Probe set for capturing mycoplasma pneumoniae whole genome feature sequence, kit and application

Abstract

The invention discloses a probe set and a kit for capturing a mycoplasma pneumoniae whole genome feature sequence and application, the probe set can efficiently and specifically capture the mycoplasma pneumoniae whole genome feature sequence, and then the mycoplasma pneumoniae whole genome is efficiently sequenced and analyzed. The mycoplasma pneumoniae typing, traceability and drug resistance gene detection accuracy can be improved, and the problem of mycoplasma pneumoniae non-culture-dependent whole genome sequencing is solved.

Description

(I) Technical field

[0001] The present invention relates to a probe set, a kit and an application thereof for capturing a characteristic sequence of a whole genome of Mycoplasma pneumoniae. (II) Background technology

[0002] Mycoplasma pneumoniae, a member of the Mycoplasma family, is a cell wall-less bacterium that is commonly associated with human respiratory infections. The microorganism was first discovered by scientists in 1944 and is named for its role in causing pneumonia. Mycoplasma pneumoniae can cause symptoms ranging from mild upper respiratory tract infections to more severe pneumonia, and is more common in children aged 5 to 15 years, but all age groups may be affected. In recent years, infections with Mycoplasma pneumoniae have been increasing, accounting for about 10% to 20% of pneumonia, and regional epidemics occur every 3 to 5 years.

[0003] Disease caused by Mycoplasma pneumoniae is commonly referred to as "mycoplasma pneumonia", a relatively common lung infection characterized by a slow onset and gradually worsening symptoms, including persistent dry cough, fever, headache, and general malaise. Compared with other types of pneumonia caused by bacteria or viruses, mycoplasma pneumonia usually has milder symptoms and a longer course.

[0004] Mycoplasma pneumoniae are special bacteria because they lack a cell wall. This feature not only makes them naturally resistant to antibiotics such as penicillins, which work by destroying the bacterial cell wall, but also affects their morphological plasticity and survival strategies. Mycoplasmas can survive inside and outside host cells, showing stubborn infectivity and high adaptability. This microorganism regulates the biological activities of host cells by adhering to the surface of host cells, interfering with cell functions, and releasing a variety of toxins and enzymes.

[0005] At the molecular level, the genome of M. pneumoniae is relatively small, approximately 800,000 to 1,000,000 base pairs, encoding approximately 700 to 800 proteins. Despite the small size of the genome, their survival strategies are extremely sophisticated, including the compact organization of genetic information and efficient genetic regulatory networks. The pathogenicity of M. pneumoniae involves multiple aspects, including the variability of cell surface proteins, which are responsible for bacterial adhesion and immune escape.

[0006] Because infection with Mycoplasma pneumoniae is highly contagious, it can be spread through droplets, i.e. tiny droplets expelled by an infected person through coughing or sneezing. In addition, the treatment and management of Mycoplasma pneumonia is also challenging because the symptoms of infection are often vague and similar to the common cold or flu, which can easily lead to misdiagnosis.

[0007] Isolation and culture is the gold standard for the diagnosis of Mycoplasma pneumoniae. However, the traditional isolation and culture method is time-consuming (2-4 weeks) and has low sensitivity. It is subject to many interference factors, and the positive rate of the traditional microbial culture method is only about 10%. Studies have shown that if there are drug-resistant or multidrug-resistant bacteria in the oropharynx, as well as bacteria with natural resistance mechanisms to certain antibiotics, false positive results will be generated in the rapid culture method of Mycoplasma pneumoniae. PCR detection can be superior to culture methods in terms of sensitivity and specificity, but PCR detection is based on the gene sequence of known pathogens, and the genomic information it can provide is limited, which cannot meet the analysis of the genome. Metagenomic sequencing (mNGS) is a new type of microbial detection technology developed and used in recent years. It directly sequences samples from infected patients to obtain information about the types of suspected pathogens. However, in the clinical diagnosis of Mycoplasma pneumoniae, for ordinary throat swab samples, due to the load of pathogens, different stages of infection, and relatively complex flora composition, the number of effective sequences detected is often small, and more in-depth and accurate analysis results cannot be obtained.

[0008] In order to prevent and control mycoplasma pneumonia infection, timely, rapid and effective diagnosis of pathogens, and monitoring of the source, lineage, transmission, drug resistance and genomic variation of mycoplasma pneumoniae are of great significance for the formulation of prevention strategies for mycoplasma pneumoniae infection. Therefore, constructing a non-culture-dependent mycoplasma pneumoniae whole genome targeted capture sequencing method, shortening the detection cycle, improving the sequencing success rate, and accurately obtaining the whole genome information of mycoplasma pneumoniae is of great value for the rapid typing, tracing and drug resistance determination of mycoplasma pneumoniae, and also provides an accurate molecular basis for clinical diagnosis and disease prevention and control. (III) Summary of the invention

[0009] The present invention aims to provide a probe set, a kit and an application for capturing characteristic sequences of the whole genome of Mycoplasma pneumoniae. The probe set can efficiently and specifically capture characteristic sequences of the whole genome of Mycoplasma pneumoniae, thereby efficiently sequencing and analyzing the whole genome of Mycoplasma pneumoniae, thereby improving the accuracy of Mycoplasma pneumoniae typing, tracing and drug resistance gene detection, and solving the problem of non-culture-dependent whole genome sequencing of Mycoplasma pneumoniae.

[0010] The technical solution adopted by the present invention is:

[0011] The present invention provides a probe set for capturing characteristic sequences of the whole genome of Mycoplasma pneumoniae, wherein the probe set comprises probes that specifically bind to one or more specific gene segments of Mycoplasma pneumoniae; the specific gene segments include but are not limited to antibiotic resistance genes, pathogenic genes or marker genes for typing of Mycoplasma pneumoniae.

[0012] Furthermore, the antibiotic resistance gene is 23S rRNA, the pathogenic gene includes the whole genome of Mycoplasma pneumoniae, and the typing marker genes include ppa, pgm, gyrB, gmk, glyA, atpA, arcC, adk, etc.

[0013] Furthermore, each probe of the probe set has at least one modification selected from biotin labeling, fluorescent labeling or magnetic labeling at one end thereof to facilitate the capture, detection or separation of the probe.

[0014] Furthermore, the nucleotide sequence of the probe set is shown as one or more of SEQ ID NO.1-SEQ ID NO.300.

[0015] The present invention also provides a kit for constructing the probe set, which comprises a whole genome extraction reagent, a metagenomic DNA library construction reagent, a hybridization reagent containing the probe set, and a nucleic acid purification reagent.

[0016] Furthermore, the metagenomic DNA library construction reagents include reagents for library construction such as fragmentation filling enzyme, fragmentation filling buffer, balancing buffer, M linker DNA, etc., preferably a metagenomic DNA library construction kit (reversible terminal termination sequencing method) (Hangzhou Jieyi Biotechnology Co., Ltd. / MD001P).

[0017] Furthermore, the hybridization reagent containing the probe set includes 2X Hyb Buffer, RNase Inhibitor, PCR Mix, Universal Blocker with cot-1 2.0, Primer Mix, Probes, capture magnetic beads, WashBuffer 1, Wash Buffer 2 and Wash Buffer 3, etc., which are reagents used for probe set hybridization capture. The probe set hybridization capture reagent is from the Mycoplasma pneumoniae (Mp) whole genome capture library construction kit (Hangzhou Jieyi Biotechnology Co., Ltd. / MR001P).

[0018] Furthermore, the nucleic acid purification reagents include purification magnetic beads, purification cleaning solution, purification eluent, etc. for library purification, and the library purification reagents are from the Nucleic Acid Purification Kit (Magnetic Bead Method) (Hangzhou Jieyi Biotechnology Co., Ltd. / MD012).

[0019] The present invention also provides an application of the probe set in capturing the whole genome characteristic sequence of Mycoplasma pneumoniae in a sample to be tested, and the application method comprises: (1) extracting whole genome DNA from the sample to be tested; (2) constructing a metagenomic DNA library, amplifying the library and purifying the library; (3) using the probe set to hybridize with the purified library; (4) using magnetic beads to capture the hybridized complex; (5) washing and eluting the library bound to the magnetic beads; and (6) sequencing the eluted library.

[0020] Further, step (2) is performed as follows: the extracted total genomic DNA containing the microorganism is fragmented, and an adenine deoxynucleotide is added to the end of the DNA after the fill-in, and connected with the universal sequencing primer through the "TA" connection method, thereby constructing a DNA library that can be used for the Illumina high-throughput sequencing platform.

[0021] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0022] The probe set for capturing and sequencing the characteristic sequence of the whole genome of Mycoplasma pneumoniae provided by the present invention can specifically bind to a specific gene segment of Mycoplasma pneumoniae, and each probe is designed with the diversity and variability of the Mycoplasma pneumoniae genome in mind to ensure that different strains can be covered. Each probe is labeled with biotin at one end during synthesis to facilitate subsequent capture and processing.

[0023] Using the probe set, the DNA of Mycoplasma pneumoniae can be specifically captured from clinical samples through hybridization capture technology, and high specificity can be guaranteed even in complex samples where multiple pathogens coexist. After hybridization, the probe-DNA complex can be easily separated from the sample by magnetic beads combined with biotin labeling. The captured DNA is used to construct a sequencing library. The library is subjected to high-throughput sequencing, such as next-generation sequencing technology, to obtain detailed genetic information of Mycoplasma pneumoniae. The data obtained by sequencing is annotated with drug resistance information using the KMA alignment tool and the CARD database, and the blastn alignment tool and the pub MLST database are used for identification and traceability of Mycoplasma pneumoniae to accurately identify the strains, traceability information and potential drug resistance of Mycoplasma pneumoniae.

[0024] The probe set of the present invention can greatly improve the detection accuracy of Mycoplasma pneumoniae, especially in identifying specific strain characteristics and drug resistance. The method can quickly and effectively extract target DNA from complex clinical samples, providing strong technical support for the rapid diagnosis and epidemic monitoring of Mycoplasma pneumoniae. (IV) Description of the drawings

[0025] Figure 1 For the experimental process.

[0026] Figure 2 Capture efficiency comparison for this method.

[0027] Figure 3 This is the whole genome coverage map of Mycoplasma pneumoniae.

[0028] Figure 4 For evolutionary analysis of Mycoplasma pneumoniae. (V) Specific implementation methods

[0029] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0030] Example 1: Probe set for capturing characteristic sequences of the whole genome of Mycoplasma pneumoniae

[0031] 1. Acquisition of the complete genome sequence of Mycoplasma pneumoniae

[0032] Download 30 Mycoplasma pneumoniae genome sequences from the NCBI public database, giving priority to those with high assembly levels and recent release times. Collect 30 Mycoplasma pneumoniae-positive respiratory samples (the samples were obtained in accordance with laboratory and relevant ethical regulations) and sequence them using mNGS technology to obtain 30 Mycoplasma pneumoniae genome sequences.

[0033] 2. Obtaining the target sequence for probe design

[0034] Select the representative genome (NZ_LR214945) on NCBI as the standard reference genome, use the mummer tool to compare the 60 genome sequences obtained in step 1 with the reference genome, and obtain multiple genomes with specific region sequences that are different from the reference genome. Integrate the reference genome sequence and the specific region sequence as the target sequence for probe design.

[0035] 3. Probe design

[0036] Use catch software to design probes for the target sequence obtained in the previous step to obtain three different probe sets.

[0037] 4. Probe Screening

[0038] Three Mycoplasma pneumoniae-positive respiratory samples were collected, and the three different probe sets obtained in the previous step were used to perform whole genome capture of Mycoplasma pneumoniae in the samples using the experimental method detailed in Example 2. The results are shown in Table 1. The probe combination with the best capture effect (highest genome coverage) was selected, namely probe set 2 (SEQ ID NO.1-SEQ ID NO.300).

[0039] Table 1. Coverage of sample genomes by different probe sets

[0040]

[0041] Example 2: Probe Set Capture Sequencing of Mycoplasma Pneumoniae-Positive Respiratory Samples

[0042] 1. Experimental methods:

[0043] Reference Figure 1 The experimental process is to capture and sequence the respiratory samples positive for Mycoplasma pneumoniae. The specific steps are as follows:

[0044] (1) Nucleic acid extraction

[0045] 16 respiratory samples positive for Mycoplasma pneumoniae were prepared (the samples were obtained in accordance with laboratory and relevant ethical regulations), and the nucleic acid was extracted using the magnetic bead method. The DNA concentration of nucleic acid was determined using the Qubit fluorometer and the kit FineQubit 1×dsDNA HSQuantitation Assay Kit (Jifan Biotechnology Co., Ltd. / NK901) as the sample DNA to be tested.

[0046] (2) Library construction

[0047] The library was constructed using a metagenomic DNA library construction kit (reversible terminal termination sequencing method, Hangzhou Jieyi Biotechnology Co., Ltd. / MD001P).

[0048] a. Prepare 16 0.2 mL DNase-free PCR tubes, place them on ice and label them. Add reagents and sample DNA (DNA loading amount 200 ng) into the tubes in sequence according to Table 2. Vortex for 5 seconds to mix, and centrifuge at 12,000 rpm for 5 seconds.

[0049] Table 2. PCR Reagents

[0050]

[0051] Note: X represents the volume of the sample DNA added to be tested, which is calculated based on the corresponding sample nucleic acid concentration and should not exceed 35 μL.

[0052] b. Place the PCR tube in step a into a PCR instrument for reaction: react at 37°C for 10 min and inactivate at 75°C for 10 min.

[0053] c. Remove the PCR tubes in step b from the PCR instrument and place them on ice. Add 5 μL of ligase to each PCR tube, and then add 30 μL of M linker DNA.

[0054] d. Centrifuge the PCR tube in step c at 12,000 rpm for 5 seconds, vortex rapidly for 5 seconds to mix thoroughly, centrifuge again at 12,000 rpm for 5 seconds, and place the PCR tube on ice.

[0055] e. Place the PCR tube in step d into a PCR instrument and perform the reaction according to the following procedure: 20°C for 15 min, and inactivate at 75°C for 5 min. The reaction solution in each PCR tube is the constructed library.

[0056] (3) Library purification

[0057] The library was purified using a nucleic acid purification (magnetic bead method) kit (Hangzhou Jieyi Biotechnology Co., Ltd. / MD012).

[0058] a. Take out the purified magnetic beads from the 2-8°C refrigerator and use a vertical mixer to mix and equilibrate at room temperature for 30 minutes.

[0059] b. Add 40 μL of the balanced purified magnetic beads to each PCR tube containing the constructed library in step (2). Vortex for 5 seconds to mix the sample and purified magnetic beads, and leave them at room temperature for 5 to 10 minutes without remixing.

[0060] c. After instant centrifugation of the PCR tube in step b, place the PCR tube on a magnetic rack. After adsorption for 10 minutes, use a pipette to discard the supernatant.

[0061] d. Add 200 μL of 80% ethanol aqueous solution to the PCR tube, let it stand for 30 seconds, and slowly aspirate the waste liquid with a pipette. Repeat this step once.

[0062] e. Remove the PCR tube from the magnetic rack, place it in a centrifuge for a short spin for 5 seconds, put it back into the magnetic rack, and use a 10μL pipette to aspirate and discard all waste liquid.

[0063] f. Open the PCR tube cap and let it stand at room temperature for 3 to 5 minutes.

[0064] g. Remove the PCR tube from the magnetic rack in step f and add 30 μL of the purified eluent. Vortex for 5 seconds to mix well and leave at room temperature for 5 minutes.

[0065] h. After instant centrifugation for 5 seconds, place the PCR tube on a magnetic rack for adsorption for 5 minutes. After the solution is clarified, pipette the supernatant into a new PCR tube, which is the purified DNA library.

[0066] (4) Library amplification

[0067] Step (3) The purified DNA library was amplified using the KAPA Library Amplification Kit (KAPA Biosystems / KK2611) in strict accordance with the kit instructions. The concentration of the amplified library was measured using the FineQubit 1×dsDNA HS Quantitation Assay Kit (Jifan Biotechnology Co., Ltd. / NK901).

[0068] (5) Library mixing

[0069] According to the concentration of the library after amplification, 16 libraries were mixed at an input amount of 800 ng per library.

[0070] (6) Library Concentration

[0071] a. Take out the purified magnetic beads from the 2-8°C refrigerator and use a vertical mixer to mix and equilibrate at room temperature for 30 minutes.

[0072] b. Add the mixed library obtained in step (5) to a centrifuge tube, and then add the balanced magnetic beads. The volume of the magnetic beads is 0.5 times the total volume of the mixed library. Vortex for 5 seconds to mix the sample and purified magnetic beads, and leave them at room temperature for 5 to 10 minutes without mixing again.

[0073] c. Perform cleaning operations according to steps (3) c to d.

[0074] d. Remove the centrifuge tube from the magnetic stand, add 5 μL of nuclease-free water and 7.5 μL of blocking solution system (QuarAcces Universal Blocker with cot-1 2.0), pipette 10 times to mix, and let stand at room temperature for 2 minutes.

[0075] e. Place the tube on a magnetic rack and let it stand for clarification, then aspirate the supernatant and transfer it to a new centrifuge tube to obtain a concentrated library.

[0076] (7) Hybridization reaction

[0077] Steps (7)-(11) all used the Mycoplasma pneumoniae (Mp) whole genome capture library construction kit (Hangzhou Jieyi Biotechnology Co., Ltd. / MR001P).

[0078] a. Preheat the 2X Hyb Buffer in Table 3 in a 65°C metal bath for 10-15 minutes and not more than half an hour. Place the probe set 2 (SEQ ID NO.1-SEQ ID NO.300) synthesized in Example 1 and RNase Inhibitor on ice to melt.

[0079] b. Prepare hybridization mixture according to the dosage of components in Table 3.

[0080] Table 3. Hybridization mixture

[0081]

[0082] c. Use a pipette to gently blow and mix the hybridization mixture 8 times, and quickly centrifuge for 5 seconds. Place the PCR tube containing the hybridization mixture into the PCR instrument and perform the hybridization reaction according to the program in Table 4, hot cover mode (105℃), set the volume to 40μL (total hybridization volume 36μL), and hybridize at 65℃ for 18 hours.

[0083] Table 4. PCR program

[0084]

[0085] (8) Preparation of magnetic beads and reagents

[0086] a. First, take out the capture beads from the 4℃ refrigerator, shake on a vortex shaker for 30 seconds until the beads are fully resuspended, and then equilibrate at room temperature for more than 30 minutes. During the 30-minute equilibration at room temperature, take out Wash Buffer 1, Wash Buffer 2, and Wash Buffer 3, set them to 65℃Hold on another PCR instrument, and set the hot cover to 70℃. Prepare 3 tubes of Wash Buffer 3, 170μL per tube, and put them into the PCR instrument for heating.

[0087] b. After equilibration at room temperature for more than 30 minutes, resuspend the captured magnetic beads by shaking them on a vortex shaker for 30 seconds. Add 150μL of Wash Buffer 1 to 50μL of captured magnetic beads and gently blow 8 times to mix the magnetic beads thoroughly. Place on a magnetic stand and let it stand to clarify, then discard the supernatant. Repeat this step twice. After washing with Wash Buffer 1 3 times, discard the supernatant and add 150μL of Wash Buffer 1 to resuspend the magnetic beads.

[0088] (9) Target sequence capture

[0089] a. After the hybridization mixture is hybridized at 65°C for 18 hours, 150 μL of the magnetic bead resuspension in step (8) b is added to the hybridization mixture on a PCR instrument and gently pipetted to mix.

[0090] b. Remove the magnetic bead hybridization mixture from the PCR instrument and place it on a vertical rotator and fix it. Rotate and incubate at room temperature for 30 minutes. After 30 minutes, remove it from the rotator, centrifuge it quickly for 5 seconds, place it on a magnetic stand for 2-5 minutes, ensure that the liquid is clear, and discard the supernatant.

[0091] c. Remove the centrifuge tube from the magnetic rack, add 150 μL of Wash Buffer 2 to resuspend the magnetic beads, gently pipette 10 times to mix, incubate at room temperature for 15 minutes, and quickly invert up and down 15 times every 5 minutes.

[0092] (10) Non-target sequence cleaning

[0093] a. After discarding the supernatant in step (9) c, place the tube on a PCR instrument at 65°C for hybridization, immediately add 150 μL Wash Buffer 3 preheated in step (8) a, gently pipette and mix on the PCR instrument, cover the tube and incubate at 65°C for 10 minutes. Remove the centrifuge tube from the PCR instrument and place it on a magnetic rack. After the liquid is clear, discard the supernatant.

[0094] b. Repeat step a, Wash Buffer 3 for a total of 3 times.

[0095] c. After three washes, centrifuge quickly for 10 seconds, place on the magnetic stand again and use a 10μL pipette to remove as much residual liquid as possible. Finally, add 20μL of nuclease-free water to resuspend the magnetic beads and gently pipette to mix.

[0096] (11) Capture library amplification

[0097] a. Prepare the reaction system according to Table 5 using 20 μL of the magnetic bead resuspension obtained in step (10) c. Mix by gently pipetting.

[0098] Table 5. Reaction system

[0099]

[0100] b. Place the PCR tube containing the above reaction solution (including magnetic beads) into a PCR instrument and perform the reaction according to the program in Table 6, hot cover mode (105°C), and set the volume to 50 μL.

[0101] Table 6. Reaction procedure

[0102]

[0103] (12) Capture library purification

[0104] The reagents used in this step are from a finished kit - Nucleic Acid Purification Kit (Magnetic Bead Method) (Hangzhou Jieyi Biotechnology Co., Ltd. / MD012).

[0105] a. After the PCR in step (11) b is completed, remove the centrifuge tube, centrifuge quickly for 5 seconds, and place it on a magnetic rack. After standing for 1 minute, transfer all the supernatant to a new PCR tube, and then add 90 μL of balanced purified magnetic beads. Vortex for 5 seconds to mix the sample and purified magnetic beads, and leave it at room temperature for 5 to 10 minutes without mixing again.

[0106] b. Perform cleaning operations according to steps (3) c to d.

[0107] c. Remove the PCR tube from the magnetic stand and add 40 μL of purification eluent. Vortex for 5 seconds to mix and place at room temperature for 5 minutes.

[0108] d. After instant centrifugation for 5 seconds, place the PCR tube on a magnetic rack for adsorption for 5 minutes. After the solution is clarified, pipette the supernatant into a new PCR tube, which is the purified DNA library.

[0109] (13) Library Quantification

[0110] The purified DNA library was quantified using the KAPA Library Quantification Kit (KAPA Biosystems / KK4824), strictly following the instructions of the quantification kit and instrument.

[0111] (14) Sequencing reaction

[0112] Using Illumina NextSeq500 sequencer and Nextseq 500 / 550 High Output Kit (75 Cycles)

[0113] (illumina / 20024906) for DNA sequence detection, and sequencing was performed strictly in accordance with the sequencing kit and instrument instructions.

[0114] Table 7. Sequencing method settings

[0115]

[0116] (15) Bioinformatics analysis

[0117] Each read sequence obtained by sequencing on the sequencing platform was aligned to the reference genome (NZ_LR214945) for analysis using kma 1.3.23 software.

[0118] 2. Result analysis:

[0119] The coverage and average sequencing depth of the sequencing data of each sample on the reference genome sequence of Mycoplasma pneumoniae (NZ_LR214945) are shown in Table 8. Taking sample No. 1 as an example, the distribution of the sequencing data of this sample on the Mycoplasma pneumoniae genome is plotted, that is, the coverage map (see Figure 3 ), it can be seen that the sequences captured in this sample are evenly distributed on the M. pneumoniae genome. Among the 16 samples, the coverage of M. pneumoniae is 98.37% to 99.41% (see Table 8 and Figure 2), indicating that this method has high efficiency in capturing M. pneumoniae in different samples and meets the requirements for whole genome analysis.

[0120] Table 8 Mycoplasma pneumoniae genome coverage and average sequencing depth after capture

[0121]

[0122] Example 3: Application of probe set

[0123] The whole genome sequence of Mycoplasma pneumoniae was captured and sequenced for 80 respiratory samples positive for Mycoplasma pneumoniae using the method of Example 2. After comparing the sequencing results with the reference genome (NZ_LR214945), a phylogenetic tree was drawn, as shown in FIG. Figure 4 As shown, samples with the "_ST" suffix are Mycoplasma pneumoniae genomes from respiratory samples, and the rest are reference genomes.

[0124] The results show that: (1) the probe set of the present invention can effectively capture the whole genome sequence of Mycoplasma pneumoniae of different samples and can be used for the evolution analysis of Mycoplasma pneumoniae; (2) in this example, Mycoplasma pneumoniae in 13 samples was ST14 type, 1 sample was ST7 type, 50 samples were ST3 type, and the specific typing of 16 samples could not be determined; the probe set of the present invention can be used for Mycoplasma pneumoniae typing.

Claims

1. A probe set for capturing characteristic sequences of the whole genome of Mycoplasma pneumoniae, characterized in that: The probe set comprises probes that specifically bind to one or more specific gene segments of Mycoplasma pneumoniae; the specific gene segments include but are not limited to antibiotic resistance genes, pathogenicity genes or marker genes for typing of Mycoplasma pneumoniae.

2. The probe set according to claim 1, characterized in that The antibiotic resistance gene is 23S rRNA, the pathogenicity gene includes the whole genome of Mycoplasma pneumoniae, and the typing marker genes include ppa, pgm, gyrB, gmk, glyA, atpA, arcC, and adk.

3. The probe set according to claim 1, characterized in that Each probe of the probe set has at one end thereof at least one modification selected from a biotin label, a fluorescent label or a magnetic label.

4. The probe set according to claim 1, characterized in that The nucleotide sequence of the probe set is shown as one or more of SEQ ID NO.1-SEQ ID NO.

300.

5. A kit for constructing the probe set according to claim 1.

6. The kit according to claim 5, characterized in that The kit comprises a whole genome extraction reagent, a metagenomic DNA library construction reagent, a hybridization reagent containing the probe set, and a nucleic acid purification reagent.

7. Use of the probe set according to claim 1 in capturing the characteristic sequence of the whole genome of Mycoplasma pneumoniae in a sample to be tested.

8. The use according to claim 7, characterized in that The application method comprises: (1) extracting whole genome DNA from a sample to be tested; (2) constructing a metagenomic DNA library, amplifying the library and purifying the library; (3) hybridizing the purified library with the probe set; (4) capturing the hybridized complex with magnetic beads; (5) washing and eluting the library bound to the magnetic beads; and (6) sequencing the eluted library.

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