A Mycoplasma pneumoniae whole genome capture method, primer set and kit

Abstract

The present invention discloses a method, primer set and kit for capturing the whole genome of Mycoplasma pneumoniae, belonging to the field of biomedicine. The present invention provides a primer set for detecting the whole genome of Mycoplasma pneumoniae, a primer set for amplifying the whole genome of Mycoplasma pneumoniae, a product for detecting the whole genome of Mycoplasma pneumoniae, a method for amplifying the whole genome of Mycoplasma pneumoniae, a method for detecting Mycoplasma pneumoniae virus in a sample, a method for constructing a library for detecting Mycoplasma pneumoniae virus in a sample, and related applications.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a method, a primer set and a kit for capturing the whole genome of Mycoplasma pneumoniae. Background Art

[0002] Mycoplasma pneumonia (MP), a small microorganism intermediate between bacteria and viruses, is the causative agent of human mycoplasma pneumonia. Mycoplasmas are widely distributed in nature, with over 80 species. M. pneumoniae belongs to a subgenus of the mycoplasma family and is so-called because of a specialized terminal structure at one end that enables it to adhere to the surface of respiratory epithelial cells, causing lung infection. M. pneumoniae is one of the leading pathogens causing community-acquired pneumonia. In addition to respiratory symptoms, severe cases can also present with extrapulmonary lesions. M. pneumoniae infection lacks specific clinical manifestations and has complex imaging findings. Currently, pathogen culture remains the gold standard for diagnosing M. pneumoniae infection, but it has limitations such as low sensitivity, making it ineffective for early diagnosis in clinical settings. Furthermore, the positive rate of M. pneumoniae serological antibody testing is low, with significant variations in sensitivity and specificity. With the development of molecular diagnostic technologies, these technologies have demonstrated significant advantages in sensitivity and specificity for M. pneumoniae detection and can be used for drug resistance detection. They may become an important tool for the rapid diagnosis of M. pneumoniae in the future.

[0003] Since its emergence in the 1960s, molecular diagnostic technology has evolved through the stages of molecular hybridization-based techniques, nucleic acid sequencing, molecular conformation-based molecular diagnostics, and quantitative PCR. Nucleic acid sequencing technology—whole genome sequencing (WGS), a commonly used genotyping technique—offers high resolution. It not only identifies single nucleotide polymorphisms (SNPs) across the genome to determine the microevolution of MPs during transmission, but also allows for the complete sequence of the target genome, enabling the prediction of resistance to most drugs. Application of NGS in MP resistance testing: NGS offers advantages such as simplicity, speed, accuracy, and reliability. Whole-genome sequencing (WGS) allows for the generation of complete MP genome sequences. Leveraging known MP resistance mechanisms, relevant resistance mutations can be detected at the genome-wide level, expanding the use of molecular biology-based resistance testing beyond the limitations of targeting one or a few first-line drugs. Whole-genome sequencing is often used in drug resistance research to discover new resistance loci and elucidate novel resistance mechanisms. Therefore, obtaining the complete MP genome is of great significance. In recent decades, significant advances have been made in the research of whole-genome amplification technologies. Whole genome amplification (WGA) technology can amplify small amounts of DNA samples without loss, bias, or bias. Based on their principles, WG amplification methods can be divided into three categories: polymerase chain reaction (PCR)-based whole genome amplification, isothermal-based whole genome amplification, and hybrid whole genome amplification. Summary of the Invention

[0004] In order to make up for the deficiencies of the prior art, the present invention provides the following technical solutions.

[0005] This patent application utilizes a single-cell amplification method called multiple displacement amplification (MDA), which is an isothermal strand displacement amplification. Under constant temperature, random primers consisting of six random bases randomly anneal with the template, followed by a strand displacement reaction under the action of phi29 DNA polymerase. The displaced single strands can then randomly bind to the primers, anneal, and extend, ultimately forming branched amplification.

[0006] The MDA amplification system, developed by improving upon traditional WGA technology, is currently the most popular isothermal amplification technique. The MDA amplification process involves the following steps: first, random 6-mer primers anneal to the template DNA at multiple sites at 30°C. Then, under the action of phi29 DNA polymerase, strand displacement amplification occurs. The resulting single-stranded product becomes a new replication template, forming a hyperbranched structure. This cycle continues, ultimately producing amplification products with a size >10 kb. The advantages of this system include low error rates and amplification bias, high amplification efficiency and fidelity, and long amplification products. This technique allows for the generation of large quantities of high-molecular-weight DNA. However, because MDA is a constant-temperature amplification method, it is subject to certain nonspecific amplification issues. Furthermore, this method places relatively high demands on template quality, and non-uniform amplification can occur under suboptimal experimental conditions.

[0007] The present invention provides a primer set for detecting the whole genome of Mycoplasma pneumoniae. The sequence of the primer set is shown in any one or more groups of CGCGAA, TAACGGTT, TAACGGTA, CGCTTAAT, CCGCTTTA, AACGCTTTA, AAACGGTT, AACGCTG, TTAACGAA, and CGGTTTTA.

[0008] Furthermore, the primer set includes a label with a detectable marker.

[0009] Furthermore, the detectable label may be directly bound to the primer or may be bound via a linker.

[0010] In the present invention, nucleic acid sequence or nucleic acid molecule refers to a deoxyribonucleotide or ribonucleotide polymer, including but not limited to cDNA, mRNA, genomic DNA and synthetic (e.g., chemically synthesized) DNA or RNA. Nucleic acid can be double-stranded (ds) or single-stranded (ss). When the nucleic acid is single-stranded, it can be a positive strand or an antisense strand. Nucleic acid can include natural nucleotides (e.g., A, T / U, C, and G) and can contain analogs of natural nucleotides, such as labeled nucleotides. Target nucleic acid molecule refers to a nucleic acid molecule intended to be detected, quantitatively detected, qualitatively detected, or a combination thereof. Nucleic acid molecules do not have to be in a purified form. Various other nucleic acid molecules can also coexist with the target nucleic acid molecule. For example, the target nucleic acid molecule can be a specific nucleic acid molecule intended to be amplified. If necessary, the purification or separation of the target nucleic acid molecule can be carried out by methods known to those skilled in the art, such as using commercially available purification kits, etc. In one embodiment, the target nucleic acid molecule is a novel coronavirus nucleic acid sequence.

[0011] Furthermore, the detectable markers include fluorescent dyes, chemiluminescent compounds, radioactive isotopes, enzymes, colored particles, and biotin.

[0012] Furthermore, the fluorescent dye includes fluorescent organic dyes, fluorescent quantum dots, fluorescent doublets, fluorescent carbon dots, graphene quantum dots or other carbon-based fluorescent nanostructures, fluorescent proteins, and fluorescent DNA origami-based nanostructures.

[0013] Furthermore, the chemiluminescent compound includes luminol, acridinium ester, terpyridine ruthenium, lucigenin, lophanine, and peroxy oxalate esters.

[0014] Furthermore, the radioisotope includes 3 H. 123 I. 125 I. 131 I. 18 F. 11 C. 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 32 P. 33 P. 35 S.

[0015] Furthermore, the enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, and acetylcholinesterase.

[0016] Furthermore, the colored particles include gold, silver, and platinum particles.

[0017] For convenience, the term "colored particles" will be used, but the present invention contemplates embodiments using other forms of detectable particles. In embodiments utilizing colored particles, the particles can be gold, silver, or platinum particles. In some embodiments, the particle diameter is from about 60 to about 120 nm.

[0018] In this document, the terms "fluorescent dye", "fluorophore", "fluorochrome", and "dye" are used interchangeably to refer to a fluorescent chemical compound or structure, and may in particular be one of the following: fluorescent organic dyes, fluorescent quantum dots, fluorescent dyads, fluorescent carbon dots, graphene quantum dots or other carbon-based fluorescent nanostructures, fluorescent proteins, and fluorescent DNA origami-based nanostructures. Among organic fluorescent dyes, the term "fluorescent dye" refers in particular to derivatives of xanthenes (e.g., fluorescein, rhodamine, Oregon Green, Texas), cyanines (e.g., cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine), derivatives, squaraine rotaxane derivatives, naphthalene, coumarin, oxadiazole, anthracene (anthraquinone, DRAQ5, DRAQ7, CyTRAK Orange), pyrene (Cascade Blue), oxazines (Nile Red, Nile Blue, Cresyl Violet, Oxazine 170), acridines (proflavine, acridine orange, acridine yellow), arylmethines (auramine, crystal violet, malachite green), tetrapyrroles (porphine, phthalocyanine, bilirubin), dipyrromethene (BODIPY, aza-BODIPY), phosphorescent dyes or luminescent dyes. The following trademark groups designate commercially available fluorescent dyes that may include dyes belonging to different chemical families: CF dyes (Biotium), DRAQ and CyTRAK probes (BioStatus), BODIPY (Invitrogen), EverFluor (Setareh Biotech), Alexa Fluor (Invitrogen), BellaFluor (Setareh Biotech), DyLight Fluor (Thermo Scientific), Atto and Tracy (Sigma-Aldrich), FluoProbes (Interchim), Abberior dyes (Abberior Dyes), Dy and MegaStokes dyes (Dyomics), Sμlfo Cy dyes (Cyandye), HiLyte Fluor (AnaSpec), Seta, SeTau, and Square dyes (SETA BioMedicals), Quasar and Cal Fluor dyes (Biosearch Technologies), SureLight dyes (Columbia Biosciences), Vio dyes (Milteny Biotec) (List modified from: https: / / en.wikipedia.org / wiki / FluorophoreIn the group of fluorescent proteins, in particular members of the green fluorescent protein (GFP) family, including GFP and GFP-like proteins (e.g., DsRed, TagRFP) and their (monomeric) derivatives (e.g., EBFP, ECFP, EYFP, Cerμlaen, mTurquoise2, YFP, EYFP, mCitrine, Venus, YPet, Superfolder GFP, mCherry, mPlum), are referred to herein as "fluorescent dyes". Furthermore, in the group of fluorescent proteins, the term "fluorescent dye" may include herein fluorescent proteins whose absorbance or emission characteristics change upon ligand binding, such as BFPms1, or in response to environmental changes, such as redox-sensitive roGFP or pH-sensitive variants. Furthermore, in the group of fluorescent proteins, the term "fluorescent dye" may include herein derivatives of cyanobacterial phycobiliproteins, such as small superred fluorescent proteins (smURFPs), and fluorescent protein nanoparticles that can be derived from smURFPs. An overview of fluorescent proteins can be found in Rodriguez et al., 2017, Trends Biochem Sci. 2017 Feb; 42(2): 111-129. The term "fluorescent dye" in this document may also refer to fluorescent quantum dots. The term "fluorescent dye" in this document may also refer to fluorescent carbon dots, fluorescent graphene quantum dots, fluorescent carbon-based nanostructures, as described by Yan et al., 2019, Microchimica Acta (2019) 186: 583 and Iravani and Varma, 2020, Environ Chem Lett. 2020 Mar 10: 1-25. The term "fluorescent dye" in this document may also refer to fluorescent polymer dots (Pdots) or nanodiamonds. The term "fluorescent dye" in this document may also refer to a fluorescent dyad, such as the dyad of Perylene Antenna and Triangelium Emitter as described in Kacenauskaite et al. 2021 J. Am. Chem. Soc. 2021, 143, 1377-1385.

[0019] As used herein, a "label" or "detectable marker" is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or any other means. For example, useful labels include radiolabeled molecules, fluorophores, luminescent compounds, electron-dense reagents, enzymes (e.g., as commonly used in ELISA), biotin, digoxigenin, or haptens and proteins, which can be made detectable, for example, by incorporating the label into a peptide or used to detect antibodies that specifically react with the peptide.

[0020] The term "fluorophore" includes any compound, composition, or molecule that is capable of emitting light in response to illumination. In many cases, fluorophores emit light in the visible region. In other cases, fluorophores can emit light in the non-visible region of light, such as ultraviolet light, near ultraviolet light, near infrared light, and infrared light. For example, but not limited to, examples of fluorophores include: quantum dots; nanoparticles; fluorescent proteins, such as green fluorescent protein and yellow fluorescent protein; heme-based proteins or their derivatives; carbocyanine-based chromophores, such as IRDye800CW, Cy 3, and Cy 5; coumarin-based chromophores, such as (7-diethylamino-3-(4'-maleimidophenyl)-4-methylcoumarin) (CPM); fluorine-based chromophores, such as fluorescein, fluorescein isothiocyanate (FITC); and many ALEXAFLUORs. TM Chromophores and ALEXAFLUOR TM Bioconjugates that absorb in the visible and near infrared spectra. Emission from the fluorophore can be detected by any number of methods, including but not limited to fluorescence spectroscopy, fluorescence microscopy, fluorometers, fluorescence plate readers, infrared scanner analysis, laser scanning confocal microscopy, automated confocal nanoscanning, laser spectrophotometers, fluorescence activated cell sorters (FACS), image-based analyzers, and fluorescence scanners (e.g., gel / membrane scanners).

[0021] The term "radioisotope" is used in this specification to refer to a specific radioisotopic substance, and indeed, multiple radioisotopes of a specific radioisotopic substance may be formed when performing the method. The present invention also does not exclude the possibility of simultaneously producing multiple radioisotopic substances, for example, by simultaneously irradiating multiple target elements. For the purposes of this specification, reference to "radioisotope" refers to "at least one" radioisotopic substance, and thus may include multiple different radioisotopic substances, and reference to "radioisotope" refers to multiple radioisotopes of the at least one substance, and thus may include multiple radioisotopes of multiple radioisotopic substances.

[0022] Unless the context clearly indicates otherwise, the term "element" as used in this specification is used in the chemical sense according to the elements listed in the Periodic Table of Elements.

[0023] For purposes of this disclosure, it is understood that the terms "biotin" or "free biotin" are used interchangeably and refer to the naturally occurring compound, ie, D(+)-biotin.

[0024] Furthermore, the primer set includes at least one modified nucleotide.

[0025] Furthermore, the modified nucleotides include 2'-modified nucleotides or 5-methylcytosine.

[0026] Furthermore, the 2'-modified nucleotides include 2'-O-methyl modified nucleotides or 2'-fluoro modified nucleotides.

[0027] Furthermore, the 5-methylcytosine includes 5-methyl-deoxycytosine.

[0028] Furthermore, the 5-methyl-deoxycytosine includes 5-Me-dC and 5-methyl-2'-deoxycytosine.

[0029] The present invention provides a primer set for amplifying the whole genome of Mycoplasma pneumoniae, the primer set comprising:

[0030] 1) a nucleic acid sequence complementary to the primer set described above;

[0031] 2) A nucleic acid sequence that hybridizes with the nucleic acid sequence described in 1) under stringent conditions.

[0032] In the present invention, complementary nucleic acid sequences are made up of two complementary chains of double-stranded DNA or RNA chains consisting of base pairs. When the bases of one nucleic acid molecule form hydrogen bonds with the bases of another nucleic acid molecule, complementary binding occurs. In general, the base adenine (A) is complementary to thymine (T) and uracil (U), while cytosine (C) is complementary to guanine (G). For example, the sequence 5'-ATCG-3' of an ssDNA molecule can form a bond with the 3'-TAGC-5' of another ssDNA to form dsDNA. In this example, the sequence 5'-ATCG-3' is reversely complementary to 3'-TAGC-5'. Nucleic acid molecules can also complement each other even when all the bases of each molecule do not fully form hydrogen bonds. For example, hybridization with complementary nucleic acid sequences can occur under conditions of different stringencies, where the complementary chains will bind at some but not all nucleotide sites.

[0033] In the present invention, stringent conditions refer to conditions under which specific hybridization is formed and non-specific hybridization is not formed. For example, the following conditions can be mentioned: conditions under which a DNA having a high identity (identity of 90% or more, preferably 95% or more) to a DNA consisting of a sequence represented by SEQ ID NOs: 1-10 hybridizes with a DNA consisting of a base sequence complementary to the DNA consisting of a sequence represented by SEQ ID NOs: 1-10. Generally, hybridization is formed at a temperature of about 5°C to about 30°C, preferably about 10°C to about 25°C, below the melting temperature (Tm) for complete hybridization.

[0034] In the present invention, hybridization means that two completely or partially complementary nucleic acid sequences combine together in a parallel or antiparallel direction under specific hybridization test conditions, thereby forming the ability of a stable structure with a double-stranded region. The two component chains of this double-stranded structure (sometimes referred to as hybrid chains) are combined together by hydrogen bonds. Although these hydrogen bonds are most commonly formed between nucleotides containing the bases adenine and thymine or uracil (A and T or U) or cytosine and guanine (C and G) on a single nucleic acid chain, base pairing can also be formed between bases that are not members of these "classical" pairs. Non-canonical base pairing is well known in the art.

[0035] The present invention provides a product for detecting the whole genome of Mycoplasma pneumoniae, which comprises the primer set described above.

[0036] Furthermore, the products include chips, nucleic acid membrane strips, and test kits.

[0037] Furthermore, the kit also includes PCR amplification buffer and amplification enzyme.

[0038] Furthermore, the amplification enzyme includes DNA polymerase and / or RNA polymerase.

[0039] Furthermore, the kit also includes a reverse transcription reaction system.

[0040] Furthermore, the reverse transcription reaction system includes primers.

[0041] Furthermore, the primers are random primers.

[0042] Furthermore, the kit also includes instructions.

[0043] Furthermore, the kit includes Thermo Qubit 1X dsDNA HS (high sensitivity) quantitative kit, 1.8 times the volume of Beijing Micro Future PCR product recovery magnetic beads (purification kit), MagMAX TM Virus / Pathogen Ultra Nucleic Acid Isolation Kit (Nucleic Acid Extraction Kit), Illumina DNA Prep Library Preparation Kit.

[0044] Furthermore, the kit contains ultra-sensitive Mycoplasma pneumoniae whole genome capture reagents, including EquiPhi29 TM DNA Polymerase (Reagent AMix), exonuclease-resistant targeted primers (Reagent B Mix), dNTP mix (Reagent C Mix), EquiPhi29 TM DNA Polymerase (Reagent D Mix), EquiPhi29TM DNA Polymerase (Reagent E Mix), Pyrophosphatase, inorganic (Reagent FMix), Ambion TM Nuclease-free water (Nuclease-free water).

[0045] In the present invention, a suitable amount of one or more primers is provided in one or more containers, or fixed on a matrix. The primer can be provided as a suspension in an aqueous solution, or as a freeze-dried or lyophilized powder, for example. The container in which the nucleic acid is provided can be any conventional container capable of accommodating the provided form, such as a microcentrifuge tube, ampoule or bottle. The kit can contain labeled or unlabeled probes for detecting digestive tract viral nucleotide sequences. The amount of the nucleic acid primer provided in the kit can be any suitable amount and can depend on the target market for the product.

[0046] The present invention provides a method for amplifying the whole genome of Mycoplasma pneumoniae, which comprises using the aforementioned primer set or the aforementioned product to perform an amplification reaction to obtain a DNA amplification product.

[0047] Furthermore, the amplification reaction is performed by the MDA method.

[0048] Furthermore, the amplification method includes sample nucleic acid extraction, whole genome capture, and product recovery.

[0049] In some embodiments, the extraction is performed according to the corresponding requirements and steps in the bacterial nucleic acid extraction kit, and the final nucleic acid elution volume is recommended to be 20-50 μl. It is recommended to use MagMAX TM Nucleic acid extraction was performed using the Viral / Pathogen Ultra Nucleic Acid Isolation Kit (Cat. No. A42356).

[0050] In some embodiments, (1) Place the components of the kit on ice or thaw at 4°C in advance, invert and mix, and then place in a centrifuge for brief centrifugation. (2) Add the following reagents to a 0.2ml thin-walled PCR tube that does not contain nucleic acids on ice: Reagent AMix 2μl, Reagent B Mix 5μl, Reagent C Mix 2μl, Reagent D Mix 2μl, DNA Template 1μl, Nuclease-free water 6μl, for a total volume of 18μl. (3) Gently mix the above reaction solution and briefly centrifuge it, and incubate it according to the following procedure: 105°C hot cover, 95°C 5min, 4°C Hold. (4) After the reaction is completed, place the PCR tube on ice and add the following reagents: 18μl of the reaction solution in the previous step, 1μl of Reagent E Mix, 1μl of Reagent F Mix, for a total volume of 18μl.

[0051] (5) Gently mix the reaction solution and centrifuge briefly. Amplify according to the following procedure: 85℃

[0052] Heat cover, 42℃ for 4h, 65℃ for 10min, hold at 4℃.

[0053] In some embodiments, the amplified product is purified using 1.8 volumes of Beijing Micro Future purification magnetic beads. The beads should be equilibrated at room temperature for 30 minutes and fully activated by vortexing. Otherwise, the purification recovery rate will be affected. The purified product can be used for subsequent library construction or stored at -20°C for future use if not directly used for subsequent experiments.

[0054] The present invention provides a method for detecting Mycoplasma pneumoniae virus in a sample, the method comprising using the aforementioned primer set, the aforementioned product, or the aforementioned method to perform amplification to obtain an amplified product, and using the amplified product for sequencing.

[0055] Furthermore, the sample includes a human sample.

[0056] Furthermore, the human sample includes tissue samples, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous humor, lymph fluid, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tumor lysate, tissue culture fluid, tissue extracts, homogenized tissue, tumor tissue, cell extracts, alveolar lavage fluid, nasal swabs, and throat swabs.

[0057] The present invention provides a method for constructing a library for detecting Mycoplasma pneumoniae virus in a sample, the construction method comprising fragmenting the DNA amplification product obtained by the aforementioned amplification method, purifying the DNA after fragmentation and labeling, amplifying the fragmented and labeled DNA, purifying the library, and performing library quality control.

[0058] Furthermore, the DNA fragmentation includes the following steps: 1.1 Qubit quantification, add 2-30μl DNA (1-500ng) to the PCR tube; *Note: For multiple samples, the concentration should preferably be greater than 50 or less than 50ng to facilitate subsequent PCR amplification. 1.2 Add pure water to the sample well to make the total volume 30μl; 1.3 Vortex BLT for 10s to check whether the magnetic beads are completely suspended; 1.4 Prepare the fragmentation master reaction solution, each reaction: BLT 11μl + TB111μl, vortex the master reaction solution to mix; 1.5 Take 20μl of the master reaction solution and add it to the PCR sample well, vortex to mix (a total of 50μl); 1.6 Place in the PCR instrument and run the TAG Program (reaction system: 50μl / well): hot cover 100℃, 55℃ for 15 minutes, hold at 10℃.

[0059] Furthermore, the purification includes step 2.1 adding 10μl TSB to the fragmentation reaction solution, gently pipetting and mixing 10 times, and resuspending the beads. *Note: TSB (check for precipitation) and TWB are stored at 15-30°C and restored to room temperature before use. 2.2 Run the program in the PCR instrument: (set the hot cover to 100°C, the system is 60μl); 37°C, 15min; store at 10°C. 2.3 Place the reaction tube in the magnetic stand, wait for the liquid to clarify (about 3 minutes), aspirate and discard the supernatant. 2.4 Remove the reaction tube from the magnetic stand, slowly add 100μl TWB, and resuspend the magnetic beads, place the reaction tube in the magnetic stand, wait for the liquid to clarify (about 3 minutes), aspirate and discard the supernatant. 2.5 Repeat step 2.4 once. 2.6 Remove the reaction tube from the magnetic rack, slowly add 100 μl of TWB, resuspend the magnetic beads, place the reaction tube in the magnetic rack, and wait for the liquid to clarify (about 3 minutes).

[0060] Further, amplify the fragmented and tagged DNA as in step 3.1. Prepare the PCR master reaction solution (for each sample): 22 μl EPM + 22 μl water, vortex to mix, and flick. *Note: EPM: Thaw on ice. Invert to mix, then briefly centrifuge. DNAAdapter: Thaw at room temperature. For tubes, vortex to mix, then briefly centrifuge. For well plates, briefly centrifuge before use. 3.2 Remove the supernatant from the magnetic rack and immediately add 40 μl of the PCR master reaction solution to each sample well and mix thoroughly. 3.3 For IndexAdapter Tubes, add 5 μl of the i7 adapter and 5 μl of the i5 adapter, respectively, and pipette to mix thoroughly. 3.4 Run the PCR program: Set the heated lid to 100°C, the volume to 50 μl, and determine the number of PCR cycles based on the total DNA input amount. For samples from 1-9 ng, use 12 cycles, 10-24 ng for 8 cycles, 25-49 ng for 6 cycles, 5 cycles for 50-99 ng, 5 cycles for 100-500 ng, and 5 cycles for blood / saliva. The cycling program is 68°C for 3 min, 98°C for 3 min, then 98°C-62°C-68°C for 45 s-30 s-2 min (X cycles), where the specific value of X depends on the total DNA input amount, and 68°C for 1 min. *Note: Safe-holding point: 4°C can be stored for up to 2 days. Alternatively, place on the PCR plate for a maximum of 24 hours.

[0061] Furthermore, the library purification includes step 4.1 centrifuging the reaction tube and placing it on a magnetic stand, waiting for the liquid to clarify (about 5 minutes). 4.2 Aspirating and transferring 45μl of supernatant to a new centrifuge tube. 4.3 Thoroughly vortexing to resuspend SPB. *Note: SPB needs to be restored to room temperature and vortexed before use. 4.4 For 100-500ng of starting DNA, perform the following steps: a. Add 40μl H2O to the reaction tube containing supernatant. b. Add 45μl SPB to the reaction tube, mix thoroughly, and incubate at room temperature for 5 minutes. c. Place the reaction tube on a magnetic stand and wait for the liquid to clarify (about 5 minutes). d. During incubation, vortex the SPB thoroughly, then take 15μl and add it to a new centrifuge tube. e. Transfer 125μl of supernatant from the reaction tube on the magnetic stand to a new centrifuge tube (containing 15μl SPB) and mix thoroughly. 4.5 For <100ng starting DNA, perform the following steps: a. Add 81μl SPB to the reaction tube containing the supernatant and mix thoroughly. 4.6 Incubate at room temperature for 5 minutes, place the reaction tube on a magnetic stand, wait for the liquid to clarify (about 5 minutes), aspirate and discard the supernatant. 4.7 Add 200μl freshly prepared 80% ethanol to the reaction tube, incubate for 30s, discard the supernatant, and repeat this step once. 4.8 Keep the reaction tube on the magnetic stand, aspirate and discard the residual ethanol in the reaction tube, and let it dry for 5 minutes. 4.9 Remove the reaction tube from the magnetic stand, add 32μl RSB to resuspend the magnetic beads, and incubate at room temperature for 2 minutes. 4.10 Place the reaction tube on a magnetic stand, wait for the liquid to clarify (about 2 minutes), and transfer 30μl supernatant to a new centrifuge tube.

[0062] Furthermore, the library quality control includes the steps of taking 5.11 μl of the constructed library and running it on an Agilent Bioanalyzer 2100 (optional). 5.2 Qubit quantification; molar concentration (nM) = mass concentration (ng / μl) * 106 / (660 * average fragment length of the library).

[0063] The present invention provides the use of the aforementioned primer set in preparing a product for amplifying or detecting Mycoplasma pneumoniae virus in a sample.

[0064] The present invention provides the use of the aforementioned primer set or the aforementioned product in detecting Mycoplasma pneumoniae virus in a sample. DETAILED DESCRIPTION

[0065] The present invention will be further described below with reference to specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the present invention. The main features of the present invention may be applied to various embodiments without departing from the scope of the present invention.

[0066] Example

[0067] 1. Experimental Materials

[0068] The primers for Mycoplasma pneumoniae are shown in Table 1.

[0069] Table 1

[0070]

[0071]

[0072] Mycoplasma pneumoniae DNA samples, sample requirements: alveolar lavage fluid, nasopharyngeal / oropharyngeal swabs, sputum, etc. The above positive samples can be used for Mycoplasma pneumoniae whole genome capture after nucleic acid extraction.

[0073] Ultra-Sensitive Mycoplasma Pneumoniae Whole Genome Capture Kit (Low Capacity), detailed component information is shown in Table 2.

[0074] Table 2

[0075]

[0076]

[0077] Instruments: Thermo Qubit instrument, Bio-Rad vortex mixer, centrifuge, PCR instrument, PCR tubes, pipettes.

[0078] Reagents: Thermo Qubit 1× dsDNA HS (High Sensitivity) Quantitation Kit, 1.8 times the volume of Beijing Micro Future PCR Product Recovery Magnetic Beads (Purification Kit) MagMAX TM Virus / Pathogen Ultra Nucleic Acid Isolation Kit (Nucleic Acid Extraction Kit), Illumina DNA Prep Library Preparation Kit.

[0079] 2. Experimental methods

[0080] 1) Sample nucleic acid extraction.

[0081] Extract according to the corresponding requirements and steps of the bacterial nucleic acid extraction kit. The final nucleic acid elution volume is recommended to be 20-50μl.

[0082] MagMAX is recommended TM Nucleic acid extraction was performed using the Viral / Pathogen Ultra Nucleic Acid Isolation Kit (Cat. No. A42356).

[0083] 2) Whole-genome capture.

[0084] (1) Thaw all components of this kit on ice or at 4°C in advance, mix thoroughly by inversion, and centrifuge briefly.

[0085] (2) Add the reagents in Table 3 to a 0.2 ml thin-walled PCR tube containing no nucleic acid on ice.

[0086] Table 3

[0087]

[0088]

[0089] Gently mix the reaction solution, centrifuge briefly, and incubate according to the procedure in Table 4.

[0090] Table 4

[0091] program temperature time - 105℃ Hot cover 1 95℃ 5min 2 4℃ Hold

[0092] After the reaction is complete, place the PCR tube on ice and add the reagents listed in Table 5.

[0093] Table 5

[0094] Components volume Previous step reaction solution 18 μl Reagent E Mix 1 μl Reagent F Mix 1 μl Total 20 μl

[0095] The reaction solution was gently mixed and centrifuged briefly, and amplification was performed according to the procedure in Table 6.

[0096] Table 6

[0097] program temperature time - 85℃ Hot cover 1 42℃ 4h 2 65℃ 10min 3 4℃ Hold

[0098] 3) Product recovery.

[0099] It is recommended to use 1.8 times the volume of Beijing Micro Future purification magnetic beads to purify the above amplified products. The purified magnetic beads should be equilibrated at room temperature for 30 minutes and fully activated by vortexing. Failure to do so will affect the purification recovery rate. The purified product can be used for subsequent library construction. If not used directly for subsequent experiments, it can be stored at -20°C for future use.

[0100] Detection methods and limitations

[0101] The test results of this kit are for clinical reference only and shall not be used as a standard for clinical diagnosis.

[0102] The sample test results are closely related to the collection, preservation, processing of the samples, as well as the extraction and preservation of nucleic acids. Any improper handling and operation may affect the accuracy of the test results.

[0103] Clinical laboratories should strictly equip equipment and operators in accordance with the requirements of the "Clinical Gene Amplification Laboratory Work Specifications" and perform standardized operations in strict accordance with the instructions for use of the test kit.

[0104] 4) Purification.

[0105] After the amplification reaction is completed, herpes zoster virus gene fragments of different sizes will be obtained in one tube of PCR reaction. After the experiment is completed, the 5 tubes of amplified products are concentrated in a 1.5ml EP tube. After purification and qubit quantification, the subsequent library construction experiment can be directly carried out.

[0106] 4.1 Add 250μl AMPure XP beads; *Note: This step is for 1,0×-1.5× AMPure XP beads.

[0107] 4.2 Oscillate at 1800 rpm for 2 min; incubate for 5 min.

[0108] 4.3 Let stand on the magnetic rack for 2 minutes or until clarified, then discard the supernatant.

[0109] 4.4 Add 500 μl of freshly prepared 80% ethanol, place on the magnetic stand for 30 seconds, and discard the supernatant.

[0110] 4.5 Repeat the above steps.

[0111] 4.6 Use a 10 μl pipette tip to remove any remaining liquid.

[0112] 4.7 Dry on a magnetic rack for 2 minutes.

[0113] 4.8 Remove from the magnetic stand and add 40 μl RSB.

[0114] 4. Oscillate at 1800 rpm for 2 min, then incubate at room temperature for 2 min.

[0115] 4.10 Let it stand on the magnetic rack until the liquid becomes clear.

[0116] 4.11 Transfer 38 μl of supernatant to a new plate.

[0117] 5) Quantification.

[0118] 1. Instrument: Qubit.

[0119] 2. Reagents: Qubit dsDNA HS (High Sensitivity) Kit (make sure all reagents are at room temperature before use).

[0120] 3. Operation process.

[0121] a) Add 1 μl dye reagent to 199 μl buffer, vortex mix to obtain the working solution, and protect from light until use.

[0122] b) Transfer 1-20 μl of library to Qubit working solution for a final volume of 200 μl.

[0123] c) Vortex for 2-3 seconds and incubate at room temperature in the dark for 2 minutes.

[0124] d) Place the Qubit on the plate and perform the assay.

[0125] 4. Reference Document: QUICK REFERENCE - Qubit TM Assays (MAN0017210 vA.0).

[0126] 3. Library construction operation process.

[0127] 1) The DNA amplification products obtained by the aforementioned amplification are subjected to DNA fragmentation.

[0128] 1.1 Using Qubit quantification, add 2-30 μl of DNA (1-500 ng) to a PCR tube. *Note: For multiple samples, the concentration should ideally be greater than 50 or less than 50 ng to facilitate subsequent PCR amplification.

[0129] 1.2 Add pure water to the sample wells to bring the total volume to 30 μl.

[0130] 1.3 Vortex BLT for 10 seconds to check whether the magnetic beads are thoroughly suspended.

[0131] 1.4 Prepare the fragmentation master reaction solution. For each reaction, add 11 μl of BLT and 11 μl of TB1. Vortex the master reaction solution to mix thoroughly.

[0132] 1.5 Add 20 μl of the main reaction solution to the PCR sample well and vortex to mix (a total of 50 μl).

[0133] 1.6 Place in a PCR instrument and run the TAG program (reaction system: 50 μl / well): heat cover at 100°C, 55°C for 15 minutes, hold at 10°C.

[0134] 2) Purification.

[0135] 2.1 Add 10 μl of TSB to the fragmentation reaction solution, gently pipette up and down 10 times to mix, and resuspend the beads. *Note: TSB (check for precipitation) and TWB should be stored at 15-30°C and brought to room temperature before use.

[0136] 2.2 Run the program in a PCR instrument: (set the heated lid to 100°C, the system is 60 μl); 37°C, 15 min; store at 10°C.

[0137] 2.3 Place the reaction tube in the magnetic rack, wait for the liquid to clarify (about 3 minutes), aspirate and discard the supernatant.

[0138] 2.4 Remove the reaction tube from the magnetic rack, slowly add 100 μl of TWB, resuspend the magnetic beads, place the reaction tube in the magnetic rack, wait for the liquid to clarify (about 3 minutes), aspirate and discard the supernatant.

[0139] 2.5 Repeat step 2.4 again.

[0140] 2.6 Remove the reaction tube from the magnetic rack, slowly add 100 μl of TWB, resuspend the magnetic beads, and place the reaction tube back into the magnetic rack. Allow the liquid to clear (approximately 3 minutes). Keep the reaction tube on the magnetic rack until you proceed to step 3.2 of the Amplify Fragmented and Tagged DNA section.

[0141] 3) Amplify the fragmented and tagged DNA.

[0142] 3.1 Prepare the PCR master reaction solution (per sample): 22 μl EPM + 22 μl water, vortex to mix, and flick gently. *Note: EPM: Thaw on ice. Invert to mix, then centrifuge briefly. DNAAdapter: Thaw at room temperature. For tubes, vortex to mix, then centrifuge briefly. For well-plates, briefly centrifuge before use.

[0143] 3.2 Remove the supernatant from the magnetic rack and immediately add 40 μl of PCR master reaction solution to the sample well and mix thoroughly.

[0144] 3.3 For Index Adapter Tubes, add 5 μl of i7 adapter and 5 μl of i5 adapter respectively and mix thoroughly by pipetting.

[0145] 3.4 Run the PCR program: set the heated lid to 100°C and the volume to 50 μl.

[0146] The number of PCR cycles is determined based on the total DNA input amount: 12 cycles for 1-9 ng, 8 cycles for 10-24 ng, 6 cycles for 25-49 ng, 5 cycles for 50-99 ng, 5 cycles for 100-500 ng, and 5 cycles for blood / saliva. The cycling program is 68°C for 3 min, 98°C for 3 min, then 98°C-62°C-68°C for 45 s-30 s-2 min (X cycles). The specific value of X depends on the total DNA input amount, and 68°C for 1 min. *Note: Safe holding point: 4°C can be stored for up to 2 days. Alternatively, the sample can be placed in the PCR machine for a maximum of 24 hours.

[0147] 4) Library purification.

[0148] 4.1 Centrifuge the reaction tube and place it on a magnetic rack to wait for the liquid to clarify (about 5 minutes).

[0149] 4.2 Pipette and transfer 45 μl of supernatant to a new centrifuge tube.

[0150] 4.3 Vortex thoroughly to resuspend SPB.

[0151] *Note: SPB needs to be returned to room temperature and vortexed before use.

[0152] 4.4 For 100-500 ng of starting DNA, perform the following steps: a. Add 40 μl of HO to the reaction tube containing the supernatant. b. Add 45 μl of SPB to the reaction tube, mix thoroughly, and incubate at room temperature for 5 minutes. c. Place the reaction tube on a magnetic rack and wait for the liquid to clear (approximately 5 minutes). d. During the incubation, vortex the SPB thoroughly and transfer 15 μl to a new microcentrifuge tube. e. Transfer 125 μl of the supernatant from the reaction tube on the magnetic rack to a new microcentrifuge tube (containing 15 μl of SPB) and mix thoroughly.

[0153] 4.5 For <100 ng starting DNA, perform the following steps: Add 81 μl SPB to the reaction tube containing the supernatant and mix thoroughly.

[0154] 4.6 Incubate at room temperature for 5 minutes. Place the reaction tube on a magnetic rack and wait for the liquid to clear (approximately 5 minutes). Aspirate and discard the supernatant.

[0155] 4.7 Add 200 μl of freshly prepared 80% ethanol to the reaction tube, incubate for 30 seconds, discard the supernatant, and repeat this step once.

[0156] 4.8 Keep the reaction tube on the magnetic rack, aspirate and discard the remaining ethanol in the reaction tube, and let it dry for 5 minutes.

[0157] 4.9 Remove the reaction tube from the magnetic stand, add 32 μl RSB to resuspend the magnetic beads, and incubate at room temperature for 2 minutes.

[0158] 4.10 Place the reaction tube on a magnetic rack, wait for the liquid to clarify (about 2 minutes), and transfer 30 μl of the supernatant to a new centrifuge tube.

[0159] 5) Library quality control.

[0160] 5.1 Take 1 μl of the constructed library and run it on Agilent Bioanalyzer 2100 (optional).

[0161] 5.2 Qubit quantification; molar concentration (nM) = mass concentration (ng / μl) * 106 / (660 * average fragment length of the library).

[0162] 4. Operate the MiSeq instrument.

[0163] Dilute and denature the library.

[0164] 1. Denature the library with NaOH.

[0165] 1) Recommended loading concentrations: For MiSeq v3 kits, the sample starting concentration is 4 nM, and the library loading concentration range is 6-20 pM. For MiSeq v2 kits, the sample starting concentration is 4 nM, and the library loading concentration range is 6-20 pM.

[0166] II) Preparation: 1N NaOH, PCR-grade water, HT1, pipettes and tips, vortex shaker, and handheld centrifuge.

[0167] 3) Operation steps: 1.20 μl 1N NaOH + 80 μl ultrapure water to obtain 0.2N NaOH.

[0168] 2. Vortex for 3-5 seconds and centrifuge briefly. Repeat once.

[0169] 3.5 μl 4 nM or 2 nM library + 5 μl 0.2 N NaOH.

[0170] 4. Vortex for 3-5 seconds and centrifuge briefly. Repeat once.

[0171] 5. Denature at room temperature for 5-10 minutes.

[0172] 6. Add 990 μl of ice-cold HT19.

[0173] 7. Vortex for 5-10 seconds and centrifuge briefly. Repeat once.

[0174] 8. Based on the library type and optimized loading concentration, perform a second dilution according to Tables 7 and 8.

[0175] a. 4 nM starting concentration library (Table 7).

[0176] Table 7

[0177]

[0178] b. 2 nM starting concentration library (Table 8).

[0179] Table 8

[0180]

[0181]

[0182] 9. Place the diluted and denatured library on ice or in a 2-8°C refrigerator, ready for use on the instrument.

[0183] 5. Experimental results

[0184] A set of primers was designed based on the Mycoplasma pneumoniae genome sequence, and 10 clinical samples were randomly selected for whole-genome capture sequencing experiments. The sequencing results were subjected to bioinformatics analysis, and the results are shown in Table 9.

[0185] Table 9

[0186]

[0187] The above experimental results show that the gene coverage of 10 randomly selected clinical samples is above 95%, indicating that the primers designed in this experiment can effectively amplify almost the entire length of the Mycoplasma pneumoniae genome, and the amplification efficiency is stable.

Claims

1. A primer set for detecting the whole genome of Mycoplasma pneumoniae, wherein the sequence of the primer set is shown as CGCGAAAA, TAACGGTT, TAACGGTA, CGCTTAAT, CCGCTTTA, AACGCTTTA, AAACGGTT, AACGCTG, TTAACGAA, and CGGTTTTA. The primer set of claim 1 , wherein the primer set is labeled with a detectable marker.

3. The primer set according to claim 2, wherein the detectable label comprises a fluorescent dye, a chemiluminescent compound, a radioactive isotope, an enzyme, a colored particle, or biotin.

4. The primer set according to claim 3, wherein the fluorescent dye comprises a fluorescent organic dye, a fluorescent quantum dot, a fluorescent doublet, a fluorescent carbon dot, a graphene quantum dot or other carbon-based fluorescent nanostructures, a fluorescent protein, or a fluorescent DNA origami-based nanostructure.

5. The primer set according to claim 3, wherein the chemiluminescent compound comprises luminol, acridinium ester, terpyridine ruthenium, lucigenin, lophanine, and peroxyoxalate esters.

6. The primer set according to claim 3, wherein the radioisotope comprises 3 H. 123 I. 125 I. 131 I. 18 F. 11 C. 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 32 P. 33 P. 35 S. 7 . The primer set according to claim 3 , wherein the enzyme comprises horseradish peroxidase, alkaline phosphatase, β-galactosidase, and acetylcholinesterase. The primer set according to claim 3 , wherein the colored particles include gold, silver, or platinum particles.

9. A product for detecting the whole genome of Mycoplasma pneumoniae, comprising the primer set according to any one of claims 1 to 8.

10. The product according to claim 9, comprising a chip, a nucleic acid membrane strip, and a test kit. The product according to claim 10 , wherein the kit further comprises a PCR amplification buffer and an amplification enzyme.

12. The product according to claim 11, wherein the amplification enzyme comprises DNA polymerase and / or RNA polymerase.

13. The product according to claim 10, wherein the kit further comprises a reverse transcription reaction system.

14. The product of claim 10, further comprising instructions.

15. A method for constructing a library for detecting Mycoplasma pneumoniae virus in a sample, the method comprising performing an amplification reaction using the primer set of any one of claims 1-8 or the product of any one of claims 9-14 to obtain a DNA amplification product, fragmenting the DNA amplification product, purifying the DNA, amplifying the fragmented and tagged DNA, purifying the library, and performing library quality control.

16. Use of the primer set according to any one of claims 1 to 8 in preparing a product for detecting Mycoplasma pneumoniae virus in a sample.

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