A2063G), where the x-axis represents the cycle number and the y-axis represents the detected fluorescence value;

[0059] Figure 12 Results of RPA-CRISPR / Cas12 fluorescence assay for detecting different Mycoplasma pneumoniae nucleic acid samples (A2064G), where the x-axis represents the cycle number and the y-axis represents the detected fluorescence value;

[0060] Figure 13 Results of RPA-CRISPR / Cas12 test strip method for detecting different Mycoplasma pneumoniae nucleic acid samples (A2063G / A2064G); 1-6: Detection results of A2063G mutation site; 7-12: Detection results of A2064G mutation site; 1, 2, 7, 8: Drug-resistant mutant wild-type Mycoplasma pneumoniae nucleic acid samples; 5, 6, 9, 10: Drug-resistant A2063G mutant Mycoplasma pneumoniae nucleic acid samples; 3, 4, 11, 12: Mycoplasma pneumoniae nucleic acid samples with drug-resistant A2064G mutant template. Detailed Implementation

[0061] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims. The following embodiments are intended to further illustrate the invention and are not intended to limit the invention.

[0062] The plasmid DNA of the Mycoplasma pneumoniae A2063G / A2064G drug resistance mutation site gene and the wild-type plasmid DNA used in this invention were synthesized by Nanjing Qingke Biotechnology Co., Ltd., and the Mycoplasma pneumoniae nucleic acid samples of the drug resistance mutation gene were provided by the Institute of Tropical Diseases, Beijing Friendship Hospital. The specific experimental methods and sequence synthesis involved are conventional methods.

[0063] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0064] Example 1: A kit for detecting drug resistance genes in Mycoplasma pneumoniae and its usage method

[0065] This embodiment provides a kit for detecting drug resistance genes in Mycoplasma pneumoniae and its usage method.

[0066] The kit contains the components shown in Table 1, wherein the LbCas12a enzyme and 10×NEB3.1 buffer were purchased from New England Biolabs (NEB), and the RNase inhibitor was purchased from NEB; the crRNA and probe are shown in Table 2, with a fluorescent group (preferably FAM group) modified at the 5' end of the reporter probe and a quenching group (preferably BHQ1 group) or biotin modified at the 3' end; according to the detection site (A2063G / A2064G), the crRNA is preferably A2063G crRNA-3 (SEQ ID NO. 11) or A2064G crRNA-3 (SEQ ID NO. 16).

[0067] Table 1 CRISPR / Cas12 detection system

[0068]

[0069] Table 2. RPA primers, crRNA sequences, and reporter probe sequences targeting the drug resistance mutation sites of Mycoplasma pneumoniae.

[0070]

[0071]

[0072] Note: In the table, the underlined crRNA sequences are anchoring sequences (sequences that bind to the Cas12 protein), and the ununderlined sequences are guide sequences (sequences that match the amplification products of the primer pairs); SEQ ID NO. 1–4 are upstream primers for RPA, SEQ ID NO. 5–8 are downstream primers for RPA, and SEQ ID NO. 9–13 are crRNAs that recognize the A2063G mutation site.

[0073] SEQ ID NO. 19 represents the crRNA that recognizes the A2064G mutation site, and SEQ ID NO. 19 represents the probe in the RPA-Cas12 detection system.

[0074] The method of using the kit (fluorescence method) is as follows: (1) Construct the CRISPR / Cas12 detection system according to Table 1; (2) Perform fluorescence detection using a fluorescence isothermal amplification instrument (Xianda Gene, GS8), with a detection reaction condition of 42℃ and a detection time of 10 minutes; (3) Obtain and interpret the detection results: On the fluorescence detector, if the detection result is a horizontal straight line that tends to flatten (without obvious amplification trend) or the fluorescence signal gain value is <300 during the detection time, it indicates that the detection result is negative, that is, the subject is not infected with A2063G / A2064G drug-resistant Mycoplasma pneumoniae; conversely, if the detection result is a curve that rises first and then reaches a plateau, it means that the detection result is positive, that is, the subject is infected with A2063G / A2064G drug-resistant Mycoplasma pneumoniae.

[0075] Example 2: RPA-CRISPR / Cas12 detection system

[0076] It should be understood that RPA technology can further increase the detection sensitivity of the kit described in Example 1. Therefore, this example provides a procedure for using the Mycoplasma pneumoniae drug resistance gene detection kit (Example 1) in conjunction with RPA technology.

[0077] 2.1 The reaction system (total volume 40 μL, see Table 3 for details) was established using the recombinase polymerase amplification (RPA) method. When the detection site is A2063G or / and A2064G, the upstream primer is preferably RPA-F3 (SEQ ID NO.3) and the downstream primer is preferably RPA-R4 (SEQ ID NO.8).

[0078] Table 3 RPA amplification system

[0079]

[0080]

[0081] 2.2 Construct a CRISPR / Cas12 detection system for use with RPA technology (total system 10 μL, see Table 4 for details). The components in the system are sourced from the same sources as described in Example 1. According to the detection site, the crRNA is preferably A2063G crRNA-3 (SEQ ID NO. 11) or A2064G crRNA-3 (SEQ ID NO. 16).

[0082] Table 4. CRISPR / Cas12 detection system used in conjunction with RPA technology

[0083]

[0084] 2.3 Establishing an RPA-CRISPR / Cas12 detection system

[0085] Add the 40 μL amplification reaction system from step 2.1 to the reaction tube (preferably a PCR tube in this embodiment), and add the 10 μL detection system from step 2.2 to the cap of the reaction tube to complete the construction of the RPA-CRISPR / Cas12 detection system.

[0086] The amplification and detection reactions were both performed at temperatures of 37–45°C (preferably 42°C in this embodiment). The amplification-detection process was conducted in the same reaction tube. First, the 50 μL RPA-CRISPR / Cas12 detection system was incubated in a metal bath or water bath at 37–45°C for 10–15 minutes (preferably 42°C for 15 minutes in this embodiment). The reaction tube was then removed and inverted 7–10 times to mix. Next, the reaction tube was inserted into a real-time PCR instrument (ThermoFisher QuantStudio 3) with a preset temperature of 37–45°C (preferably 42°C in this embodiment) for 5–10 minutes (preferably 10 minutes in this embodiment) to acquire fluorescence signals. The total detection reaction time was 15–25 minutes. The results were interpreted as described in Example 1.

[0087] Example 3: Design of RPA primers and crRNA for detecting drug-resistant mutation sites A2063G and A2064G in Mycoplasma pneumoniae, and construction of drug-resistant mycoplasma standards.

[0088] 3.1 Design of RPA primers and crRNA

[0089] Based on the NCBI website (https: / / www.ncbi.nlm.nih.gov / ) and the location of drug resistance mutation sites in the Mycoplasma pneumoniae 23S RNA gene, a partial region of the 23S RNA gene containing the A2063G and A2064G mutation sites was selected as the target sequence. Based on the primer design principles of recombinase polymerase amplification (RPA) technology, primers were designed to be 28–35 bp in length, with amplified fragments not exceeding 500 bp and GC content between 30% and 60%. Using Peimer Premier 5, RPA primers targeting genes containing A2063G and A2064G mutation sites and crRNAs targeting different mutation sites were designed and sequenced. Four upstream and downstream primers with the lowest probability of secondary structure, hairpin structure, and primer dimer formation were selected as candidate RPA primers. Five crRNA (CRISPR RNA) primers were selected. A fluorescent group (preferably FAM) was modified at the 5' end of the reporter probe, and a quenching group (preferably BHQ1) or biotin was modified at the 3' end (RPA primer, crRNA, and probe sequences are shown in Table 2). All RPA primers, crRNA, and probes were synthesized by General Biotechnology (Anhui) Co., Ltd. The RPA-Cas12 detection system will be used to screen and validate the primers and crRNAs mentioned above.

[0090] 3.2 Construction of plasmid template (standard)

[0091] Based on the drug resistance mutation sites of the Mycoplasma pneumoniae 23S RNA gene, a partial region (SEQ ID NO. 21-22) of the 23S RNA gene containing the A2063G / A2064G mutation sites was selected as the target amplification segment. The above regions were constructed into the vector pUC57 to prepare positive plasmids containing the Mycoplasma pneumoniae drug resistance mutation sites A2063G / A2064G. Similarly, a plasmid containing the wild-type 23S RNA gene fragment (SEQ ID NO. 23) was prepared as a negative control. All three DNA plasmids were synthesized by Nanjing Qingke Biotechnology Co., Ltd., and the selected partial sequences of the 23S RNA gene are shown in Table 5.

[0092] Table 5. Local 23S RNA sequences of different subtypes of Mycoplasma pneumoniae

[0093]

[0094] Note: In the table, the underlined sites are sites 2063 and 2064. The bolded bases in SEQ ID NO. 21-22 are the mutation sites. Specifically, the A2063G mutation refers to the mutation of the "A" base to the "G" base on the 2063rd ribonucleotide of the 23S RNA (SEQ ID NO. 21), and the A2064G mutation refers to the mutation of the "A" base to the "G" base on the 2064th ribonucleotide of the 23S RNA (SEQ ID NO. 22).

[0095] 3.3 Preparation of Standards at Different Concentrations

[0096] The concentrations of the synthesized wild-type Mycoplasma pneumoniae 23S RNA plasmid and the 23S RNA plasmid containing the A2063G / A2064G mutation sites were measured using a NanoDrop micro-spectrophotometer, and then diluted to 10 ng / μL with TE buffer. The full-length of the three plasmids was approximately 3,000 bp, and a rough estimate of 10 ng / μL was approximately 2.5 × 10⁻⁶. 10 Copies / μL, and based on this concentration, perform serial (10×) dilutions to sequentially dilute the three plasmids to 2.5×10⁻⁶. 9 Copies / μL, 2.5 × 10 8 Copies / μL, 2.5 × 10 7 Copies / μL, 2.5 × 10 6 Copies / μL, 2.5 × 10 5 Copies / μL, 2.5 × 10 4 Copies / μL, 2.5 × 10 3 Copies / μL, 2.5 × 10 2 Negative and positive control standards were prepared by diluting the samples to 25 copies / μL and 2.5 copies / μL.

[0097] Example 4: Screening of Cas12 protein

[0098] To improve the detection sensitivity and accuracy of the kit, this embodiment uses different Cas12 proteins (Lb5Cas12a protein and AsCas12a protein) to screen for different mutation sites of Mycoplasma pneumoniae 23S RNA. The crRNA consists of an anchor sequence and a guide sequence. On one hand, different Cas12 proteins use different anchor sequences. Specifically, when the Cas protein is Lb5Cas12a, the anchor sequence is 5'-UAAUUUCUACUAAGUGUAGAU-3'; when the Cas protein is AsCas12a, the anchor sequence is 5'-UAAUUUCUACUCUUGUAGAU-3'. On the other hand, when detecting the A2063G mutation site, the guide sequence used is 5'-ACGGGGUCUUCCCGUCCCGU-3'; when detecting the A2064G mutation site, the guide sequence used is 5'-ACGGGGUCUCUCCGUCCCGU-3'. The remaining operational steps are the same as described in Examples 1 and 8-9. Specific detection results are shown in Table 6.

[0099] Table 6. Effects of different Cas12 proteins on detection sensitivity and accuracy.

[0100]

[0101] The results show that the Lb5Cas12a protein has better sensitivity and accuracy than the AsCas12a protein in detecting the A2063G and A2064G mutation sites, so the Lb5Cas12a protein is preferred.

[0102] Example 5: Screening of crRNA

[0103] This embodiment uses the Lb5Cas12a / Cas12 detection system to screen crRNAs targeting different mutation sites of Mycoplasma pneumoniae 23S RNA, thereby improving the detection efficiency and accuracy of the kit. The specific operating steps are the same as described in Example 1, wherein the synthesized A2063G / A2064G mutant plasmid and wild-type plasmid are the samples to be tested (as described in Example 3), the mutant plasmid is the positive template, and the wild-type plasmid is the negative template, and the sample concentration is 2.5 × 10⁻⁶. 8 Copy / μL (template preparation method as described in Example 3), specific detection results are shown in [link to specific results]. Figures 1-4 .

[0104] On a fluorescence detector, a negative fluorescence signal is required to be a flat horizontal straight line, indicating no obvious amplification trend and thus the specificity of the test result; a positive fluorescence signal is required to be a curve that initially increases linearly before reaching a plateau. Generally, the steeper the slope, the higher the detection efficiency. Figures 1-4It is known that different crRNAs have varying efficiencies and accuracy in detecting negative and positive templates. However, in general, A2063G crRNAs (SEQ ID NO. 9–13) and A2064G crRNAs (SEQ ID NO. 14–18) exhibit high detection specificity and a certain level of detection efficiency. Furthermore, based on the results of negative / positive detection experiments and the aforementioned criteria, the detection efficiency of A2063G crRNA, ranked from highest to lowest, is: 3 (SEQ ID NO. 11) > 2 (SEQ ID NO. 10) > 4 (SEQ ID NO. 12), while for A2064G crRNA it is: 3 (SEQ ID NO. 16) > 1 (SEQ ID NO. 14) > 2 (SEQ ID NO. 15). In conclusion, it can be considered that A2063G crRNA-3 (SEQ ID NO. 11) and A2064G crRNA-3 (SEQ ID NO. 16) have the highest efficiency in detecting the corresponding mutation sites, and therefore these two crRNAs were used for subsequent screening experiments.

[0105] Example 6: Screening of RPA primer pairs for detecting the drug resistance mutation site A2063G in Mycoplasma pneumoniae

[0106] 6.1 Screening of RPA primer pairs using RPA-CRISPR / Cas12 detection method

[0107] Different primer pairs (see Table 7) were formed by combining any upstream primer (SEQ ID NO. 1–4) and any downstream primer (SEQ ID NO. 5–8) to specifically amplify a specific region of Mycoplasma pneumoniae 23S RNA using RPA, along with A2063GcrRNA-3 (SEQ ID NO. 11), and added to the RPA-CRISPR / Cas12 detection system constructed in Example 2. The fluorescent reporter probe / test strip reporter probe was used as described above, and the concentration was increased to 2.5 × 10⁻⁶. 3 A plasmid containing the A2063G mutation site was used as a nucleic acid template at a concentration of 1 copy / μL for primer screening experiments.

[0108] Table 7. RPA primer pairs used to detect the drug resistance mutation sites A2063G / A2064G in Mycoplasma pneumoniae.

[0109]

[0110]

[0111] 6.2 Detection reaction

[0112] The amount of nucleic acid template in the RPA-CRISPR / Cas12 fluorescence detection system was 4 μL. The reaction temperature of the real-time PCR instrument (ThermoFisher QuantStudio 3) was 42℃. The detection time was 6 minutes, and fluorescence was collected every 10 seconds. The remaining amplification and detection steps were the same as those described in Example 2.

[0113] 6.3 Screening Results

[0114] The fluorescence signal of the positive sample increases linearly with a certain slope. As the reaction time is delayed, it eventually reaches a plateau. The higher the slope of the fluorescence signal growth, the higher the detection efficiency of A2063G crRNA-3 and the corresponding primer pair combination under the same template concentration. Therefore, primer pairs with a high slope of fluorescence signal growth are preferred.

[0115] Based on the above principles, from Figure 5 As can be seen from the fluorescence curves, the fluorescence signal growth rate of primer pair S12 (RPA-F3 (SEQ ID NO.3) and RPA-R4 (SEQ ID NO.8)) for detecting the A2063G site was significantly higher than that of other primer combinations, i.e., the fluorescence growth slope was the highest. This was followed by primer pair S1 (RPA-F1 (SEQ ID NO.1) and RPA-R1 (SEQ ID NO.5)). Therefore, primer pair S12 was preferentially chosen for detecting the A2063G mutant Mycoplasma pneumoniae. Example 7: Screening of RPA primer pairs for detecting the drug-resistant mutation site A2064G in Mycoplasma pneumoniae.

[0116] This embodiment screened RPA primer pairs for detecting the A2064G mutation site in Mycoplasma pneumoniae. The primer combinations are as shown in Table 7. In this embodiment, except for using A2064G crRNA-3 (SEQ ID NO.16) and a plasmid containing the A2064G mutation site as nucleic acid templates, the remaining detection steps are the same as described in Example 6. The detection results are shown in Table 7. Figure 6 .

[0117] from Figure 6The fluorescence curves show that the S12 primer pair (RPA-F3 (SEQ ID NO.3) and RPA-R4 (SEQ ID NO.8)) exhibited a significantly higher fluorescence signal growth rate than other primer pairs for detecting the A2064G mutation site, meaning it had the highest fluorescence growth slope. This was followed by the S6 primer pair (RPA-F2 (SEQ ID NO.2) and RPA-R2 (SEQ ID NO.6)). Based on the results of Example 6, the S12 primer pair can be used for the detection of both the A2063G and A2064G mutation sites in Mycoplasma pneumoniae, and it demonstrates the highest detection efficiency. It is noteworthy that although the A2063G and A2064G sites are very close together, the detection sensitivity of the same primer pair for these two sites is not identical. Only the S12 primer pair can simultaneously detect both the A2063G and A2064G sites with the highest sensitivity.

[0118] Example 8: Sensitivity and specificity test of RPA-CRISPR / Cas12 method for detecting drug resistance mutation site A2063G in Mycoplasma pneumoniae

[0119] The sensitivity and specificity of the A2063G mutation site were tested based on the reaction system of Example 2, the 2063G crRNA-3 (SEQ ID NO. 11) and primer pair S12 (SEQ ID NO. 3 and SEQ ID NO. 8) screened in Examples 5 and 6, respectively.

[0120] 8.1 Sensitivity Test

[0121] The positive templates were of different concentrations (5×10). 2 Plasmids containing the A2063G mutation site (copies / μL, 25 copies / μL, 2.5 copies / μL) were used (as described in Example 1), with water as a negative control. The sensitivity of the detection system was tested using the RPA-CRISPR / Cas12 method. The detection system and experimental procedure were the same as described in Example 3, except that the loading volume of the same concentration of plasmid was set to 2 / 4 μL. The detection results of different positive sample volumes on the fluorescence isothermal amplification instrument (SindaGene, GS8) are as follows: Figure 7 As shown.

[0122] Based on the judgment principle described in Example 1, it can be concluded that the sensitivity of the RPA-CRISPR / Cas12 method for detecting the drug resistance mutation site A2063G of Mycoplasma pneumoniae based on the primer pair S12-A2063GcrRNA-3 combination reaches 10 copies / test.

[0123] 8.2 Specificity Test

[0124] 2.5×10 5Wild-type plasmid (as described in Example 1) copies / μL, 2.5 × 10 5 The plasmid containing the A2064G mutation site (as described in Example 1), 75 ng / μL of human genomic DNA (Beina Biotechnology, BNCC372384), and 120 ng / μL of E. coli nucleic acid were used as detection targets, and 2.5 × 10⁻⁶ μL of plasmid containing the A2064G mutation site were used as detection targets. 2 A plasmid containing the A2063G mutation site (as described in Example 1) was used as a positive control. Cross-reactivity was tested using the RPA-CRISPR / Cas12 method. The detection system and experimental procedures were the same as described in Example 3. The loading volume of each nucleic acid template was 2 μL. The results of different templates were detected using a fluorescence isothermal amplification instrument (Syndagene, GS8). Figure 8 As shown.

[0125] Based on the judgment principle described in Example 1, it can be concluded that in the RPA-CRISPR / Cas12 system of S12 primer pair -A2063G crRNA-3, the test result is positive only when the target sample is a sample containing the Mycoplasma pneumoniae drug resistance mutation site A2063G, while the other samples do not have cross-reaction, indicating that the detection specificity of the system is high and there will be no false positive results.

[0126] Example 9: Sensitivity and specificity test of RPA-CRISPR / Cas12 method for detecting the drug resistance mutation site A2064G in Mycoplasma pneumoniae

[0127] The sensitivity and specificity of the A2064G mutation site were tested based on the reaction system of Example 2, the 2064G crRNA-3 (SEQ ID NO. 16) and primer pair S12 (SEQ ID NO. 3 and SEQ ID NO. 8) screened in Examples 5 and 7, respectively.

[0128] The specific operation is the same as described in Example 8, and the test results are as follows: Figures 9-10 .

[0129] Based on the principles of the judgment results described in Example 1, it can be concluded that the sensitivity of the RPA-CRISPR / Cas12 method for detecting the drug resistance mutation site A2064G in Mycoplasma pneumoniae based on the S12 primer pair-A2064GcrRNA-3 combination reaches 5 copies / test. Figure 9 Furthermore, this system has high detection specificity and will not produce false positive results. Figure 10 ).

[0130] Example 10: Sample Validation by Fluorescence Method

[0131] This embodiment utilizes an RPA-CRISPR / Cas12 detection system (fluorescence method) based on the S12 primer pair -A2063G crRNA-3 / A2064G crRNA-3 combination to validate the samples. The samples included three types of confirmed Mycoplasma pneumoniae nucleic acid samples (drug-resistant mutant wild-type Mycoplasma pneumoniae nucleic acid, drug-resistant A2063G / A2064G mutant Mycoplasma pneumoniae nucleic acid samples) provided by the Tropical Diseases Research Institute of Beijing Friendship Hospital. The detection system and steps were the same as described in Example 2, except that the sample loading volume was 4 μL, and the detection instrument was a fluorescence isothermal amplification instrument (SindaGene, GS8). The detection results are as follows: Figures 11-12 As shown.

[0132] from Figure 11 and Figure 12 It can be seen that the RPA-CRISPR / Cas12 detection system can only detect samples containing sites targeted by crRNA (A2063G crRNA-3 / A2064G crRNA-3). Specifically, in the RPA-CRISPR / Cas12 system with the S12 primer pair -A2063G crRNA-3 / 2064G crRNA-3 combination, only samples of drug-resistant A2063G / A2064G mutant Mycoplasma pneumoniae nucleic acid are positive, further demonstrating the detection accuracy of the kit provided by this invention.

[0133] Example 11: Sample Validation of the Test Strip Method

[0134] This embodiment utilizes the RPA-CRISPR / Cas12 test strip detection method to detect the three nucleic acid samples described in Example 10. The test strip is a lateral flow assay strip (CRISPR) (Syndagene, TS104), and the detection system is the same as described in Example 2. The operation procedure of the test strip method is as follows: Take the supernatant of the sample to be tested for detection; dilute the sample with sample diluent, the dilution factor being determined according to the concentration of the target analyte; take out the test strip and place it on a horizontal table (use as soon as possible), being careful not to touch the NC membrane; use a pipette or dropper to slowly add 30-60 μL of the sample to be tested dropwise onto the sample area of ​​the test strip; let it stand for 7-10 minutes to interpret the results; after use, put the amplification product and the test strip into a sealed bag and dispose of them properly. The detection results are shown in […]. Figure 13 .

[0135] When two bands appear on the test strip, the test result is positive; when only one band appears, the result is negative. The test results of the strip method are consistent with those of the fluorescence method (Example 10), that is, the RPA-CRISPR / Cas12 detection system can only detect samples containing crRNA (A2063G crRNA-3 / A2064G crRNA-3) targeting sites, and the detection of other samples is negative, further confirming the accuracy and reliability of the test results of the system.

[0136] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or basic characteristics. Therefore, the embodiments should be considered exemplary and non-limiting in all respects. The scope of the invention is defined by the appended claims rather than the foregoing description. Therefore, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention, and no reference numerals in the claims should be construed as limiting the scope of the claims.

Claims

1. A reagent for detecting drug resistance mutation sites in Mycoplasma pneumoniae 23S rRNA, characterized in that, The reagent comprises crRNA and / or an amplification primer pair; the crRNA comprises an anchor sequence and a guide sequence, the anchor sequence binding to a Cas12 protein, the Cas12 protein being selected from at least one of Cas12a, Cas12b, Cas12f, and Cas12j; the guide sequence matches the sequence amplified by the primer pair, the guide sequence being either 5'-ACGGGGUCUUCCCGUCCCGU-3' or 5'-ACGGGGUCUCUCCGUCCCGU-3'.

2. The reagent as described in claim 1, characterized in that, The mutation sites are A2063G and / or A2064G.

3. The reagent as described in claim 2, characterized in that, When detecting the A2063G mutation site, the guide sequence used is 5'-ACGGGGUCUUCCCGUCCCGU-3'; when detecting the A2064G mutation site, the guide sequence used is 5'-ACGGGGUCUCUCCGUCCCGU-3'.

4. The reagent according to claim 1, wherein the Cas12 protein is Lb5Cas12a with an anchoring sequence of 5'-UAAUUUCUACUAAGUGUAGAU-3'; or the Cas protein is AsCas12a with an anchoring sequence of 5'-UAAUUUCUACUCUUGUAGAU-3'.

5. The reagent as described in claim 1, characterized in that, The primer pair includes an upstream primer and a downstream primer, wherein the upstream primer is selected from SEQ ID NO.1 to 4 in the sequence listing, and the downstream primer is selected from SEQ ID NO.5 to 8 in the sequence listing.

6. The reagent as described in claim 5, characterized in that, The upstream primer contains the sequence shown in SEQ ID NO.3 of the sequence list, and the downstream primer contains the sequence shown in SEQ ID NO.8 of the sequence list.

7. The reagent as described in claim 1, characterized in that, The reagent also includes a reporter probe and the Cas12 protein; the reporter probe is modified with a fluorescent group at its 5' end and with a quencher group or biotin at its 3' end.

8. The reagent as described in claim 1, characterized in that, The reagent also includes at least one of the following: an isothermal amplification system containing recombinase, Exonuclease III, endonuclease IV, APE1, RNase inhibitor, and dNTPs.

9. A method for detecting drug resistance genes in Mycoplasma pneumoniae, characterized in that, The detection was performed using the reagents described in any one of claims 1 to 8.

10. The method as described in claim 9, characterized in that, The method includes the following steps: (1) Sample pretreatment: nucleic acid extraction or sample lysis; (2) The detection system is prepared using the reagents described in any one of claims 1 to 8; (3) Conduct the detection reaction, obtain and interpret the detection results.

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