Mycoplasma pneumoniae nucleic acid detection quality control product based on recombinant artificial chromosome and application of mycoplasma pneumoniae nucleic acid detection quality control product

Abstract

The invention relates to the technical field of gene detection, and provides a mycoplasma pneumoniae nucleic acid detection quality control product based on a recombinant artificial chromosome and application of the mycoplasma pneumoniae nucleic acid detection quality control product. Aiming at the problems of difficulty in integration of multi-target full-length genes, accurate drug-resistant mutation, insufficient stability and the like of an existing quality control product, a recombinant artificial chromosome carrying full-length sequences of mycoplasma pneumoniae key target genes (16S rRNA, 23S rRNA, a P1 adhesion protein gene and a community-acquired respiratory distress syndrome toxin gene toxin) is constructed; important clinical drug-resistant mutation sites (A2063G, A2064G, C2617T and A2067G) are accurately introduced into the 23S rRNA gene, and the 23S rRNA gene is stably integrated into escherichia coli engineering bacteria by using a bacterial artificial chromosome system. The quality control product exists in a low-copy form, has the advantages of multi-target coverage, accurate simulation of drug-resistant mutation, high genetic stability and no biological safety risk, and is suitable for performance analysis verification and quality control of a detection kit.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and gene detection technology, specifically to a quality control product for Mycoplasma pneumoniae nucleic acid detection based on recombinant artificial chromosomes and its application. Background Technology

[0002] Mycoplasma pneumoniae (Mp) is a major pathogen causing community-acquired pneumonia, particularly prevalent in children and adolescents. Accurate, rapid, and sensitive detection of Mycoplasma pneumoniae infection is crucial for clinical diagnosis, epidemiological investigations, and rational drug use. Molecular diagnostic techniques, especially nucleic acid amplification-based methods (such as PCR and isothermal amplification), have become the mainstream approach for Mp detection due to their high sensitivity and specificity. To ensure the reliability, comparability, and inter-laboratory consistency of these molecular detection results, high-quality nucleic acid detection quality control materials (QCMs) are indispensable. An ideal Mp nucleic acid detection QCM should stably mimic the characteristics of the target nucleic acid in clinical samples and effectively monitor the performance of the entire process from nucleic acid extraction and amplification efficiency to final detection. Currently, commonly used clinical and laboratory Mp nucleic acid detection QCMs primarily rely on naturally cultured Mp strains or their genomic DNA, artificially synthesized DNA oligonucleotide fragments or in vitro transcribed RNA, and multiplex PCR amplification products. However, these existing quality control products have significant limitations: obtaining natural Mp strains or their DNA is difficult and costly, poses biosafety risks, exhibits large batch-to-batch variability, is difficult to include specific clinically relevant drug resistance mutations, and nucleic acid stability is greatly affected by storage conditions; while artificially synthesized oligonucleotide fragments can be customized, their length is usually short (tens to hundreds of bases), making it difficult to completely cover long target gene regions or simulate the real genome structure containing multiple spacer sequences, and cannot effectively assess the detection performance involving long fragment amplification or complex secondary structures, let alone simulate the efficiency of the nucleic acid extraction process; in vitro transcribed RNA is mainly used for quality control of RNA pathogen detection and is not suitable for the detection of DNA targets like Mp; multiplex PCR products as quality control products have problems such as complex preparation processes, uneven concentrations, high copy numbers making them prone to contamination, poor stability, and also cannot include precise drug resistance mutations. Of particular note is that, with the development of molecular diagnostic technology and the increasing demands for detection reliability, the detection of Mycoplasma pneumoniae (Mp) is increasingly employing multi-target combined detection strategies (such as simultaneously targeting the 16S rRNA gene, 23S rRNA gene, adhesion protein P1 gene, and community-acquired respiratory distress syndrome toxin gene) to improve the sensitivity and specificity of detection. Simultaneously, it detects key 23S rRNA gene mutation sites (such as A2063G, A2064G, C2617T, A2067G) to rapidly assess drug resistance and guide the rational use of macrolide antibiotics. Existing quality control systems struggle to integrate these multi-target full-length gene sequences distributed across different genomic locations, along with precise drug resistance point mutations, into a unified, homogeneous, and stable substance. This makes it difficult to accurately reflect the performance of multi-target detection reagents, particularly in evaluating the limit of detection (LOD) for sequences containing specific point mutations and the ability to distinguish between wild-type and mutant strains.Therefore, there is an urgent need to develop a novel quality control product for Mp nucleic acid detection that can overcome the aforementioned shortcomings. This quality control product should stably carry multiple key full-length target genes of Mp (such as 16S rRNA, 23S rRNA, P1, and toxin), and accurately integrate clinically relevant drug resistance mutation sites (such as sites 2063, 2064, 2617, and 2067 of the 23S rRNA gene). It should possess high homogeneity, stability, and safety, and be able to simulate the extraction and amplification behavior of target nucleic acids in real samples. This would provide a comprehensive and reliable quality control tool for multi-target molecular detection of Mp and its drug resistance detection, meeting the increasingly high demands of modern precision molecular diagnostics for quality control products. Summary of the Invention

[0003] To address the significant shortcomings of existing Mycoplasma pneumoniae (Mp) nucleic acid detection quality control products in terms of coverage of multi-target full-length genes, integration of precise drug resistance mutations, stability, and safety, this invention provides a multi-target and drug resistance gene-based engineered bacterial nucleic acid detection quality control product based on genetically engineered recombinant artificial chromosomes, its preparation method, and its application. The specific technical solution is as follows:

[0004] The core of this invention lies in constructing a recombinant artificial chromosome carrying the full-length sequences of key universal detection target genes for Mycoplasma pneumoniae (16S rRNA gene, 23S rRNA gene, P1 adhesion protein gene, and community-acquired respiratory distress syndrome toxin gene). Furthermore, a clinically relevant macrolide resistance mutation site (the 23S rRNA gene contains at least one A2063G, A2064G, C2617T, or A2067G resistance mutation site) is precisely introduced into the 23S rRNA gene. This chromosome is then efficiently integrated and stably replicated into engineered bacteria using a bacterial artificial chromosome system, forming a uniform, stable, nuclease-resistant, easily preserved, biosafety-free recombinant engineered bacterial quality control product that realistically simulates the nucleic acid extraction and amplification behavior of clinical samples. The nucleotide sequence of the 16S rRNA gene is shown in SEQ ID NO:1; the nucleotide sequence of the 23S rRNA gene is shown in SEQ ID NO:2-6, wherein SEQ ID NO:2 is the wild-type sequence of the 23S rRNA gene, and SEQ ID NO:3-6 are the mutant drug-resistant sequences of the 23S rRNA gene; the nucleotide sequence of the P1 adhesion protein gene is shown in SEQ ID NO:7; and the nucleotide sequence of the toxin gene is shown in SEQ ID NO:8.

[0005] This invention utilizes the shuttle plasmid vector pBac-N-EGFP (Beyotime, catalog number D2805) and DH10Bac competent cells (Beyotime, catalog number D0346) system from Beyotime Biotechnology to construct artificial chromosomes via Tn7 transposon-mediated homologous recombination. The original application of this system was to transfer recombinant artificial chromosomes into insect cells and package them to produce recombinant baculoviruses. However, this invention only involves generating recombinant artificial chromosomes carrying the target gene through shuttle plasmid-mediated homologous recombination, representing another application of this system. It should be noted that the construction of recombinant artificial chromosomes in this invention is not limited to using the shuttle plasmid vector pBac-N-EGFP and DH10Bac competent cells; other shuttle plasmids and artificial chromosome systems can also achieve this technical objective. The engineered bacterium is Escherichia coli, and the recombinant artificial chromosome is integrated into the engineered bacterium through a bacterial artificial chromosome system. It exists in a low-copy form and can be stably replicated and propagated within the bacteria. Due to its large capacity (up to hundreds of kb), it can completely accommodate all target genes and their flanking sequences. Each engineered bacterium carries 1-2 copies of the artificial chromosome.

[0006] This invention also provides a method for preparing the above-mentioned nucleic acid detection quality control material, comprising the following steps:

[0007] (a) Design and construct a shuttle vector containing the full-length nucleotide sequences of a synthetic Mycoplasma pneumoniae 16S rRNA gene, 23S rRNA gene, P1 gene and toxin gene, wherein the 23S rRNA gene contains at least one drug resistance mutation site of A2063G, A2064G, C2617T or A2067G.

[0008] (b) The shuttle vector from step (a) is transformed into engineered Escherichia coli, and then integrated into the bacterial artificial chromosome via homologous recombination to obtain engineered bacteria that stably carry the recombinant artificial chromosome.

[0009] This invention also provides applications of the above-mentioned nucleic acid detection quality control products for at least one of the following scenarios:

[0010] (a) As a positive control or calibrator, evaluate the analytical sensitivity (LOD), specificity, repeatability and reproducibility of the Mycoplasma pneumoniae nucleic acid detection kit, which includes multi-target combined detection reagents and drug resistance mutation detection reagents based on PCR, qPCR, isothermal amplification or digital PCR.

[0011] (b) Used for internal quality control (IQC) or external quality assessment (EQA) in clinical laboratories to monitor the reliability of the entire process of nucleic acid extraction, amplification and analysis;

[0012] (c) As a standard material, it supports the standardization of molecular diagnostic methods for Mycoplasma pneumoniae or the registration of reagent kits.

[0013] (d) Evaluate the integrity and extraction efficiency of nucleic acid extraction reagents for long DNA fragments of target genes.

[0014] The genetically engineered bacterial quality control products provided by this invention have the following significant advantages and inventiveness: (1) Multi-target full coverage: It integrates the complete full-length sequences of the four most critical universal target genes (16S, 23S, P1, and toxin) in Mp molecular diagnostics at one time, which can comprehensively evaluate the extraction efficiency, amplification efficiency, and detection sensitivity for different targets, and is especially suitable for the quality control of multi-target combined detection reagents; (2) Precise drug resistance mutation simulation: In 23S Four clinically relevant drug resistance mutation sites (2063, 2064, 2617, 2067) were precisely introduced into the rRNA gene, providing a reliable evaluation benchmark for the specificity, sensitivity, and wild-type / mutant differentiation ability of the drug resistance mutation detection reagent; (3) High stability and uniformity: The recombinant artificial chromosome exists stably in the engineered bacteria with a low copy number. Each engineered bacteria carries only 1-2 copies of the artificial chromosome, resulting in good batch-to-batch consistency. This overcomes the genetic variability of natural pathogen culture and the instability of synthetic fragments, and is more uniform and stable compared to the hundreds of copies of general plasmids. It is also worth emphasizing that when a single bacterium is used as the smallest detection unit, the low copy number of this design accurately simulates the genome copy number of natural Mycoplasma pneumoniae (single copy chromosome). Compared with conventional plasmids (usually hundreds of copies / bacterium) which lead to an artificially high and distorted target gene concentration in the smallest detection unit, the quality control results of this design can more accurately reflect the sensitivity of the nucleic acid detection kit to clinical low-load samples (such as analytical sensitivity LOD assessment) and avoid batch-to-batch differences caused by copy number fluctuations. This is particularly crucial for the detection of drug-resistant mutations—a low-copy environment can verify the reagent's ability to recognize real drug-resistant strains, rather than relying on the false high sensitivity generated by amplifying high concentrations of mutant sequences. (4) No biosafety risks: The engineered bacteria are Escherichia coli, which do not carry any infectious Mp components, and the operation safety level is low (usually BSL-1), requiring no special protection; (5) Realistic simulation of nucleic acid extraction: The DNA it releases is a chromosome-level macromolecule containing natural spacer sequences and secondary structures, which can truly reflect the efficiency of the clinical sample DNA extraction process, overcoming the limitations of short-fragment synthetic oligonucleotides and conventional plasmids; (6) Flexibility and scalability: The vector design allows for the convenient addition or replacement of other target genes or mutation sites to adapt to the updating and iteration of diagnostic technologies. (7) Nuclease resistance: The presence of the cell wall and cell membrane structure of the engineered bacteria of this invention provides an extra protective layer for the internal nucleic acids, making them less susceptible to degradation by exogenous nucleases during preparation, which is far superior to naked DNA. Attached Figure Description

[0015] Figure 1Electrophoresis images of bacterial culture PCR identification of the plasmid vectors pBac-Mp-16S-M, pBac-Mp-23S-M, pBac-Mp-P1-M, and pBac-Mp-toxin-M in Example 2 of this invention.

[0016] Figure 2 The image shows the pBac-Mp-16S-M plasmid vector constructed in Example 2 of this invention, with only elements relevant to this application labeled.

[0017] Figure 3 The image shows the pBac-Mp-23S-M plasmid vector constructed in Example 2 of this invention, with only elements relevant to this application labeled.

[0018] Figure 4 The image shows the pBac-Mp-23S2063-M plasmid vector constructed in Example 2 of this invention, with only elements relevant to this application labeled.

[0019] Figure 5 The image shows the pBac-Mp-23S2064-M plasmid vector constructed in Example 2 of this invention, with only elements relevant to this application labeled.

[0020] Figure 6 The image shows the pBac-Mp-23S2617-M plasmid vector constructed in Example 2 of this invention, with only elements relevant to this application labeled.

[0021] Figure 7 The image shows the pBac-Mp-23S2067-M plasmid vector constructed in Example 2 of this invention, with only elements relevant to this application labeled.

[0022] Figure 8 The image shows the pBac-Mp-P1-M plasmid vector constructed in Example 2 of this invention, with only elements relevant to this application labeled.

[0023] Figure 9 The image shows the pBac-Mp-toxin-M plasmid vector constructed in Example 2 of this invention, with only elements relevant to this application labeled.

[0024] Figure 10 The results of nuclease treatment were obtained for the engineered Mycoplasma pneumoniae quality control and control plasmids in Example 6 of this invention. The results showed that the Ct values ​​of the engineered bacteria quality control did not differ significantly before and after nuclease treatment; however, the Ct value of the control plasmid changed significantly after nuclease treatment due to degradation.

[0025] Figure 11This is the verification result of the extraction monitoring capability of the engineered Mycoplasma pneumoniae bacteria quality control sample and the control plasmid (E6 copies / mL) in Example 7 of the present invention. The results show that the Ct value of the engineered bacteria quality control sample is significantly different before and after extraction; while the Ct value of the control plasmid is not different under the conditions of extraction and no extraction.

[0026] Figure 12 The results show the statistical values ​​of Ct values ​​for the stability of the engineered Mycoplasma pneumoniae bacteria quality control sample in Example 8 of this invention under storage conditions of 4°C, 25°C, and 37°C. The results show that the Ct values ​​of the quality control sample did not change significantly at any of the three storage temperatures.

[0027] Figure 13 The results show the repeatability of the 23S and P1 genes of the engineered Mycoplasma pneumoniae in Example 10. The results show that the amplification curves highly overlap, indicating excellent repeatability.

[0028] Figure 14 The results show the identification of the drug resistance gene 23S A2063G in the engineered Mycoplasma pneumoniae strain in Example 11. The results indicate that the ARMS-PCR mutant detection system showed a strong positive signal for the A2063G mutant quality control sample; while the wild-type detection system did not produce a significant fluorescence signal, demonstrating good specificity. Detailed Implementation

[0029] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0030] definition

[0031] In this invention, the range of values ​​represented by “value A ~ value B” or “value A - value B” refers to the range that includes the endpoint values ​​A and B.

[0032] In this invention, the numerical range indicated by "above" or "below" refers to the numerical range that includes the stated number.

[0033] In this invention, the word "may" has both the meaning of performing a certain process and the meaning of not performing a certain process. In this specification, "optional" or "optionally" means that the event or situation described below may or may not occur, and the description includes both the case where the event occurs and the case where the event does not occur.

[0034] In this invention, the terms "a", "an", or "the" may refer to "one", "one or more", "at least one", or "one or more".

[0035] In this invention, the terms "comprising," "having," "including," or "containing" can mean included or open-ended, and do not exclude additional, uncited elements or method steps. At the same time, "comprising," "having," "including," or "containing" can also mean closed-ended, excluding additional, uncited elements or method steps.

[0036] In this invention, the term "about" can mean that a value includes the standard deviation of the error of the apparatus or method used to determine that value. The numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains a standard deviation due to the aforementioned testing apparatus or method. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this invention are modified with "about". Here, "about" generally means that the actual value is within ±10%, ±5%, ±1%, or ±0.5% of a particular value or range.

[0037] In this invention, "target gene" refers to the gene targeted by nucleic acid detection. For example, when nucleic acid detection is performed using PCR, the target gene includes the gene fragment to be amplified by PCR.

[0038] In this invention, a "vector" is a replicon, such as a plasmid, bacteriophage, virus, artificial chromosome, or granule, to which another DNA segment (i.e., an "insertion") may be attached to the replicon to induce replication of the attached segment in the cell. Further, the vector may include, for example, a collection of genetic elements that regulate gene expression, such as promoters and enhancers; (2) a structural or coding sequence transcribed into mRNA and translated into protein; and (3) a transcriptional subunit containing appropriate transcription and translation initiation and termination sequences.

[0039] In this invention, an artificial chromosome is constructed via Tn7 transposon-mediated homologous recombination using the shuttle plasmid vector pBac-N-EGFP (Beyotime, catalog number D2805) and DH10Bac competent cells (Beyotime, catalog number D0346) system derived from Beyotime Biotechnology. The original application of this system was to transfer recombinant artificial chromosomes into insect cells and package them to produce recombinant baculoviruses. However, this invention only involves generating a recombinant artificial chromosome carrying the target gene through shuttle plasmid-mediated homologous recombination, representing another application of this system. It should be noted that the construction of recombinant artificial chromosomes in this invention is not limited to using the shuttle plasmid vector pBac-N-EGFP and DH10Bac competent cells; other shuttle plasmids and artificial chromosome systems can also achieve this technical objective. Furthermore, "engineered bacteria carrying artificial chromosomes" refers to *E. coli* engineered bacteria carrying an artificial chromosome with a homologous sequence of the *Mycoplasma pneumoniae* (Mp) target gene.

[0040] In this invention, "consistent sequence" or "optimized sequence" or "consistent optimized sequence" refers to a representative nucleotide sequence generated by performing site-specific statistical analysis on a set of functional or evolutionarily related nucleotide sequences using multiple sequence alignment (MSA) technology.

[0041] In this invention, a "standard curve conforming to the requirements of quantitative PCR detection specifications" refers to a linear relationship curve between the cycle threshold (Ct value) and the logarithm of the initial template concentration, established after serially diluting a known concentration of standard (such as plasmid DNA or purified PCR product) and amplifying it by fluorescent PCR. This standard curve must meet the requirements of the specification "Performance Evaluation Requirements for Biotechnology Nucleic Acid Target Sequence Quantification Methods: qPCR and dPCR" (GB / T 42077-2022), namely, a linear correlation coefficient (R²). 2 The amplification efficiency (E) was greater than 0.99 and within the range of 0.9 to 1.1, to verify the accuracy, sensitivity and quantitative reliability of the detection system.

[0042] Unless otherwise defined, other technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0043] The Mycoplasma pneumoniae 16S rRNA sequence (SEQ ID NO:1) in this application.

[0044] The Mycoplasma pneumoniae 23S rRNA (wild-type) homologous sequence (SEQ ID NO:2) in this application.

[0045] The identical sequence (SEQ ID NO:3) of Mycoplasma pneumoniae 23S rRNA (mutant A2063G) in this application.

[0046] The identical sequence (SEQ ID NO:4) of Mycoplasma pneumoniae 23S rRNA (mutant A2064G) in this application.

[0047] The identical sequence of Mycoplasma pneumoniae 23S rRNA (mutant C2617T) in this application (SEQ ID NO:5).

[0048] The identical sequence (SEQ ID NO:6) of Mycoplasma pneumoniae 23S rRNA (mutant A2067G) in this application.

[0049] The Mycoplasma pneumoniae P1 gene sequence (SEQ ID NO:7) in this application.

[0050] The Mycoplasma pneumoniae toxin gene sequence (SEQ ID NO:8) in this application.

[0051] The nucleotide sequence of primer Mp-16S-MF (SEQ ID NO:9).

[0052] The nucleotide sequence of primer Mp-16S-MR (SEQ ID NO:10).

[0053] The nucleotide sequence of primer Mp-23S-MF (SEQ ID NO:11).

[0054] The nucleotide sequence of primer Mp-23S-MR (SEQ ID NO:12).

[0055] The nucleotide sequence of primer Mp-P1-MF (SEQ ID NO:13).

[0056] The nucleotide sequence of primer Mp-P1-MR (SEQ ID NO:14).

[0057] The nucleotide sequence of primer Mp-toxin-MF (SEQ ID NO:15).

[0058] The nucleotide sequence of primer Mp-toxin-MR (SEQ ID NO:16).

[0059] pUC / M13-F primer sequence (SEQ ID NO:17).

[0060] pUC / M13-R primer sequence (SEQ ID NO:18).

[0061] The upstream primer sequence for fluorescent PCR of the 23S rRNA gene, a universal detection target for Mycoplasma pneumoniae (SEQ ID NO:19).

[0062] Downstream primer sequence for fluorescent PCR of the 23S rRNA gene, a universal detection target for Mycoplasma pneumoniae (SEQ ID NO:20).

[0063] Probe sequence for fluorescent PCR of the 23S rRNA gene, a universal detection target for Mycoplasma pneumoniae (SEQ ID NO:21).

[0064] The upstream primer sequence for fluorescent PCR of the P1 gene, a universal detection target for Mycoplasma pneumoniae (SEQ ID NO:22).

[0065] The downstream primer sequence for fluorescent PCR of the P1 gene, a universal detection target for Mycoplasma pneumoniae (SEQ ID NO:23).

[0066] Probe sequence for fluorescent PCR of the P1 gene, a universal detection target for Mycoplasma pneumoniae (SEQ ID NO:24).

[0067] The upstream primer sequence (SEQ ID NO: 25) for fluorescent PCR of the A2063G mutant 23S rRNA of Mycoplasma pneumoniae drug resistance gene.

[0068] The downstream primer sequence for fluorescent PCR of the A2063G mutant 23S rRNA of Mycoplasma pneumoniae drug resistance gene (SEQ ID NO: 26).

[0069] The probe sequence for fluorescent PCR of the A2063G mutant 23S rRNA of Mycoplasma pneumoniae drug resistance gene (SEQ ID NO:27).

[0070] The probe sequence for fluorescent PCR of the A2064G mutant 23S rRNA of Mycoplasma pneumoniae drug resistance gene (SEQ ID NO:28).

[0071] The method for preparing artificial chromosomes of engineered bacteria is as follows:

[0072] (1) Obtaining the full-length identical sequences of the 16S rRNA gene, 23S rRNA gene, P1 adhesion protein gene, and community-acquired respiratory distress syndrome toxin gene of Mycoplasma pneumoniae, and the enzyme digestion and recovery of pBac vector.

[0073] First, the full-length genes of Mp's 16S rRNA, 23S rRNA, P1 adhesion protein, and community-acquired respiratory distress syndrome toxin were optimized and analyzed. The resulting homogeneous sequences were named Mp-16S-M, Mp-23S-M (23S rRNA (wild type)), Mp-23S-2063-M (23S rRNA (mutant drug-resistant type A2063G)), Mp-23S-2064-M (23S rRNA (mutant drug-resistant type A2064G)), Mp-23S-2617-M (23S rRNA (mutant drug-resistant type C2617T)), Mp-23S-2067-M (23S rRNA (mutant drug-resistant type A2067G)), Mp-P1-M, and Mp-toxin-M, i.e., the sequences shown in SEQ ID NO: 1-8, and were sent to a gene synthesis company for synthesis. Fragments were amplified using specific primer pairs (named Mp-16S-MF / R, Mp-23S-MF / R, Mp-P1-MF / R, and Mp-toxin-MF / R, sequences shown in SEQ ID NO: 9-16) and Q5 enzyme (NEB, catalog number M0493L). Simultaneously, the pBac-N-EGFP vector (Beyotime Biotech, catalog number D2805) was digested with EcoRI and Hind III restriction endonucleases. The digestion system consisted of 5 μL of 10×M buffer, 1 μL each of EcoRI and Hind III restriction endonucleases, 1.5 μg of plasmid, and ddH2O added to a final volume of 50 μL. Digestion was carried out at 37°C for 3 h. The fragments were then recovered using a Takara gel extraction kit (Takara Bio). Inc. (Catalog No. 9762) purified and recovered amplified fragments Mp-16S-M, Mp-23S-M, Mp-23S-2063-M, Mp-23S-2064-M, Mp-23S-2617-M, Mp-23S-2067-M, Mp-P1-M, Mp-toxin-M, and the pBac-N-EGFP linear vector.

[0074] (2) Ligation of the target gene fragment with the pBac-N-EGFP linear vector

[0075] The purified and recovered Mp-16S-M, Mp-23S-M, Mp-23S-2063-M, Mp-23S-2064-M, Mp-23S-2617-M, Mp-23S-2067-M, Mp-P1-M, and Mp-toxin-M fragments were seamlessly ligated into the pBac linear vector to construct the pBac-Mp-16S-M, pBac-Mp-23S-M, pBac-Mp-23S-2063-M, pBac-Mp-23S-2064-M, pBac-Mp-23S-2617-M, pBac-Mp-23S-2067-M, pBac-Mp-P1-M, and pBac-Mp-toxin-M plasmid vectors. The ligation system consisted of 2 μL of 5×In-Fusion HD Enzyme Premix, 2 μL of pBac linear vector, 2 μL of gel-purified fragment, and 4 μL of ddH2O, forming a 10 μL reaction system. Ligation was carried out at 50 °C for 15 min. (The In-Fusion HD Enzyme Premix reagent was from Takara Bio Inc., catalog number 638911).

[0076] (3) Transformation and extraction of pBac-Mp-16S-M, pBac-Mp-23S-M, pBac-Mp-23S-2063-M, pBac-Mp-23S-2064-M, pBac-Mp-23S-2617-M, pBac-Mp-23S-2067-M, pBac-Mp-P1-M and pBac-Mp-toxin-M plasmid vectors

[0077] DH5α was selected as the competent strain (Takara Bio Inc., catalog number 9057). This competent strain was transformed using the aforementioned ligation product and inoculated into ampicillin (Amp)-resistant LB agar plates, incubated overnight at 37°C. Clones that survived and amplified due to the Amp resistance tag on the plasmid were considered successfully transformed. The plasmid was isolated using alkaline lysis extraction, purified by ion-exchange column chromatography, TE buffer elution, and endotoxin removal. Positive clones were identified using PCR. After successful sequencing identification, the next experimental step was performed.

[0078] (4) Construction of artificial chromosomes of engineered bacteria

[0079] The plasmid vector containing the Mycoplasma pneumoniae target gene was added to *E. coli* DH10Bac competent cells (Beyotime Biotechnology Co., Ltd., catalog number D0346) containing the vector bMON14272 and helper plasmid (used to express the transposable protein necessary for transposition). Transforming this strain with the pBac recombinant plasmid resulted in transposition between the mini-Tn7 element in the pBac recombinant plasmid and the mini-attTN7 element on the vector bMON14272, thus forming a recombinant artificial chromosome. Site-specific transposition disrupts the host's lacZα gene, allowing for blue-white screening of recombinant clones. Homologous recombination was then performed in DH10Bac bacterial cells after a treatment involving ice bath-heat shock-ice bath followed by shaking at 37°C for 4 hours. The cultured bacterial suspension was evenly spread onto LB fixation medium containing kanamycin, tetracycline, gentamicin, X-Gal, and IPTG (Shanghai Sangon Biotech Co., Ltd., catalog number B541007), and incubated at an incubator to screen for homologous recombinant artificial chromosome clones. Positive white monoclonal colonies were picked and incubated on LB liquid medium containing kanamycin, tetracycline, and gentamicin at 37°C to obtain engineered bacteria carrying homologous recombinant artificial chromosomes.

[0080] DNA was extracted from engineered bacteria carrying artificial chromosomes using the Omega BAC / PAC Large Basic Plasmid Extraction Kit (Omega Bio-Tek, catalog number D2156). The DNA was used as a template, and PCR amplification and sequencing of the bacterial strain were performed using pUC / M13-F (sequence shown in SEQ ID NO:17) and pUC / M13-R (sequence shown in SEQ ID NO:18) as primers. After successful PCR identification and sequencing alignment, the bacterial strain was preserved, and glycerol-containing bacteria were prepared for further experiments.

[0081] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified, specific conditions in the examples are performed under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, all materials and instruments used are conventional products that can be purchased.

[0082] Example 1: Mycoplasma pneumoniae sequence optimization

[0083] 1.1 Sequence selection and design for 16S rRNA, 23S rRNA, P1 adhesion protein, and community-acquired respiratory distress syndrome (ARDS) toxin genes of Mycoplasma pneumoniae (Mp): First, bioinformatics methods were used to obtain the 16S rRNA, 23S rRNA, P1 adhesion protein, and ARDS toxin genes from different geographical isolates of Mycoplasma pneumoniae from the authoritative database NCBI. Multiple sequence alignment tools MAFFT and ClustalOmega software were used to perform multiple alignments on the selected sequences, eliminating obvious insertion and deletion regions to ensure the accuracy and consistency of the alignment. Attention was paid to avoiding cross-correlation with host or other non-target pathogen sequences in characteristic conserved regions.

[0084] 1.2 Sequence Selection Design for Drug Resistance Mutants in Mycoplasma pneumoniae (Mp) 23S rRNA Gene: Based on typical mutation sites in the Mycoplasma pneumoniae 23S rRNA gene known to be associated with macrolide resistance (e.g., A2063G, A2064G, C2617T, A2067G), mutant sequences of the 23S rRNA gene from different geographical isolates were collected by searching the NCBI database. Combined with publicly available information on drug resistance mutation sites, representative mutant drug resistance sequences were screened. MAFFT and Clustal Omega multiple sequence alignment software were used to perform comparative analysis of the 23S rRNA gene mutant sequences, focusing on verifying the consistency of mutations at each drug resistance site and the integrity of upstream and downstream conserved regions. Abnormal sequences with large deletions or insertions were removed to ensure that the obtained sequences accurately represent the characteristics of the mutant drug resistance, while avoiding cross-reactions with host or other non-target pathogen sequences. Finally, the full-length homologous sequences of drug resistance at different mutation sites (A2063G, A2064G, C2617T, A2067G) of the 23S rRNA gene were identified and designed, providing a reliable target sequence basis for subsequent synthesis, construction and detection method development.

[0085] 1.3 Synthesis of Mycoplasma pneumoniae target gene sequences: Full-length sequences of the 16S rRNA, 23S rRNA, P1 adhesion protein, and community-acquired respiratory distress syndrome (ARDS) toxin gene from multiple Mycoplasma pneumoniae isolates were aligned to obtain the final optimized full-length sequences of the Mycoplasma pneumoniae 16S rRNA, 23S rRNA, P1 adhesion protein, and ARDS toxin gene, respectively. These sequences were abbreviated as Mp-16S-M, Mp-23S-M (23S rRNA (wild-type)), Mp-23S-2063-M (23S rRNA (mutant drug-resistant A2063G)), Mp-23S-2064-M (23S rRNA (mutant drug-resistant A2064G)), and Mp-23S-2617-M (23S rRNA (wild-type)). The genomes of Mycoplasma pneumoniae 16S rRNA (mutant drug-resistant C2617T) Mp-23S-2067-M (23S rRNA (mutant drug-resistant A2067G)) Mp-P1-M and Mp-toxin-M were synthesized according to the start and stop sites of the Mycoplasma pneumoniae 16S rRNA gene, 23S rRNA gene, P1 adhesion protein gene and community-acquired respiratory distress syndrome toxin gene toxin. The resulting full-length, homologous target sequences (SEQ ID NO: 1-8) with lengths of 1732bp, 2805bp, 2805bp, 2805bp, 2805bp, 5149bp and 1782bp were obtained. These sequences were then synthesized by GenScript Biotech Co., Ltd., yielding Mp-23S-2067-M containing Mycoplasma pneumoniae 16S rRNA gene, 23S rRNA gene, and toxin gene. Conventional pUC plasmids containing the full-length target fragments of rRNA, P1 adhesion protein, and community-acquired respiratory distress syndrome toxin gene (named pUC57-Mp-16S-M, pUC57-Mp-23S-M, pUC57-Mp-23S-2063-M, pUC57-Mp-23S-2064-M, pUC57-Mp-23S-2617-M, pUC57-Mp-23S-2067-M, pUC57-Mp-P1-M, and pUC57-Mp-toxin-M) are used to target rRNA, P1 adhesion protein, and community-acquired respiratory distress syndrome toxin gene.

[0086] Example 2: Construction of plasmid vectors for Mycoplasma pneumoniae pBac-Mp-16S-M, pBac-Mp-23S-M, pBac-Mp-23S-2063-M, pBac-Mp-23S-2064-M, pBac-Mp-23S-2617-M, pBac-Mp-23S-2067-M, pBac-Mp-P1-M, and pBac-Mp-toxin-M

[0087] 2.1 PCR amplification and purification recovery of optimized Mycoplasma pneumoniae sequences

[0088] Table 2.1 Nucleotide sequences of amplification primers for the Mp designed sequence

[0089]

[0090]

[0091] Specific primer pairs (named Mp-16S-MF / R, Mp-23S-MF / R, Mp-P1-MF / R, and Mp-toxin-MF / R, sequences shown in SEQ ID NO:9-16) were designed for plasmid vector construction, as shown in Table 2.1. PCR amplification was performed using the primers in Table 2.1 to obtain the synthesized target fragment products, as shown in SEQ ID NO:1-8. The PCR reaction system is shown in Table 2.2 below.

[0092] Table 2.2 PCR reaction system

[0093]

[0094] The PCR reaction procedure is shown in Table 2.3 below.

[0095] Table 2.3 PCR reaction procedure

[0096]

[0097]

[0098] After the reaction, the amplification products were subjected to agarose gel electrophoresis, such as... Figure 1 As shown. The target fragment was purified and recovered using the Takara gel recovery kit.

[0099] 2.2 Preparation of the linear vector pBac-N-EGFP for plasmids

[0100] The plasmid pBac-N-EGFP was double-digested with restriction endonucleases Hind III and EcoRI. The digestion reaction system is shown in Table 2.4 below.

[0101] Table 2.4 Enzyme digestion reaction system

[0102] Components Added amount pBac1-N-EGFP plasmid 1.5μg EcoRI restriction endonuclease 1.0μL HindIII restriction endonuclease 1.0μL 10×M buffer 5.0μL <![CDATA[ddH2O]]> Add to 50μL

[0103] The enzyme digestion reaction procedure is shown in Table 2.5 below.

[0104] Table 2.5 Enzyme digestion reaction procedure

[0105] temperature time 37℃ 3h

[0106] The linear vector pBac-N-EGFP was purified and recovered using a Takara gel recovery kit and used for fragment ligation.

[0107] 2.3 Ligation of linear vector with target fragment

[0108] Using Takara's homologous recombination kit HD Cloning Plus was used to seamlessly ligate the target fragments (SEQ ID NO: 1-8) into the linear vector pBac-N-EGFP. The 10 μL reaction mixture is shown in Table 2.6 below, and ligation was performed at 50°C for 15 minutes.

[0109] Table 2.6 Connection Reaction System

[0110] Components Added amount 5×In-FusionHDEnzymePremix 2μL Target fragment 50-100ng linear vector pBac-N-EGFP 50-100ng

[0111] 2.4 Transformation

[0112] The ligation product was added to Takara competent E. coli DH5α competent cells and mixed gently. After incubating on ice for 10-30 minutes, the cells were placed in a 42°C water bath for 30-60 seconds, then immediately removed and placed on ice for 2-5 minutes. 700 μL of SOC medium (pre-incubated at 37°C) was added, and the cells were incubated at 37°C with shaking for 1 hour (180 rpm). An appropriate amount was then spread onto LB solid medium (ampicillin, Amp, final concentration 100 μg / mL), and the plates were incubated upside down at 37°C overnight.

[0113] 2.5 Identification of positive plasmids

[0114] Four monoclonal bacteria were selected for culture PCR. The primers are shown in Table 2.7 below:

[0115] Table 2.7 Primer Sequences

[0116]

[0117] The PCR reaction system is shown in Table 2.8 below:

[0118] Table 2.8 PCR reaction system

[0119]

[0120]

[0121] The PCR reaction procedure is shown in Table 2.9 below:

[0122] Table 2.9 PCR reaction procedure

[0123]

[0124] After the reaction, a small amount of the amplification product was taken for agarose gel electrophoresis. The results showed that the target band was present in all samples. Figure 1 Clones that were positive for bacterial culture PCR were extracted and identified by plasmid extraction and sequencing analysis. The sequences obtained were identical to the designed sequence and were named pBac-Mp-16S-M, pBac-Mp-23S-M, pBac-Mp-23S-2063-M, pBac-Mp-23S-2064-M, pBac-Mp-23S-2617-M, pBac-Mp-23S-2067-M, pBac-Mp-P1-M, and pBac-Mp-toxin-M plasmid vectors, respectively. The diagram is shown below. Figure 2-9 As shown.

[0125] Example 3: Construction and PCR Verification of Engineered Bacteria Carrying Artificial Chromosomes

[0126] 3.1 Construction of engineered bacteria carrying artificial chromosomes:

[0127] Add 100 ng of pBac-Mp-16S-M, pBac-Mp-23S-M, pBac-Mp-23S-2063-M, pBac-Mp-23S-2064-M, pBac-Mp-23S-2617-M, pBac-Mp-23S-2067-M, pBac-Mp-P1-M, and pBac-Mp-toxin-M plasmids to competent DH10Bac cells, respectively, and incubate on ice for 30 min. Heat shock in a 42℃ water bath for 50-100 s, then immediately place on ice for 2-5 min. Add 700 μL of LB medium and incubate at 37℃ with a shaker at 200 rpm for 2-4 h. Take 200 μL of the cultured bacterial suspension and spread it evenly onto LB fixation medium containing 50 μg / mL kanamycin, 7 μg / mL tetracycline, 10 μg / mL gentamicin, 40 μg / mL X-Gal, and 40 μg / mL IPTG. Invert the plate and incubate at 37°C for 24–72 h.

[0128] Select positive white monoclonal colonies and place them into 3-5 mL of LB liquid medium containing 50 μg / mL kanamycin, 7 μg / mL tetracycline, and 10 μg / mL gentamicin. Incubate at 37°C and 200 rpm for 20-30 h. Use the Omega BAC / PAC large basic plasmid extraction kit to extract artificial chromosome DNA (i.e., DNA containing the sequences Mp-16S-M, Mp-23S-M, Mp-23S-2063-M, Mp-23S-2064-M, Mp-23S-2617-M, Mp-23S-2067-M, Mp-P1-M, and Mp-toxin-M).

[0129] 3.2 PCR verification of engineered bacteria using artificial chromosomes:

[0130] Using 1 μg of engineered bacterial DNA as a template, PCR verification and sequencing were performed using pUC / M13-F (5'-CCCAGTCACGACGTTGTAAAACG-3') (SEQ ID NO:17) and pUC / M13-R (5'-AGCGGATAACAATTTCACACAGG-3') (SEQ ID NO:18) as primers. After confirming the PCR identification and sequencing accuracy, the next step of the experiment was carried out. The PCR verification system is shown in Table 3.1 below, and the PCR amplification reaction conditions are shown in Table 3.2 below.

[0131] Table 3.1 PCR reaction system

[0132]

[0133] Table 3.2 PCR reaction conditions

[0134]

[0135] PCR verification results of the engineered bacteria's artificial chromosome DNA showed that the target band size was correct and contained the target gene and part of the vector linker region sequence. Sequencing of the PCR product yielded correct results, and the sequence obtained was identical to the designed sequence, indicating that the engineered bacteria carrying the artificial chromosome were successfully constructed.

[0136] Example 4: Preservation and Use of Engineered Bacterial Strains Carrying Artificial Chromosomes

[0137] 4.1 Preservation of engineered bacterial strains carrying artificial chromosomes:

[0138] First, prepare the bacterial culture preservation solution: aliquot and sterilize glycerol and LB liquid medium to prepare LB medium with a final concentration of 50 μg / mL kanamycin, 7 μg / mL tetracycline, and 10 μg / mL gentamicin, and use it to dilute the glycerol (ratio 1:1); take 500 μL of the bacterial culture for preservation, add 500 μL of diluted glycerol, mix well, place in a 1.5 mL EP tube, and label with the strain number, strain name, preservation date, operator, and other information; then store the glycerol culture backup in a -80℃ freezer.

[0139] 4.2 Instructions for using engineered bacteria carrying artificial chromosomes:

[0140] Remove the glycerol bacteria and rewarm them at room temperature or 2–8°C until thawed. Spread a small amount of the bacterial suspension onto a plate and incubate it in LB broth containing 50 μg / mL kanamycin, 7 μg / mL tetracycline, 10 μg / mL gentamicin, 40 μg / mL X-Gal, and 40 μg / mL IPTG. Invert the plate and incubate at 37°C for 24–48 hours. Pick positive white monoclonal colonies and transfer them to 3–5 mL of LB liquid broth containing 50 μg / mL kanamycin, 7 μg / mL tetracycline, and 10 μg / mL gentamicin. Incubate at 37°C and 200 rpm for 20–30 hours. The bacterial suspension is used to extract artificial chromosome DNA for subsequent quality control testing.

[0141] Example 5: Preparation and Production of Raw Materials and Finished Products for Engineered Microorganism Quality Control Products

[0142] 5.1 Heat inactivation of engineered bacterial cultures carrying artificial chromosomes

[0143] Place the centrifuge tubes containing the engineered bacterial culture into a 60°C water bath. Ensure a tight seal to prevent leakage during heating. After heating for 1 hour, remove the inactivated culture as raw material for quality control of the engineered bacteria.

[0144] 5.2 Extraction and preparation of bacterial artificial chromosome DNA

[0145] The engineered bacterial control samples were serially diluted with phosphate-buffered saline to obtain sufficient concentrations of engineered bacteria. Nucleic acid was extracted from the engineered bacterial control samples at different concentrations using the MagaBio plus Viral DNA / RNA Purification Kit (Hangzhou Bori Technology Co., Ltd., catalog number BSC86S1E). Following the kit instructions, purified nucleic acid solutions were finally obtained.

[0146] 5.3 Digital PCR (ddPCR) Assay

[0147] Using the extracted nucleic acid as a template, prepare a reaction system containing the nucleic acid template, ddPCR reaction premix, primers, and probes, referring to the finished kit. Ensure the proportions of each component are accurate. Transfer the prepared reaction system to a digital PCR instrument for testing (ddPCR reaction system and conditions are shown in Tables 5.1 and 5.2). Directly obtain the absolute concentration of the target nucleic acid in the sample, and then calculate the nucleic acid concentration in the engineered bacterial quality control sample.

[0148] Table 5.1 ddPCR reaction system

[0149] Components Added amount PCR reaction solution 19μL enzyme mixture 1μL sample 5μL

[0150] Table 5.2 ddPCR reaction conditions

[0151]

[0152] 5.4 Preparation of engineered microbial quality control products

[0153] The raw materials for Mycoplasma pneumoniae quality control were diluted with known concentrations. According to the selection of common target genes of Mycoplasma pneumoniae, the 16S rRNA gene, P1 adhesion protein gene and community-acquired respiratory distress syndrome toxin gene toxin were first mixed to obtain an intermediate mixture. Then, the 23S rRNA gene and its mutant drug-resistant type were mixed with the intermediate mixture respectively to finally obtain the finished Mycoplasma pneumoniae engineered bacteria quality control products containing clinically relevant drug-resistant mutation sites at different concentrations.

[0154] Example 6: Verification of nuclease tolerance in engineered bacterial quality control samples

[0155] The engineered bacterial control sample prepared in Example 5 and the conventional pUC57-Mp-P1-M plasmid from Example 1 (hereinafter referred to as plasmids) (both E8 copies / mL) were digested with Thermo#EN0521 DNase I at 37℃ for 30 min (working concentration: 0.1 U per ng of nucleic acid), followed by inactivation at 70℃ for 10 min. A control without any treatment was also provided. Each group was tested in duplicate. The samples were then cumulatively diluted 100-fold and subjected to Q-PCR detection (Q-PCR reaction system, conditions, and primers / probes are shown in Tables 6.1, 6.2, and 6.3). Each sample was tested twice. The results are as follows: Figure 10 As shown in Table 6.3, there was no difference in the Q-PCR detection results of the engineered bacterial control samples before and after DNaseI digestion, while the Ct value of the plasmid increased significantly after DNaseI digestion, indicating that the engineered Mycoplasma pneumoniae control samples prepared in this invention have superior nuclease tolerance, which is much higher than that of conventional plasmids.

[0156] Table 6.1 Q-PCR reaction system

[0157] Components Added amount PCR reaction solution 19μL enzyme mixture 1μL sample 5μL

[0158] Table 6.2 Q-PCR reaction conditions

[0159]

[0160] Table 6.3 Primer and probe sequences

[0161]

[0162] Table 6.4 Data on the quality control of Mycoplasma pneumoniae 23S rRNA gene and the results of plasmid nuclease treatment.

[0163]

[0164] Note: NoCt was not detected; C T An increase in the value indicates that the template has been degraded by nucleases.

[0165] Table 6.5 Data on the quality control of Mycoplasma pneumoniae P1 gene and the results of plasmid nuclease treatment.

[0166]

[0167] Note: NoCt was not detected; C T An increase in the value indicates that the template has been degraded by nucleases.

[0168] Example 7: Verification of the ability of engineered bacterial quality control samples to monitor nucleic acid extraction

[0169] Nucleic acid extraction and non-extraction comparison tests were performed on the engineered Mycoplasma pneumoniae quality control samples at concentrations of 5.00E+06 copies / mL and 5.00E+03 copies / mL, and the pUC57-Mp-P1-M plasmid, respectively (qPCR reaction conditions were the same as in Example 6). The results are shown in Tables 7.1, 7.2, and 7.2. Figure 11 As shown, the quality control product of engineered Mycoplasma pneumoniae can only be detected after nucleic acid extraction; it cannot be detected without nucleic acid extraction. However, the plasmid can be detected regardless of whether nucleic acid extraction has been performed, and the Ct value is the same. This indicates that the quality control product of engineered Mycoplasma pneumoniae prepared in this invention can effectively monitor the nucleic acid extraction process, which is not possible with conventional plasmids.

[0170] Table 7.1 Data on the proficiency testing results of quality control samples and plasmid extraction monitoring of Mycoplasma pneumoniae 23S rRNA engineered bacteria

[0171]

[0172] Note: NoCt was not detected.

[0173] Table 7.2 Data Table of Quality Control Products and Plasmid Extraction Monitoring Results of Mycoplasma pneumoniae P1 Engineered Bacteria

[0174]

[0175] Note: NoCt was not detected.

[0176] Example 8: Stability test of engineered microbial quality control samples

[0177] Referring to standard JJF1343-2022 "Assignment and Homogeneity and Stability Assessment of Standard Reference Materials", stability was assessed by storing the sample at three different temperatures: 4℃, 25℃, and 37℃ at three time points: 0 days, 3 days, and 5 days. Two units were sampled from each temperature at each time point, and qPCR was used for detection (qPCR reaction conditions were the same as in Example 6). Each unit was tested twice. The test results are shown in Tables 8.1, 8.2, and 8.3. Figure 12 The quality control samples of Mycoplasma pneumoniae engineered bacteria showed no significant differences in detection results after storage at different temperatures. This indicates that the quality control samples of Mycoplasma pneumoniae engineered bacteria prepared in this invention have outstanding and excellent stability.

[0178] Table 8.1 Statistical Results of Ct Values ​​for the Stability of Engineered Mycoplasma pneumoniae Bacteria at 4℃

[0179]

[0180] Table 8.2 Statistical Results of Ct Values ​​for the Stability of Engineered Mycoplasma pneumoniae Bacteria at 25℃

[0181]

[0182] Table 8.3 Statistical Results of Ct Values ​​for the Stability of Engineered Mycoplasma pneumoniae Bacteria at 37℃

[0183]

[0184] Example 9: Repeatability test of engineered microbial quality control samples

[0185] After nucleic acid extraction from the engineered Mycoplasma pneumoniae bacteria quality control sample at a concentration of 5.00E+03 copies / mL, 16 parallel qPCR tests were performed using primers and probes targeting the Mycoplasma pneumoniae 23S gene and P1 gene (see Example 6 for details). The test results are detailed in Table 10 and [Table 10 is missing from the original text]. Figure 13 The Ct values ​​of the engineered bacterial quality control samples showed good consistency across repeated tests, and the coefficient of variation (CV) of the Ct values ​​for each repeated test was no greater than 5%. These results indicate that the engineered Mycoplasma pneumoniae quality control samples developed in this invention exhibit good repeatability when used for testing and can meet the quality control requirements for nucleic acid detection.

[0186] Table 10. Repeatability test results of engineered microbial quality control samples

[0187]

[0188] Example 10: Specificity identification of quality control samples based on A2063G drug-resistant mutant engineered bacteria

[0189] The engineered bacterial control material carrying the drug-resistant mutant genes A2063G and A2064G of Mycoplasma pneumoniae 23S rRNA as described in claim 1 (preparation method as described in Examples 2-3) was used as the test object, and specificity was verified by fluorescent PCR. Two sets of primer and probe systems were set up for the experiment: the first set was a specific detection system targeting the A2063G drug resistance mutation based on ARMS-PCR (Amplification Refractory Mutation System PCR) (forward primer F: 5'-GTGAAATCCAGGTACGGGTGAA-3' (SEQ ID NO:25); mutant reverse primer R: 5'-GTAAAGCTTCACGGGGTCGTC-3' (SEQ ID NO:26); mutant probe: 5'-FAM-CGTCCCGTTGCGCCTAACGG-BHQ1-3' (SEQ ID NO:27)). The second set was a detection system targeting the 23S rRNA A2064G drug resistance mutation based on Taqman MGB (minor groove binder) method (primer F: 5'-AATCCAGGTACGGGTGAAGACA-3' (SEQ ID NO:27)). NO:19); Primer R: 5'-TGCTCCTACCTATTCTCTACATGATAATG-3' (SEQ ID NO:20); Wild-type probe: 5'-VIC-CGCAACGGGACGGAAA-MGB-3' (SEQ ID NO:21); A2064 mutant probe: 5'-FAM-AACGGGACGGAGAG-MGB-3' (SEQ ID NO:28). The engineered bacterial control samples were serially diluted to 1E6 copies / mL and amplified using two different detection systems. The Q-PCR reaction system and conditions are shown in Tables 11.1 and 11.2.

[0190] Table 11.1 Q-PCR reaction system

[0191] Components Added amount PCR reaction solution 19μL enzyme mixture 1μL sample 5μL

[0192] Table 11.2 Q-PCR reaction conditions

[0193]

[0194] The results showed that the ARMS-PCR mutant detection system exhibited a strong positive signal for the quality control sample; while the wild-type detection system did not produce a significant fluorescence signal (Ct value > 38, judged as negative) (results are shown in Table 11.3 and...). Figure 14The Taqman-MGB mutant detection system can distinguish between wild-type and A2064G mutants with Ct values ​​differing by at least 4 cycle numbers (results shown in Table 11.4), indicating that these two different template types are significantly different and can be specifically distinguished. This embodiment confirms that the genetically engineered bacterial quality control sample can accurately simulate the A2063G and A2064G mutation characteristics of clinical drug-resistant strains and is only recognized by primer-probe systems that specifically target drug resistance sites, meeting the standardized requirements for performance verification of drug resistance mutation detection reagents.

[0195] Table 11.3 Identification Results of Quality Control Samples of Mycoplasma pneumoniae 23S rRNA Drug-Resistant Mutant A2063G Engineered Bacteria

[0196]

[0197] Note: NoCt was not detected; a large Ct value indicates low detection efficiency.

[0198] Table 11.4 Identification Results of Quality Control Samples of Wild-type and Drug-resistant Mutant Mycoplasma pneumoniae 23S rRNA

[0199]

[0200] Note: A large Ct value indicates low detection efficiency.

Claims

1. A quality control product for Mycoplasma pneumoniae nucleic acid detection, characterized in that, The invention comprises engineered bacteria carrying a recombinant artificial chromosome, wherein the artificial chromosome integrates the full-length sequences of the Mycoplasma pneumoniae 16S rRNA gene, 23S rRNA gene, P1 adhesion protein gene, and community-acquired respiratory distress syndrome toxin gene, and the 23S rRNA gene contains at least one drug resistance mutation site of A2063G, A2064G, C2617T or A2067G. The nucleotide sequence of the 16S rRNA gene is shown in SEQ ID NO:1; the nucleotide sequence of the 23S rRNA gene is shown in SEQ ID NO:2-6, wherein SEQ ID NO:2 is the wild-type sequence of the 23S rRNA gene, and SEQ ID NO:3-6 are the mutant drug-resistant sequences of the 23S rRNA gene; the nucleotide sequence of the P1 adhesion protein gene is shown in SEQ ID NO:7; and the nucleotide sequence of the toxin gene is shown in SEQ ID NO:

8.

2. The nucleic acid detection quality control product according to claim 1, characterized in that, The engineered bacteria are Escherichia coli, and the recombinant artificial chromosome is integrated into the engineered bacteria through a bacterial artificial chromosome system, and replicates stably in the form of a low-copy large plasmid. Each engineered bacterium carries 1-2 copies of the artificial chromosome.

3. The nucleic acid detection quality control product according to claim 1 or 2, characterized in that, The drug resistance mutation site is precisely located at nucleotides 2063, 2064, 2617, or 2067 of the 23S rRNA gene.

4. A method for preparing the nucleic acid detection quality control product according to any one of claims 1-3, characterized in that, Includes the following steps: (a) Design and construct a shuttle vector containing the full-length nucleotide sequences of a synthetic Mycoplasma pneumoniae 16S rRNA gene, 23S rRNA gene, P1 gene and toxin gene, wherein the 23S rRNA drug resistance mutation gene contains at least one A2063G, A2064G, C2617T or A2067G drug resistance mutation site. (b) The shuttle vector from step (a) is transformed into engineered Escherichia coli, and engineered bacteria carrying recombinant artificial chromosomes are obtained by homologous recombination and integration into the bacterial artificial chromosome.

5. An application of the nucleic acid detection quality control product according to any one of claims 1-3, characterized in that, For use in at least one of the following scenarios: (a) As a positive control or calibrator, evaluate the analytical sensitivity (LOD), specificity, repeatability and reproducibility of the Mycoplasma pneumoniae nucleic acid detection kit, which includes multi-target combined detection reagents and drug resistance mutation detection reagents based on PCR, qPCR, isothermal amplification or digital PCR. (b) Used for internal quality control (IQC) or external quality assessment (EQA) in clinical laboratories to monitor the reliability of the entire process of nucleic acid extraction, amplification and analysis; (c) As a standard material, it supports the standardization of molecular diagnostic methods for Mycoplasma pneumoniae or the registration of reagent kits. (d) Evaluate the extraction efficiency and integrity of nucleic acid extraction reagents for long target gene DNA fragments.

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