Mycoplasma pneumoniae whole genome capturing method, primer group and kit

Abstract

The invention discloses a mycoplasma pneumoniae whole genome capturing method, a primer group and a kit, belongs to the field of biological medicine, provides a primer group for detecting a mycoplasma pneumoniae whole genome, and provides a primer group for amplifying the mycoplasma pneumoniae whole genome. The invention provides a product for detecting a mycoplasma pneumoniae whole genome, an amplification method of the mycoplasma pneumoniae whole genome, a method for detecting mycoplasma pneumoniae viruses in a sample and a construction method of a library for detecting the mycoplasma pneumoniae viruses in the 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) is a small microorganism between bacteria and viruses and is the pathogen of human mycoplasma pneumonia. Mycoplasma is widely distributed in nature, with more than 80 species. Mycoplasma pneumonia belongs to a category of mycoplasma. It is called mycoplasma pneumonia because it has a special terminal structure at one end that allows mycoplasma to adhere to the surface of respiratory mucosal epithelial cells and cause lung infection. Mycoplasma pneumonia is one of the main pathogens causing community-acquired pneumonia. In addition to causing respiratory symptoms, severe cases may also have extrapulmonary damage. Mycoplasma pneumonia infection lacks specific clinical manifestations and has complex imaging manifestations. At present, pathogen culture is the gold standard for diagnosing mycoplasma pneumonia infection, but it has limitations such as low sensitivity and cannot assist in the early diagnosis of clinical patients; in addition, the positive rate of mycoplasma pneumonia serological antibody detection is also low, and the difference in sensitivity and specificity is huge. With the development of molecular diagnostic technology, it has shown obvious advantages in the sensitivity and specificity of mycoplasma pneumonia detection, and can be used for drug resistance detection. In the future, it can be used as an important technology for the rapid diagnosis of mycoplasma pneumonia.

[0003] Since the emergence of molecular diagnostic technology in the 1960s, it has roughly gone through stages such as molecular technology based on molecular hybridization, nucleic acid sequencing technology, molecular diagnostic technology based on molecular conformation, and quantitative PCR technology. Nucleic acid sequencing technology - whole genome sequencing (WGS) as a commonly used genotyping technology has the characteristics of high resolution. It can not only identify single nucleotide polymorphisms (SNPs) on the genome to determine the microevolution of MP during the transmission process, but also obtain the entire sequence of the target genome and predict the resistance of most drugs. Application of NGS in MP resistance detection: NGS has the advantages of simplicity, rapidity, accuracy and reliability. The whole gene sequence of MP is obtained through whole genome sequencing technology, and the related resistance mutations are detected at the whole gene level using the known MP resistance mechanism, so that the resistance detection based on molecular biology is no longer limited to one or several first-line drugs. Whole genome sequencing technology is often used in scientific research on drug resistance to discover new resistance sites and explain new resistance mechanisms. Therefore, it is of great significance to obtain the whole genome of MP. In recent decades, the research on whole genome amplification technology has made great progress. Whole genome amplification technology can amplify a small amount of DNA samples without loss, bias, and faithfulness. According to different amplification principles, WG amplification methods can be divided into three categories: the first category is whole genome amplification technology based on polymerase chain reaction (PCR), the second category is isothermal whole genome amplification technology, and the third category is hybrid whole genome amplification technology. 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 uses a single-cell amplification method - multiple displacement amplification (MDA) is an isothermal strand displacement amplification. Under constant temperature conditions, random primers consisting of 6 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 randomly bind, anneal, and extend with the primers, eventually forming branch amplification.

[0006] The MDA amplification system is formed by improving the traditional WGA technology and is currently the most popular isothermal amplification technology. The amplification steps of MDA include: first, at 30°C, random 6-base primers anneal and bind to template DNA at multiple sites, and then under the action of phi29 DNA polymerase, a chain displacement amplification reaction occurs, and the single-stranded product produced by the displacement becomes a new replication template, forming a hyperbranched structure, and so on. Finally, an amplification product with a fragment size of >10kb is produced. The advantages of this system are low error rate and amplification deviation, high amplification efficiency and fidelity, and long amplification products. A large amount of high molecular weight DNA can be obtained using this technology. Since MDA is a constant temperature amplification, there is a certain problem of nonspecific amplification. In addition, this method has relatively high requirements for template quality, and non-balanced amplification can also result in non-optimal experimental conditions.

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

[0008] Furthermore, the primer set includes labeling 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 sense strand or an antisense strand. Nucleic acid can include natural nucleotides (e.g., A, T / U, C, and G), and can include analogs of natural nucleotides, such as labeled nucleotides. Target nucleic acid molecule refers to a nucleic acid molecule intended to be detected, quantitatively, 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 target nucleic acid molecules. For example, a 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 performed 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 dyads, fluorescent carbon dots, graphene quantum dots or other carbon-based fluorescent nanostructures, fluorescent proteins, and nanostructures based on fluorescent DNA origami.

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

[0014] Furthermore, 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.

[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 about 60 to about 120 nm.

[0018] In this document, the terms "fluorescent dye", "fluorophore", "fluorochrome", "dye" are used interchangeably to refer to fluorescent chemical compounds or structures, and may particularly 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, 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, Oxadiazoles, Anthracenes (Anthraquinone, DRAQ5, DRAQ7, CyTRAK Orange), Pyrene (Cascade Blue), Oxazines (Nile Red, Nile Blue, Cresyl Violet, Oxazine 170), Acridines (Proflavin, 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 / Fluorophore).In 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 in this document by the term "fluorescent dye". Furthermore, in the group of fluorescent proteins, the term "fluorescent dye" may include in this document fluorescent proteins whose absorbance or emission characteristics change upon binding to a ligand, such as BFPms1, or change 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 in this document derivatives of cyanobacterial phycobiliproteins, small super red fluorescent proteins smURFP, and fluorescent protein nanoparticles that may be derived from smURFP. 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 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 ALEXAFLUOR TM Chromophore and ALEXAFLUOR TM Bioconjugates that absorb in the visible and near infrared spectra. Emission from fluorophores 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 will be formed when performing the method. The present invention also does not exclude the possibility of producing multiple radioisotopic substances simultaneously, for example by irradiating multiple target elements simultaneously. For the purposes of the specification, reference to "radioisotope" therefore 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-deoxycytidine includes 5-Me-dC and 5-methyl-2'-deoxycytidine.

[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 formed by two complementary chains of double-stranded DNA or RNA chains 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 a 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 bases of each molecule do not fully form hydrogen bonds. For example, hybridization with complementary nucleic acid sequences can occur under conditions of different stringency, wherein the complementary chains will bind at certain 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 cited: conditions under which a DNA having high identity (identity of 90% or more, preferably 95% or more) with respect to a DNA consisting of a sequence shown in SEQ ID NO: 1-10 and a DNA consisting of a base sequence complementary to the DNA consisting of a sequence shown in SEQ ID NO: 1-10 hybridize. Generally, it refers to the situation where hybridization is formed at a temperature of about 5°C to about 30°C, preferably about 10°C to about 25°C, which is lower than the melting temperature (Tm) for complete hybridization.

[0034] In the present invention, hybridization means that two completely or partially complementary nucleic acid sequences are combined 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, and the product 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 an ultra-sensitive Mycoplasma pneumoniae whole genome capture reagent, including EquiPhi29 TM DNA Polymerase (ReagentAMix), exonuclease-resistant targeted primer (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, suitable amounts of one or more primers are provided in one or more containers, or fixed on a substrate, and the primers can be provided suspended in an aqueous solution, or, for example, as a freeze-dried or lyophilized powder. Wherein, the container provided with the nucleic acid can be any conventional container capable of accommodating the provided form, such as a microcentrifuge tube, an ampoule or a bottle. The kit can contain labeled or unlabeled probes for detecting digestive tract viral nucleotide sequences. The amount of the nucleic acid primers provided in the kit can be any suitable amount and can depend on the target market targeted by the product.

[0046] The present invention provides a method for amplifying the whole genome of Mycoplasma pneumoniae, which comprises using the primer set or the product described above 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. MagMAX is recommended TM Virus / Pathogen Ultra Nucleic Acid Isolation Kit (Cat. No. A42356) was used for nucleic acid extraction.

[0050] In some embodiments, (1) Place the components of the kit on ice or thaw at 4°C in advance, invert to mix, and then place in a centrifuge for brief centrifugation. (2) Add the following reagents to a 0.2 ml thin-walled PCR tube that does not contain nucleic acids on ice: ReagentAMix 2μl, Reagent B Mix 5μl, Reagent C Mix 2μl, Reagent D Mix 2μl, DNATemplate 1μl, Nuclease-free water 6μl, a total volume of 18μl. (3) Gently mix the above reaction solution and briefly centrifuge, and incubate 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, a total volume of 18μl.

[0051] (5) Gently mix the above reaction solution and centrifuge briefly, and then amplify according to the following procedure: 85℃

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

[0053] In some embodiments, the product is recovered using 1.8 times the volume of Beijing Micro Future Purification Magnetic Beads to purify the above amplified products. The purified magnetic beads need to be placed at room temperature for 30 minutes and shaken and mixed to fully activate them, otherwise it will affect the purification recovery rate. The purified product can be used for subsequent library construction. If the subsequent experiment is not directly carried out, it can also be stored at -20°C for future use.

[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 amplification product, and using the amplification product for sequencing.

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

[0056] Furthermore, the human samples include 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 lysates, 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 amplification method described above, purifying, amplifying the fragmented and labeled DNA, purifying the library, and quality controlling the library.

[0058] Further, the DNA fragmentation includes the steps of 1.1 Qubit quantification, adding 2-30μl DNA (1-500ng) to the PCR tube; *Note: For multiple samples, the concentration is preferably greater than 50 or less than 50ng, which is convenient for subsequent PCR amplification. 1.2 Add pure water to the sample well to make the total volume to 30μl; 1.3 Vortex BLT10s to check whether the magnetic beads are completely suspended; 1.4 Prepare the fragmentation main reaction solution, each reaction: BLT 11μl + TB111μl, vortex the main reaction solution to mix; 1.5 Take 20μl of the main 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℃for15minutes, Hold at 10℃.

[0059] Further, the purification includes step 2.1 adding 10μl TSB to the fragmentation reaction solution, gently blowing and mixing 10 times, and suspending the beads again. *Note: TSB (check whether it is precipitated) 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 rack, wait for the liquid to clarify (about 3 minutes), and aspirate and discard the supernatant. 2.4 Remove the reaction tube from the magnetic rack, slowly add 100μl TWB, and resuspend the magnetic beads, place the reaction tube in the magnetic rack, wait for the liquid to clarify (about 3 minutes), and 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 TWB and 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 labeled DNA including step 3.1 Prepare PCR master reaction solution (for each sample): 22μl EPM + 22μl water, vortex to mix, and flick. *Note: EPM: Thaw on ice. Invert to mix and then centrifuge briefly. DNAAdapter: Thaw at room temperature, vortex to mix and then centrifuge briefly for test tubes. Centrifuge briefly before use for well plates. 3.2 Remove the supernatant on the magnetic stand and immediately add 40μl PCR master reaction solution to the sample well and mix. 3.3 For IndexAdapter Tubes, add 5μl i7 adapter and 5μl i5 adapter respectively and pipette to mix. 3.4 Run PCR program: Set the hot cover to 100℃, the system to 50μl, and determine the number of PCR cycles according to the different total DNA starting amounts, among which 1-9ng is cycled 12 times, 10-24ng is cycled 8 times, 25-49ng is cycled 6 times, 50-99ng is cycled 5 times, 100-500ng is cycled 5 times, and blood / saliva is cycled 5 times. The cycle program is 68℃ for 3min, 98℃ for 3min, 98℃-62℃-68℃ for 45s-30s-2min for X times, and the specific value of X is determined according to the total DNA starting amount, 68℃ for 1min. *Note: Safe stop point, 4℃ can be stored for up to 2 days. Or place it on the PCR for no more than 24h.

[0061] Further, the library purification includes the steps of 4.1 centrifuging the reaction tube and placing it in a magnetic rack, and waiting for the liquid to clarify (about 5 minutes). 4.2 aspirating and transferring 45 μl of supernatant to a new centrifuge tube. 4.3 thoroughly vortexing and resuspending SPB. *Note: SPB needs to be restored to room temperature before use and vortexed to mix. 4.4 For 100-500 ng of starting DNA, perform the following steps: a. Add 40 μl of H 2O. b. Add 45μl SPB to the reaction tube, mix thoroughly, and incubate at room temperature for 5 minutes. c. Place the reaction tube on the magnetic rack 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 supernatant from the reaction tube on the magnetic rack 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 supernatant and mix thoroughly. 4.6 Incubate at room temperature for 5 minutes, place the reaction tube on the magnetic rack, 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 rack, 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 rack, add 32μl RSB to resuspend the magnetic beads, and incubate at room temperature for 2 minutes. 4.10 Place the reaction tube on the magnetic rack, wait for the liquid to clarify (about 2 minutes), and transfer 30μl of the supernatant to a new centrifuge tube.

[0062] Further, the library quality control includes the step of taking 5.11 μl of the constructed library and running it on 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 above-mentioned 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 above-mentioned primer set or the above-mentioned product in detecting Mycoplasma pneumoniae virus in a sample. DETAILED DESCRIPTION

[0065] The invention will be further described below in conjunction with 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 invention. The main features of the invention may be used in a variety of embodiments without departing from the scope of the 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 load), 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) 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.

[0079] 2. Experimental methods

[0080] 1) Sample nucleic acid extraction.

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

[0082] MagMAX is recommended TM Virus / Pathogen Ultra Nucleic Acid Isolation Kit (Cat. No. A42356) was used for nucleic acid extraction.

[0083] 2) Whole genome capture.

[0084] (1) Thaw the 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 that does not contain nucleic acid on ice.

[0086] Table 3

[0087]

[0088]

[0089] The reaction solution was gently mixed and centrifuged briefly, and then incubated according to the procedure in Table 4.

[0090] Table 4

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

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

[0093] Table 5

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

[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℃ Heat 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 amplification products. The purified magnetic beads need to be placed at room temperature for 30 minutes and shaken to fully activate them, otherwise it will affect the purification recovery rate. The purified product can be used for subsequent library construction. If the subsequent experiment is not carried out directly, it can also be stored at -20℃ for future use.

[0100]

Test 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 be equipped with equipment and operators in strict 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 amplification products are concentrated in a 1.5ml EP tube. After purification and qubit quantification, the subsequent library construction experiment can be carried out directly.

[0106] 4.1 Add 250μl AMPure XP beads; *Note: This step is 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 min or until clarified, then discard the supernatant.

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

[0110] 4.5 Repeat the above steps.

[0111] 4.6 Remove the remaining liquid using a 10 μl pipette tip.

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

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

[0114] 4.9 Oscillate at 1800 rpm for 2 min, then incubate at room temperature for 2 min.

[0115] 4.10 Let 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 procedures.

[0121] a) 1 μl dye reagent + 199 μl buffer, vortex mix to get the working solution, protect from light and set aside.

[0122] b) Add 1-20 μl of library to Qubit working solution, the final volume is 200 μl.

[0123] c) Vortex for 2-3 seconds and place at room temperature away from light 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 product obtained by the above amplification is subjected to DNA fragmentation.

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

[0129] 1.2 Add pure water to the sample well to make the total volume up to 30 μl.

[0130] 1.3 Vortex BLT10s 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 well.

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

[0133] 1.6 Place in PCR instrument and run 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 TSB to the fragmentation reaction solution, gently pipette and mix 10 times, and resuspend the beads. *Note: TSB (check for precipitation) and TWB are stored at 15-30℃ and restored to room temperature before use.

[0136] 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.

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

[0138] 2.4 Remove the reaction tube from the magnetic rack, slowly add 100 μl TWB and 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 once more.

[0140] 2.6 Remove the reaction tube from the magnetic rack, slowly add 100 μl TWB, resuspend the magnetic beads, and place the reaction tube in the magnetic rack until the liquid clarifies (about 3 minutes). The reaction tube remains on the magnetic rack until step 3.2 of Amplifying the fragmented and labeled DNA is performed.

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

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

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

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

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

[0146] The number of PCR cycles is determined according to the different total DNA starting amounts, among which 1-9ng is cycled 12 times, 10-24ng is cycled 8 times, 25-49ng is cycled 6 times, 50-99ng is cycled 5 times, 100-500ng is cycled 5 times, and blood / saliva is cycled 5 times. The cycle program is 68℃3min, 98℃3min, 98℃-62℃-68℃ respectively for 45s-30s-2min cycles X times, the specific value of X is determined according to the total DNA starting amount, 68℃1min. *Note: safe stop point, 4℃ can be stored for up to 2 days. Or placed on PCR for no more than 24h.

[0147] 4) Library purification.

[0148] 4.1 Centrifuge the reaction tube and place it in 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. 2 O. b. Add 45μl SPB to the reaction tube, mix thoroughly, and incubate at room temperature for 5 minutes. c. Place the reaction tube on the magnetic rack and wait for the liquid to clarify (about 5 minutes). d. During the incubation, vortex the SPB thoroughly, then take 15μl and add it to a new centrifuge tube. e. Transfer 125μl of the supernatant from the reaction tube on the magnetic rack to a new centrifuge tube (containing 15μl SPB) and mix thoroughly.

[0153] 4.5 For starting DNA <100 ng, 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 the magnetic rack, wait for the liquid to clarify (about 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 residual 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 the 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 MiSeq on the instrument.

[0163] Dilute and denature the library.

[0164] 1. Denaturation of library with NaOH.

[0165] 1) Recommended concentrations: MiSeq v3 reagents and types, sample starting concentration 4nM, library concentration range 6-20pM; MiSeq v2 reagents and types, MiSeq v2 reagents and types, 4nM, library concentration range 6-20pM.

[0166] 2) Experimental preparation: 1N NaOH, PCR grade water, HT1, pipettes and tips, vortex oscillator, handheld centrifuge.

[0167] III) 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 90 μl of ice-cold HT19.

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

[0174] 8. According to the library type and optimized loading concentration, perform the 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 machine.

[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 in any one or more of CGCGAA, TAACGGTT, TAACGGTA, CGCTTTAAT, CCGCTTTA, AACGCTTTA, AAACGGTT, AACGCTTTG, TTAACGAA, and CGGTTTTA.

2. The primer set of claim 1, wherein the primer set comprises a detectable marker; Preferably, the detectable marker comprises a fluorescent dye, a chemiluminescent compound, a radioisotope, an enzyme, a colored particle, or biotin; Preferably, the fluorescent dye comprises fluorescent organic dye, 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; Preferably, the chemiluminescent compound includes luminol, acridinium ester, terpyridine ruthenium, lucigenin, lophanine, and peroxy oxalate esters; Preferably, 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; Preferably, the enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, acetylcholinesterase; Preferably, the colored particles include gold, silver, or platinum particles.

3. The primer set of claim 1, comprising at least one modified nucleotide; Preferably, the modified nucleotides include 2'-modified nucleotides or 5-methylcytosine; Preferably, the 2'-modified nucleotides include 2'-O-methyl modified nucleotides or 2'-fluoro modified nucleotides; Preferably, the 5-methylcytosine comprises 5-methyl-deoxycytosine; Preferably, the 5-methyl-deoxycytidine includes 5-Me-dC and 5-methyl-2'-deoxycytidine.

4. A primer set for amplifying the whole genome of Mycoplasma pneumoniae, the primer set comprising: 1) a nucleic acid sequence complementary to the primer set according to any one of claims 1 to 3; 2) A nucleic acid sequence that hybridizes with the nucleic acid sequence described in 1) under stringent conditions.

5. A product for detecting the whole genome of Mycoplasma pneumoniae, comprising the primer set according to any one of claims 1 to 4; Preferably, the product includes a chip, a nucleic acid membrane strip, and a test kit; Preferably, the kit further comprises a PCR amplification buffer and an amplification enzyme; Preferably, the amplification enzyme comprises DNA polymerase and / or RNA polymerase; Preferably, the kit further comprises a reverse transcription reaction system; Preferably, the reverse transcription reaction system includes primers; Preferably, the primers are random primers; Preferably, the kit further comprises instructions.

6. A method for amplifying the whole genome of Mycoplasma pneumoniae, the method comprising using the primer set according to any one of claims 1 to 4, or the product according to claim 5 to perform an amplification reaction to obtain a DNA amplification product; preferably, the amplification reaction is performed by an MDA method.

7. A method for detecting Mycoplasma pneumoniae virus in a sample, the method comprising amplifying using the primer set according to any one of claims 1 to 4, the product according to claim 5, or the method according to claim 6 to obtain an amplified product, and sequencing the amplified product; Preferably, the sample comprises a human sample; Preferably, 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 extract, homogenized tissue, tumor tissue, cell extract, alveolar lavage fluid, nasal swab, and throat swab.

8. 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 amplification method of claim 6, purifying, amplifying the fragmented and labeled DNA, purifying the library, and quality controlling the library.

9. Use of the primer set according to any one of claims 1 to 4 in preparing a product for amplifying or detecting Mycoplasma pneumoniae virus in a sample.

10. Use of the primer set according to any one of claims 1 to 4 or the product according to claim 5 in detecting Mycoplasma pneumoniae virus in a sample.

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