Kit for detecting drug-resistant mutation of mycoplasma pneumoniae based on RPA-CRISPR and application of kit

Abstract

The invention provides a kit for detecting drug-resistant mutation of mycoplasma pneumoniae based on RPA-CRISPR and application of the kit. The invention provides an RPA primer pair and a crRNA sequence for detecting the drug-resistant mutation A2063G site of the mycoplasma pneumonia 23s rRNA gene, an RPA-CRISPR / Cas13a detection system is established, the detection system formed by the primer pair and the crRNA can accurately detect the drug-resistant mutation A2063G site of the mycoplasma pneumonia 23s rRNA gene within 30-40 minutes under an isothermal condition, the sensitivity and the specificity are very high, and the detection system can be used for detecting the drug-resistant mutation A2063G site of the mycoplasma pneumonia 23s rRNA gene. Amplification detection can be completed only by performing isothermal amplification at the temperature of 39 DEG C without using a variable-temperature instrument like PCR (Polymerase Chain Reaction); in addition, a lateral flow immunochromatography test strip is further combined, so that the on-site instant detection of the mycoplasma pneumoniae drug-resistant mutation can be realized.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a kit for detecting drug-resistant mutations of Mycoplasma pneumoniae based on RPA-CRISPR and its application. Background Art

[0002] Mycoplasma pneumoniae (MP) is a respiratory tract infection that can be transmitted through droplets or aerosols. Children and adolescents are the primary susceptible population for M. pneumoniae, making it a common pathogen causing community-acquired pneumonia in children. Symptoms of M. pneumoniae in children are complex and diverse, including atelectasis, pleural effusions, and, in severe cases, acute respiratory failure, necrotizing pneumonia, and other life-threatening conditions. M. pneumoniae is a cell-wall-less microorganism. Commonly used clinical treatments include tetracyclines, quinolones, and macrolides. However, due to the potential impact of quinolones and tetracyclines on children's development, macrolides have become the preferred treatment for children with M. pneumoniae. In recent years, the incidence of M. pneumoniae infection has continued to increase, and drug resistance has become an increasingly serious issue. The prevalence of macrolide-resistant M. pneumoniae infections has rapidly increased worldwide, contributing to severe and / or difficult-to-treat cases, complications, and sequelae of M. pneumoniae infection. The binding site for macrolide drugs is located in region V of the 23S RNA domain of the Mycoplasma pneumoniae ribosomal structure. Nucleotide mutations in this region can impair drug binding to the ribosome and confer drug resistance. Currently, the main mutation sites identified include 2063, 2064, 2067, and 2617. Among them, the A2063G mutation has the highest frequency and is associated with high drug resistance. Therefore, establishing a simple, rapid, and accurate method for detecting the A2063G mutation in Mycoplasma pneumoniae is of great significance for the early differential diagnosis of Mycoplasma pneumoniae infection and guiding clinical treatment.

[0003] Common laboratory diagnostic methods for Mycoplasma pneumoniae include culture, serological testing, and nucleic acid detection. Culture is time-consuming and costly, making it insufficient for current clinical applications. Serological testing is widely used due to its simplicity. However, due to the base composition of the Mycoplasma pneumoniae genome, the preparation of highly specific antibodies is difficult, which compromises the sensitivity and specificity of this method. PCR nucleic acid detection offers high sensitivity and specificity, but requires specialized equipment and trained operators, limiting its application in primary care laboratories and point-of-care testing. The development of targeted high-throughput sequencing technology allows for more accurate detection of Mycoplasma pneumoniae, but the costly equipment and cumbersome operation significantly limit its clinical application.

[0004] In recent years, the application of isothermal amplification technology combined with CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR) gene editing technology in pathogen diagnosis has developed rapidly. It can quickly and efficiently amplify nucleic acids at a constant temperature, has low equipment requirements and is simple to operate, giving it significant advantages in pathogen diagnosis, especially on-site instant detection. Summary of the Invention

[0005] In view of the shortcomings of the prior art described above, the object of the present invention is to provide a primer pair, crRNA and kit for detecting the A2063G mutation of the 23srRNA gene of Mycoplasma pneumoniae, which are used to solve the problems in the prior art.

[0006] To achieve the above-mentioned purpose and other related purposes, the present invention is achieved through the following technical solutions.

[0007] In a first aspect of the present invention, a reagent for detecting the A2063G mutation in the 23s rRNA gene of Mycoplasma pneumoniae is provided. The reagent comprises an amplification reagent, the amplification reagent comprising a primer for detecting the A2063G mutation in the 23s rRNA gene of Mycoplasma pneumoniae, and the nucleotide sequence of the primer is as follows:

[0008] F1:GAAATTAATACGACTCACTATAGGGTCTCTTGACTGTCTCGGCTATAGACTCG GTGAAATC;

[0009] R3: TCCTACCTATTCTCTACATGATAATGTCCTG.

[0010] In some embodiments of the present invention, the reagent further includes a CRISPR-Cas detection reagent, which includes a crRNA for detecting the A2063G mutation in the 23s rRNA gene of Mycoplasma pneumoniae, wherein the DNA template nucleic acid sequence of the crRNA is: GATTTAGACTACCCCAAAAACGAAGGGGACTAAAACGGTCTTCCGGT CCCGTTGCGCCTAACGG; the DNA template sequence is used to transcribe and generate crRNA.

[0011] In some embodiments of the present invention, the CRISPR-Cas detection reagent further comprises a Cas protein and a nucleic acid probe.

[0012] In the reagent, "Cas protein" refers to the abbreviation of CRISPR-associated protein, which is a DNA nuclease guided by RNA (such as CRISPR RNA); clustered regularly interspaced short palindromic repeats (CRISPR) and associated proteins (Cas) constitute the CRISPR-Cas system, which is an anti-phage immune system present in many bacteria and most archaea. In some embodiments of the present invention, the Cas protein is selected from Cas12a and / or Cas13a, for example, selected from one or more of AsCas12a, BbCas12a, BoCas12a, FnCas12a, HkCas12a, Lb4Cas12a, Lb5Cas12a, LbCas12a, OsCas12a, TsCas12a, LwaCas13a, LbaCas13a, CamCas13a, LbuCas13a, LshCas13a, RcaCas13a, HheCas13a, PprCas13a, LseCas13a, LbmCas13a or LbnCas13a.

[0013] In some embodiments of the present invention, the nucleic acid probe can be cleaved by its corresponding Cas protease and generate a signal through a signal generating molecule.

[0014] In the reagent, "probe" refers to a nucleotide sequence that can be cleaved by the CRISPR-Cas system and can generate a detectable light signal after cleavage, which can serve as a reporter molecule. It should be noted that since the trans-cleavage activity of the Cas protein is activated and can cleave any nucleotide sequence, the nucleotide sequence of the probe is not particularly limited.

[0015] In some embodiments of the present invention, the signal generating molecule is selected from the group consisting of: one or more of a fluorescent molecule, a radioisotope, a chromophore, an enzyme, an enzyme substrate, a chemiluminescent moiety, a bioluminescent moiety, a non-metallic isotope, a paramagnetic metal ion, or a ferromagnetic metal;

[0016] In some embodiments of the present invention, the fluorescent molecule can be selected from fluorescein dyes, rhodamine dyes and cyanine dyes.

[0017] In some embodiments of the present invention, the fluorescent molecule is selected from AMCA, Pacific Blue, Atto 425, BODI PYFL, FAM, Alexa Fluor 488, TET, JOE, Yakima Yellow, VIC, HEX, Quasar 570, Cy3, NED, TAMRA, ROX, AquaPhluor593, Texas Red, Atto 590, Cy5, Quasar 670, Cy5.5 and Cy5.5.

[0018] In some embodiments of the present invention, the nucleic acid probe further comprises a signal regulating molecule; wherein the signal generating molecule and the signal regulating molecule are located at both ends of the nucleic acid probe.

[0019] In some preferred embodiments of the present invention, the signal regulating molecule preferably includes a quencher molecule or biotin.

[0020] In some embodiments of the present invention, the quencher molecule is at least one of BHQ1, BHQ2, BHQ3, Dabcyl, Eclipse and MGB.

[0021] In some embodiments of the present invention, the kit further comprises reagents for RPA amplification, such as amplification reaction buffer, RPA reaction enzyme (enzyme components include recombinase, single-strand binding protein, strand displacement DNA polymerase), Mg 2+ .

[0022] In some embodiments of the present invention, the reagent further comprises at least one of a buffer suitable for the Cas nuclease cleavage reaction system, an RNase Inhibitor, dNTPs / rNTPs, and an RNA polymerase;

[0023] In some embodiments of the invention, the RNA polymerase comprises T7 RNA polymerase.

[0024] In some embodiments of the present invention, the reagent further comprises a nucleic acid extraction reagent.

[0025] The second aspect of the present invention provides a kit, which comprises the reagent described in the first aspect of the present invention.

[0026] In some embodiments of the present invention, the kit further comprises a substance for detecting the signal generating molecule, and the substance comprises a lateral flow test strip or a fluorescent detection substance.

[0027] In some embodiments of the present invention, the kit is used to detect the A2063G mutation in the 23s rRNA gene of Mycoplasma pneumoniae.

[0028] In some embodiments of the invention, the detecting comprises the following steps:

[0029] S1. Extracting nucleic acid from the sample to be tested;

[0030] S2, amplifying the nucleic acid in the sample to be tested using an amplification reagent to obtain an amplification product;

[0031] S3, adding the product from step S2 to the CRISPR-Cas detection reagent to perform a CRISPR reaction;

[0032] S4. Use fluorescence detection method or nucleic acid detection test strips to display the test results.

[0033] In some embodiments of the present invention, the nucleic acid detection test strip comprises a lateral flow test strip.

[0034] In some embodiments of the present invention, the sample includes but is not limited to one or more of saliva, serum, plasma, blood, urine, alveolar lavage fluid, tongue swab, throat swab, nasal swab, and cerebrospinal fluid.

[0035] In some embodiments of the present invention, the amplification temperature in step S2 is 35-42°C.

[0036] In some embodiments of the present invention, the amplification time in step S2 is 5 to 30 minutes.

[0037] In some embodiments of the present invention, the detection temperature in step S3 is 35-42°C.

[0038] In some embodiments of the present invention, the temperature is detected in step S3 for 20 to 60 minutes.

[0039] In some embodiments of the present invention, the final concentration of the upstream primer or the downstream primer in the amplification reaction system is 0.2-0.5 μM.

[0040] In some embodiments of the present invention, the final concentration of the crRNA in the CRISPR reaction system is 0.5 to 1 ng / μl.

[0041] In some embodiments of the present invention, the final concentration of the nucleic acid probe in the CRISPR reaction system is 0.2 to 1 μM.

[0042] In some embodiments of the present invention, the final concentration of Cas13a in the CRISPR reaction system is 3 to 6 ng / μl.

[0043] When using fluorescence to detect signal-generating molecules, fluorescence detection can be performed on a microplate reader. When the A2063G mutation in the 23s rRNA gene of Mycoplasma pneumoniae is present in the CRISPR / Cas detection system, the endonuclease activity of the CRISPR / Cas is activated under the mediation of specific crRNA. The activated CRISPR / Cas cleaves the probe labeled with a fluorophore and a quencher, causing the fluorophore to release fluorescence, which is detected by the microplate reader at a high intensity.

[0044] When the lateral flow test strip detects signal-generating molecules, the control line and test line on the test strip have streptavidin and secondary antibodies, respectively, and the colloidal gold conjugate pad adjacent to the sample addition area has a colloidal gold-labeled anti-FAM antibody. Because the nucleic acid probe is modified with biotin and FAM groups, when the sample to be tested after CRISPR / Cas cleavage is added to the lateral flow test strip, the signal-generating molecule will bind to the gold-labeled antibody, and the complex will move from the control line to the test line in the direction of liquid flow. When the target to be tested is not present in the sample, the streptavidin at the control line saturates and captures the biotin-labeled nucleic acid molecule, and a control line band is displayed. When the Mycoplasma pneumoniae A2063G mutation site is present in the sample, the nucleic acid probe labeled with FAM and biotin will be cleaved and separated, so that the FAM-labeled nucleic acid probe will be captured by the secondary antibody on the test line and develop color.

[0045] If both the test line (T line) and the quality control line (C line) on the test strip are colored, it indicates that the test result is positive and the test is valid; if the T line does not show color and the C line shows color, it indicates that the test result is negative and the test is valid.

[0046] The third aspect of the present invention provides the use of the above reagent or kit in any of the following:

[0047] (1) preparing a product for identifying drug resistance of Mycoplasma pneumoniae;

[0048] (II) preparing products for identifying drug-resistant mutations in Mycoplasma pneumoniae;

[0049] Among them, the drug-resistant mutation of Mycoplasma pneumoniae is the A2063G mutation of the 23s rRNA gene of Mycoplasma pneumoniae.

[0050] Beneficial effects:

[0051] The present invention proposes an RPA primer pair and crRNA sequence for detecting the A2063G site of the Mycoplasma pneumoniae 23s rRNA gene resistance mutation based on the RPA-CRISPR / Cas13a technology, and establishes an RPA-CRISPR / Cas13a detection system. The primer pair of the present invention cooperates with the detection system composed of crRNA to accurately detect the A2063G site of the Mycoplasma pneumoniae 23s rRNA gene resistance mutation only under isothermal conditions within 30-40 minutes. It has high sensitivity and specificity and does not require a temperature-variable instrument like PCR to achieve amplification detection. It only needs to be isothermal amplified at 39 ° C to complete the detection. Further combined with lateral flow immunochromatographic test strips, on-site real-time detection of Mycoplasma pneumoniae resistance mutations can be achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 : Electrophoresis gel image of RPA primer negative sample.

[0053] Figure 2 : Electrophoresis gel image of RPA primer amplification sensitivity analysis. M: Marker, 1: Negative control, 2: 1000 copies / μl, 3: 10,000 copies / μl, 4: 100,000 copies / μl.

[0054] Figure 3 : Fluorescence intensity diagram of crRNA screening detection.

[0055] Figure 4 : Fluorescence intensity graphs of the sensitivity (A) and specificity (B) of RPA-CRISPR / Cas13a in detecting drug-resistant mutations of Mycoplasma pneumoniae.

[0056] Figure 5 : Schematic diagram of the detection principle of RPA-CRISPR / Cas13a system combined with lateral flow test strips.

[0057] Figure 6 : Fluorescence intensity diagram of Mycoplasma pneumoniae clinical samples detected by RPA-CRISPR / Cas13a mutation detection system.

[0058] Figure 7 Figure 2: Lateral flow immunochromatographic strip image of Mycoplasma pneumoniae clinical samples using the RPA-CRISPR / Cas13a mutation detection system. NTC: negative control, 1-3: wild-type samples, 4-10: mutant samples. DETAILED DESCRIPTION

[0059] The following describes the implementation of the present invention through specific embodiments. People skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0060] Before further describing the specific embodiments of the present invention, it should be understood that the scope of the present invention is not limited to the specific embodiments described below. It should also be understood that the terminology used in the examples is intended to describe specific embodiments and is not intended to limit the scope of the present invention. The experimental procedures in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or according to the conditions recommended by the respective manufacturers.

[0061] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

[0062] Sources of experimental materials used in the present invention: Cas13a protein (LwaCas13a) was purchased from Shanghai Huicheng Biotechnology Co., Ltd., model E-003; RPA amplification kit was purchased from TwistDx, plasmid extraction kit was purchased from Takara, T7 high-yield RNA synthesis kit and Monarch RNA purification kit were purchased from New England Biolabs, colloidal gold universal detection kit Milenia GenLine HybriDetect was purchased from Milenia Biotec, Mycoplasma genome extraction kit QIAamp UCP Pathogen Mini Kit was purchased from Qiagen, and clinical samples were throat swab samples from children with positive Mycoplasma pneumoniae.

[0063] Example 1

[0064] (1) Preparation of Mycoplasma pneumoniae 23s wild-type and mutant plasmid standards

[0065] Partial wild-type and mutant nucleic acid fragment sequences at position 2063 of the 23S rRNA gene of Mycoplasma pneumoniae (shown in SEQ ID NO. 4 and SEQ ID NO. 5) were synthesized by Sangon Biotech (Shanghai) Co., Ltd. and cloned into the pUC57 plasmid, conferring ampicillin resistance. The plasmid was transformed into the competent DH5α strain and inoculated at a 1% inoculum into LB medium containing 100 μg / mL ampicillin. The cells were cultured overnight at 37°C and 220 rpm in a shaking incubator. Plasmids were extracted using a Takara plasmid miniprep kit, and the concentration was measured to calculate the copy number and perform serial dilutions.

[0066] The wild-type partial sequence of the Mycoplasma pneumoniae 23s rRNA gene is shown in SEQ ID NO.1:

[0067] TATGCCAAACCGTAAGGTGATGTATATGGGGTGACACCTGCCCAGTGCTGGAAGGTTAAAGAAGGAGGTTAGCGCAAGCGAAGCTTTTAACTGAAGCCCCAGTGAACGGCGGCCGTAACTATAACGGTCCTAAGGTAGCGAAATTCCTAGTCGGGTAAATTCCGTCCCGCTTGAATGGTGTAACCATCTCTTGACTGTCTCGGCTATAGACTCGGTGAAATCCAGGTACGGGTGAAGACACCCGT TAGGCGCAACGGGACGGAAAGACCCCGTGAAGCTTTACTGTAGCTTAATATTGATCAGGACATTATCATGTAGAGAATAGGTAGGAGCAATCGATGCAAGTTCGCTAGGACTTGTTGATGCGAAAGGTGGAATACTACCCTTGGTTGTGTGCTGTTCTAATTGGTAACTGTTATCCAGTTTCAAGACAGTGTTAGGTGGGCAGTTTGACTGGGGCGGTCGCCTCCTAAAAGGTAACGGAGGCGTA CAAAGGTACC;

[0068] The mutant partial sequence of the Mycoplasma pneumoniae 23s rRNA gene is shown in SEQ ID NO.2:

[0069] TATGCCAAACCGTAAGGTGATGTATATGGGGTGACACCTGCCCAGTGCTGGAAGGTTAAAGAAGGAGGTTAGCGCAAGCGAAGCTTTTAACTGAAGCCCCAGTGAACGGCGGCCGTAACTATAAC GGTCCTAAGGTAGCGAAATTCCTAGTCGGGTAAATTCCGTCCCGCTTGAATGGTGTAACCATCTCTTGACTGTCTCGGCTATAGACTCGGTGAAATCCAGGTACGGGTGAAGACACCCGTTAGGC GCAACGGGACGGGAAGACCCCGTGAAGCTTTACTGTAGCTTAATATTGATCAGGACATTATCATGTAGAGAATAGGTAGGAGCAATCGATGCAAGTTCGCTAGGACTTGTTGATGCGAAAGGTGGAATACTACCCTTGGTTGTGTGCTGTTCTAATTGGTAACTGTTATCCAGTTTCAAGACAGTGTTAGGTGGGCAGTTTGACTGGGGCGGTCGCCTCCTAAAAGGTAACGGAGGCGTACAAAGGTACC.

[0070] (2) The wild-type and mutant plasmid standards of Mycoplasma pneumoniae 23s rRNA gene fragments were diluted in series to obtain 1.0x10 1 Copies / μL, 1.0x10 2 Copies / μL, 1.0x10 3 Copies / μL Mycoplasma pneumoniae 23s rRNA gene fragment wild-type plasmid standard and 23s rRNA gene fragment mutant plasmid standard.

[0071] (3) Design and screening of primers for RPA amplification

[0072] According to the RPA primer design principles, multiple RPA amplification primers for the 2063 site fragment of the Mycoplasma pneumoniae 23s rRNA gene were designed using the NCBI Primer-BLAST primer website and Oligo software and synthesized by Nanjing GenScript Biotechnology Co., Ltd.

[0073] The prepared positive plasmid was diluted and used as a template for RPA amplification according to the RPA amplification kit instructions. To an RPA powder tube (containing recombinase, polymerase, and single-stranded binding protein), 50 μL of the amplification system was added: 29.5 μL of RPA amplification reaction buffer, 2.4 μL of each upstream and downstream primer at a 10 μM concentration, 2 μL of different dilutions of a standard Mycoplasma pneumoniae mutant plasmid template, and 11.2 μL of RNase-free water. 2.5 μL of 280 mM magnesium acetate was added and immediately placed on a metal bath. The reaction temperature was set at 39°C and the reaction time was 30 minutes. The RPA amplification product was purified by sodium acetate precipitation. The purified DNA product was subjected to agarose gel electrophoresis to compare the amplification efficiency of the primer pairs.

[0074] The RPA primers were screened twice. The first time, the amplification primers designed according to the software showed different degrees of non-specific amplification bands. A primer pair with fewer non-specific bands and obvious target bands was selected. The upstream sequence of the primer pair remained unchanged, and the downstream sequence was shifted forward or backward by 5-10 bases. After resynthesis, they were paired with each other and amplified and screened again. According to the gel electrophoresis results, three pairs of amplification bands with relatively single sequences were obtained. Among them, the upstream primer (F1) not only contained a sequence that matched the target fragment, but also contained a sequence recognized by T7 RNA polymerase (underlined). The specific sequence is shown in Table 1; and the primers were amplified as a negative control (clear water control). The results are shown in Table 1. Figure 1 , it can be seen that the three pairs of primers do not involve nonspecific amplification; then the amplification sensitivity of the primers was analyzed, and the results are shown in Figure 2 ,from Figure 2 It can be seen that the amplification effect of primer pair F1R3 is better than the other two pairs. -6 The plasmid could amplify the target band; therefore, the F1R3 primer pair was selected for subsequent experiments.

[0075] Table 1

[0076]

[0077] Example 2

[0078] (1) Design crRNA DNA template sequence and in vitro transcription synthesis

[0079] According to the RPA primer amplification fragment sequence screened out, based on the feature of Cas13a protein without PAM sequence restriction, it is conducive to design crRNA in 23s rRNA gene A2063G mutation site.According to existing reports, Cas13a activity is affected by binding target sequence and mutation type, and some sequence single mispairings may only cause Cas13a partial activity to decline, and two or more mispairings can significantly suppress the activity of Cas13a.When designing crRNA sequence, the present invention uses A2063G mutation site as origin, and is respectively separated by 0-5 bases and artificially adds another mutant base, and the DNA template sequence of each crRNA is shown in Table 2.

[0080] Table 2

[0081]

[0082] A single-stranded DNA template containing a complementary crRNA and a T7 promoter was synthesized and annealed to form a double-stranded DNA template. The annealing reaction was performed as follows: 1 μL of each 100 μM crRNA DNA template, 1 μL of 10× Taq buffer, and 7 μL of DNase- / RNase-free water were added to a PCR tube. The reaction was then incubated in a PCR amplifier at 95°C for 10 minutes, followed by a temperature ramp of 0.1°C every 8 seconds until room temperature. Transcription was then performed overnight at 37°C using the annealed product as a template according to the HiScribe T7 High-Yield RNA Synthesis Kit instructions. The transcript was digested with DNase I for 30 minutes and purified using the Monarch RNA Purification Kit. The crRNA concentration was then determined.

[0083] (2) Preparation of RPA-CRISPR / Cas13a detection system for crRNA screening

[0084] After research and optimization, the 20μL reaction system of RPA-CRISPR / Cas13a for detecting Mycoplasma pneumoniae was established as follows: 29.5μL RPA amplification reaction buffer, 1.6μL of upstream and downstream primers at a concentration of 10μM, and 7.3μL of RNase-free water were mixed evenly in a 1.5mL EP tube and added to the RPA powder tube. After dissolution, 10μL was drawn and added to 4 reaction units respectively, and 1μL of Mycoplasma pneumoniae DNA template and 1μL of 240mM magnesium acetate were added immediately to the metal bath. The reaction temperature was set to 39°C and the reaction time was 5min. Then, 8μL of CRISPR / Cas13a reaction mixture was quickly added, where the CRISPR / Cas13a reaction solution consisted of 0.5μL 40U / μl RNase Inhibitor, 1.5μL 10ng / μl crRNA, and 1.0μL 10 μM RNA fluorescent reporter molecule (sequence as follows: FAM-TUUUUUUC-BHQ1), 0.2 μL 25 mM rNTP, 0.1 μL 40 U / μl T7 RNA polymerase, 1.5 μL 60 ng / μl Cas13a, and RNase-free water to 8 μL.

[0085] The RPA and CRISPR / Cas13a reaction solutions were mixed evenly and reacted on a fluorescence detector at 39°C for 60 min, with fluorescence collected every 1 min.

[0086] Test results such as Figure 3 As shown, it can be seen that the fluorescence intensity of the crRNA-2-containing detection system when detecting wild-type templates is significantly lower than that of other crRNAs, and the fluorescence intensity is higher when detecting mutant templates, indicating that crRNA-2 can well detect mutant Mycoplasma pneumoniae, while there is no obvious nonspecific amplification of wild-type Mycoplasma pneumoniae. Subsequent experiments are carried out based on crRNA-2.

[0087] (3) Sensitivity and specificity experiments

[0088] Sensitivity experiment:

[0089] After the RPA-CRISPR / CAS13a detection system was established, fluorescence detection was performed using a gradient-diluted DNA plasmid of the Mycoplasma pneumoniae 23s mutant as a template. The results showed that amplification signals were generated within 30 minutes, and the detection sensitivity could reach 1.0×10 1 Copies / reactions, such as Figure 4 A.

[0090] Specificity experiments:

[0091] The specificity of the mutation detection system was verified using a variety of non-pneumoniae strains or viruses, with a strain or virus concentration of 106 -10 7 copies / ml, mutant plasmid DNA was used as a positive control, and the results showed that the system did not cross-react with non-Mycoplasma pneumoniae pathogens, indicating that the detection was highly specific, such as Figure 4 B.

[0092] Example 3

[0093] (1) Principle of combined detection of RPA-CRISPR / Cas13a system and lateral flow test strips

[0094] The nucleic acid lateral flow test strip is an immunochromatographic technology that uses gold nanoparticles (AuNPs) to detect pathogen nucleic acids. TM The analytical solution in the HybriDetect kit is mixed and the test strip is placed in the reaction solution. When the test result is positive, the probe labeled with FAM and biotin is cut by the activated Cas13a enzyme, the FAM end and the biotin end are separated, and the analyte solution combines with the gold-labeled-FAM specific antibody in the sample application area of the test strip to form a colloidal gold complex. The complex moves along the membrane driven by capillary force. The intact FAM-biotin probe gold particles will bind when passing through the immobilized biotin ligand molecule line, and produce a control band (C) over time. The gold particles bound to the cut probe FAM end migrate to the antibody capture area and are captured by the species-specific antibody, and produce a detection band (T) over time, as shown in FIG. Figure 5 shown.

[0095] (2) Application of RPA-CRISPR / CAS13a Mycoplasma pneumoniae mutation site detection system in clinical sample detection

[0096] To verify the practicality of the established RPA-CRISPR / CAS13a Mycoplasma pneumoniae and mutation detection system, 10 throat swab samples from children who were positive for Mycoplasma pneumoniae were collected and tested using the system. The genomic DNA of the 10 samples was extracted using a Mycoplasma genome extraction kit. The 23s gene partial sequence was amplified by PCR, and the sequence was compared after sequencing to analyze the mutation of the samples. Among them, 3 were wild-type samples and 7 were A2063G mutant samples. The 10 samples were tested using the RPA-CRISPR / CAS13a Mycoplasma pneumoniae mutation site detection system, and the results are shown in the table. Figure 6 , it can be seen that the test results are consistent with the sample mutation.

[0097] The RNA fluorescent probe in the RPA-CRISPR / CAS13a fluorescence detection system was replaced with a LF-RNA probe labeled with a FAM fluorescent group and biotin. The sequence is as follows: FAM-UUUUUUUUUUUUUU-Biotin (SEQ ID NO. 13). The other components remained unchanged. The LF-RPA-CRISPR / CAS13a detection method was established by combining it with the test strip and testing clinical samples. Figure 7 ,The results showed that this method can accurately and quickly detect Mycoplasma pneumoniae nucleic acid and mutation sites.

[0098] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. A reagent for detecting the A2063G mutation in the 23s rRNA gene of Mycoplasma pneumoniae, comprising an RPA amplification reagent, wherein the RPA amplification reagent comprises a primer for detecting the A2063G mutation in the 23s rRNA gene of Mycoplasma pneumoniae, wherein the nucleotide sequence of the primer is as follows: F1:GAAATTAATACGACTCACTATAGGGTCTCTTGACTGTCTCGGCTATAGACTCGGTGAAATC; R3: TCCTACCTATTCTCTACATGATAATGTCCTG.

2. The reagent according to claim 1, characterized in that The reagents also include a CRISPR-Cas detection reagent, which includes a crRNA for detecting the A2063G mutation of the 23s rRNA gene of Mycoplasma pneumoniae, wherein the DNA template nucleic acid sequence of the crRNA is: GATTTAGACTACCCCAAAAACGAAGGGGACTAAAACGGTCTTCCGGTCCCGTTGCGCCTAACGG.

3. The reagent according to claim 2, characterized in that The CRISPR-Cas detection reagent also includes a Cas protein and a nucleic acid probe; Preferably, the Cas protein is selected from Cas12a and / or Cas13a; Preferably, the nucleic acid probe further comprises a signal generating molecule and a signal regulating molecule; Preferably, the signal generating molecule is selected from one or more of a fluorescent molecule, a radioisotope, a chromophore, an enzyme, an enzyme substrate, a chemiluminescent moiety, a bioluminescent moiety, a non-metallic isotope, a paramagnetic metal ion or a ferromagnetic metal; Preferably, the signal modulating molecule comprises a quencher molecule or biotin.

4. The reagent according to claim 3, characterized in that The amplification reagent also includes amplification reaction buffer, recombinase, DNA polymerase, single-strand binding protein, Mg 2+ At least one of; Preferably, the CRISPR-Cas detection reagent further comprises at least one of a buffer suitable for the Cas nuclease cleavage reaction system, an RNase Inhibitor, dNTPs, rNTPs, and RNA polymerase; Preferably, the reagents further comprise a nucleic acid extraction reagent.

5. A kit comprising the reagent according to any one of claims 1 to 4.

6. The kit according to claim 5, characterized in that The kit further comprises a substance for detecting the signal generating molecule, wherein the substance comprises a nucleic acid detection test strip or a fluorescence detection substance.

7. The kit according to claim 6, characterized in that The kit is used to detect the A2063G mutation in the 23srRNA gene of Mycoplasma pneumoniae.

8. The kit according to claim 7, characterized in that The detection comprises the following steps: S1. Extracting nucleic acid from the sample to be tested; S2, amplifying the nucleic acid in the sample to be tested using an amplification reagent to obtain an amplification product; S3, adding the product from step S2 to the CRISPR-Cas detection reagent to perform a CRISPR reaction; S4. Display the test results using fluorescence detection or nucleic acid test strips; Preferably, the nucleic acid detection test strip comprises a lateral flow immunochromatography test strip.

9. The kit according to claim 8, characterized in that The sample includes one or more of saliva, serum, plasma, blood, urine, alveolar lavage fluid, tongue swab, throat swab, nasal swab, and cerebrospinal fluid; and / or, The amplification temperature in step S2 is 35-42°C; and / or, the amplification time in step S2 is 5-30 minutes; and / or, the detection temperature in step S3 is 35-42°C; and / or, the detection temperature in step S3 is 20-60 minutes.

10. Use of the reagent according to any one of claims 1 to 4 or the kit according to any one of claims 5 to 9 in any of the following: (1) preparing a product for identifying drug resistance of Mycoplasma pneumoniae; (II) preparing products for identifying drug-resistant mutations in Mycoplasma pneumoniae; Among them, the drug-resistant mutation of Mycoplasma pneumoniae is the A2063G mutation of the 23s rRNA gene of Mycoplasma pneumoniae.

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