Method for detecting mycoplasma pneumoniae 23s rRNA gene a2063g drug resistance mutation based on CRISPR-LbCas12a system

Abstract

The application discloses a mycoplasma pneumoniae 23S rRNA gene A2063G drug resistance mutation detection method based on a CRISPR-LbCas12a system and belongs to the technical field of molecular diagnosis. The detection method can accurately distinguish single base differences such as A2063 and G2063, effectively avoids mismatch, and ensures the accuracy of results. Relying on a one-step reaction of RPA and CRISPR, the total time consumption is less than 45 minutes, and the waiting time is significantly shortened. The whole reaction is carried out under constant temperature conditions, does not need to rely on complex equipment such as a thermal cycler, is easy to operate, is very suitable for the application of primary medical institutions and instant test scenes. The cost control advantage is outstanding, the reagent cost is about 1 / 50 of second-generation sequencing and 1 / 5 of fluorescent PCR, and the economy is extremely good. In addition, the application technology shows good multiplex detection potential and wide application prospect.

Description

Technical Field

[0001] This invention relates to a method for detecting drug resistance mutations in the A2063G gene of Mycoplasma pneumoniae 23S rRNA based on the CRISPR-LbCas12a system, belonging to the field of molecular diagnostic technology. Background Technology

[0002] Mycoplasma pneumoniae ( Mycoplasma pneumoniae Mycoplasma pneumoniae is a major pathogen of community-acquired pneumonia (CAP), with an infection rate as high as 20%-40% in children and adolescents. Macrolide antibiotics (such as erythromycin and azithromycin) are first-line drugs for the treatment of Mycoplasma pneumoniae infection.

[0003] However, with the widespread use of macrolide antibiotics, the problem of Mycoplasma pneumoniae resistance is becoming increasingly serious. Studies have shown that the A2063G mutation in the V domain of the 23S rRNA gene is the main mechanism leading to macrolide resistance. This mutation reduces the affinity of 23S rRNA for antibiotics, resulting in high levels of resistance (MIC ≥ 64 μg / mL). In Asia, the resistance rate of Mycoplasma pneumoniae has exceeded 90%, making rapid and accurate resistance detection crucial for guiding clinical medication.

[0004] Currently used methods for detecting drug resistance each have their own characteristics and limitations. Traditional drug susceptibility testing, which combines in vitro culture with minimum inhibitory concentration (MIC) determination, is the gold standard, but suffers from long culture periods (2–4 weeks), low positive rates, and the inability to distinguish between different types of drug resistance mutations. PCR-RFLP technology relies on restriction enzyme digestion analysis, which is cumbersome and has limited sensitivity. While fluorescent PCR can achieve rapid detection, it relies on expensive instruments and has limited single-base resolution. Sanger sequencing, as a classic direct sequencing method, has low sensitivity, typically requiring a mutation load of over 20% for detection, and is also time-consuming and costly. Next-generation sequencing, while offering high throughput, still faces challenges such as high detection costs and complex data analysis, limiting its widespread application in routine clinical testing.

[0005] Therefore, there is an urgent need to develop a rapid, sensitive, and specific method for detecting drug resistance mutations in the Mycoplasma pneumoniae 23S rRNA gene A2063G. Summary of the Invention

[0006] To address the shortcomings of the existing technologies, this invention provides a method for detecting the A2063G drug resistance mutation of the Mycoplasma pneumoniae 23S rRNA gene based on the CRISPR-LbCas12a system. The aim is to solve the problems of low sensitivity, long cycle time, and high cost in the existing technologies for detecting the A2063G mutation of Mycoplasma pneumoniae.

[0007] The first technical solution provided by the present invention is a crRNA that specifically recognizes the A2063G mutation, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] The second technical solution provided by the present invention is a composition containing the crRNA described in the first technical solution.

[0009] The third technical solution provided by the present invention is a detection system for drug-resistant mutant genes of Mycoplasma pneumoniae, wherein the detection system contains crRNA as described in the first technical solution or the composition as described in the second technical solution.

[0010] In some embodiments, the detection system further includes the Cas12 protein, wherein the Cas12 protein is LbCas12a.

[0011] In some embodiments, the detection system further includes a reporter probe, the two ends of which are modified with a fluorescent group or biotin, wherein the fluorescent group dye is SYTO. 13. SYTO 82. FAM, FITC, SYBR Green I, SYTO 13. SYTO 82. VIC, HEX, JOE, TAMRA, TET, Cy3, ROX, TEXAS One of Red or Cy5.

[0012] In some embodiments, the detection system further includes an RPA amplification system composition.

[0013] In some embodiments, the RPA amplification system composition further includes primer pairs, A Buffer, and B Buffer.

[0014] The fourth technical solution provided by the present invention is a method for detecting drug-resistant mutant genes of Mycoplasma pneumoniae in vitro, wherein the method uses the detection system described in the third technical solution to detect the sample.

[0015] The fifth technical solution provided by this invention is the application of the crRNA described in the first technical solution, the composition described in the second technical solution, or the detection system described in the third technical solution in the preparation of products for detecting drug-resistant mutation genes in Mycoplasma pneumoniae. In some embodiments, the product includes reagents and kits.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: This detection method boasts excellent single-base resolution, accurately distinguishing between single-base differences such as A2063 and G2063, effectively avoiding mismatches and ensuring accurate results. The detection process is highly efficient and rapid, relying on a one-step reaction using RPA and CRISPR, with a total time of less than 45 minutes, significantly reducing waiting time. The entire reaction is carried out under isothermal conditions, eliminating the need for complex equipment such as thermal cyclers, making it simple to operate and ideal for applications in primary healthcare institutions and point-of-care testing (POCT) scenarios. It also offers significant advantages in cost control, with reagent costs approximately 1 / 50th that of next-generation sequencing (NGS) and 1 / 5th that of fluorescent PCR, making it extremely economical. Furthermore, this technology demonstrates excellent potential for multiplexing, and can be easily expanded to simultaneous screening of other drug resistance sites such as A2064G and C2617A in the future, showing broad application prospects. Attached Figure Description

[0017] Figure 1 This is a sequence fragment of the V domain of the Mycoplasma pneumoniae 23S rRNA gene.

[0018] Figure 2 This is a diagram showing the detection results of Embodiment 2 of the present invention. Detailed Implementation

[0019] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0020] Example 1: Screening and Optimization of crRNA crRNA or sgRNA was designed near the A2063G mutation, with its detection sequence encompassing the mutation site. Spacer1 and Spacer2 are traditional CRISPR detection site design methods. Using PAM sites as the screening criteria for detection sites, it is expected that CRISPR will be able to distinguish between WT and MT sequences through point mutation sites in the spacer sequences. Figure 1 As shown, MT-Spacer uses a non-traditional site design. Because the A2063G mutation causes a change in the PAM site position (from TTTC to TTCC), the detection site of the mutant sequence will have a one-base shift compared to the wild type.

[0021] Two candidate sgRNA / crRNAs were designed and screened for the A2063G mutation site, as shown in Table 1.

[0022] Table 1 Candidate sgRNA / crRNA

[0023] As shown in Table 1, the Tm of MT-crRNA is lower than that of MT-sgRNA, but its secondary structure ΔG is closer to 0 than that of MT-sgRNA. This means that the backbone of MT-crRNA is "naked" and can freely bind to target sequences or proteins (such as Cas12).

[0024] Synthetic oligonucleotides were used as targets (wild-type: A2063; mutant: G2063). Fluorescence signals of the two candidate sgRNA / crRNAs were detected, and the results are shown in Table 2.

[0025] Table 2 Filtering Results

[0026] As shown in Table 2, MT-crRNA (i.e., the crRNA-MUT described in this invention) has the highest fluorescence signal and the best recognition ability for the A2063G mutation.

[0027] Example 2 Detection System A detection system was constructed using the sgRNA / crRNA from Example 1. The detection principle of this system is as follows: First, Mycoplasma pneumoniae DNA was extracted from the sample, and then the 23S rRNA gene fragment was isothermally amplified using RPA technology. The amplification product activates the LbCas12a-crRNA complex, which specifically recognizes the A2063G drug-resistant mutation. Once recognition is successful, the LbCas12a protein initiates trans-cleavage activity, non-specifically cleaving the fluorescent reporter probe in the system, thereby releasing a strong fluorescent signal. Finally, the presence or absence of the fluorescent signal is detected to determine whether a drug-resistant mutation exists in the sample, achieving rapid diagnosis of Mycoplasma pneumoniae drug resistance status. The specific components of the detection system are shown in Table 3. FAM fluorescence was collected every 30 seconds at 37℃ for 45 min.

[0028] Table 3 Composition of the detection system

[0029] Following the above system and method, MT sg / cr-RNA was tested using WT and MT plasmids respectively. The construction methods of WT and MT plasmids are as follows: Based on the drug resistance mutation site of the Mycoplasma pneumoniae 23S RNA gene, a partial region of the 23S RNA gene containing the A2063G mutation site (SEQ ID NO.3) was selected as the target amplification segment, and the above region was constructed into the vector pUC57 to prepare a positive plasmid containing the Mycoplasma pneumoniae drug resistance mutation site A2063G; similarly, a plasmid containing the wild-type 23S RNA gene fragment (SEQ ID NO.4) was prepared as a negative control, and the vector pUC57 was used as a blank control.

[0030] The results are as follows Figure 2 As shown, after simultaneously testing the structures of AapCas12b and LbCas12a, it was found that although the detection sites were completely identical, the detection efficiency of AapCas12b binding to sgRNA was significantly lower than that of LbCas12a binding to crRNA. Furthermore, AapCas12b could not distinguish between wild-type and mutant templates. However, LbCas12a could distinguish between wild-type and mutant genes.

[0031] In summary, for the A2063G target mutation, the wild-type and mutant MP samples can be distinguished by LbCas12a binding to the mutant MT-crRNA sequence and based on the effect of the mutation on the PAM site.

[0032] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A crRNA that specifically recognizes the A2063G mutation, characterized in that, The nucleotide sequence of the crRNA is shown in SEQ ID NO.

1.

2. A composition, characterized in that, The composition contains the crRNA as described in claim 1.

3. A detection system for drug-resistant mutant genes in Mycoplasma pneumoniae, characterized in that, The detection system contains the crRNA of claim 1 or the composition of claim 2.

4. The detection system according to claim 3, characterized in that, The detection system also includes the Cas12 protein, which is LbCas12a.

5. The detection system according to claim 3, characterized in that, The detection system also includes a reporter probe, the two ends of which are modified with a fluorescent group or biotin, wherein the fluorescent group dye is SYTO.

13. SYTO 82. FAM, FITC, SYBR Green I, SYTO 13. SYTO 82. VIC, HEX, JOE, TAMRA, TET, Cy3, ROX, TEXAS One of Red or Cy5.

6. The detection system according to claim 3, characterized in that, The detection system also includes an RPA amplification system composition.

7. The detection system according to claim 6, characterized in that, The RPA amplification system composition also includes primer pairs, A Buffer, and B Buffer.

8. A method for detecting drug-resistant mutant genes in Mycoplasma pneumoniae in vitro, characterized in that, The method involves using the detection system described in any one of claims 3 to 7 to detect the sample.

9. The use of the crRNA of claim 1, the composition of claim 2, or the detection system of any one of claims 3 to 8 in the preparation of products for detecting drug-resistant mutant genes of Mycoplasma pneumoniae.

10. The application according to claim 9, characterized in that, The products include reagents and kits.

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