Chlamydia whole genome detection method and application thereof

Abstract

The invention provides a chlamydia whole genome detection method and application thereof, and further provides a probe for capturing a chlamydia whole genome, and the probe is designed according to a chlamydia genome sequence as a reference sequence; the probes are 3-10 layers of probes designed in an imbricated manner, the length of each probe is 90-120nt, and the total number of the probes is 50,000-200,000. A probe for capturing a chlamydia whole genome is used for performing hybrid capture and high-throughput sequencing analysis on a to-be-detected sample to obtain a whole genome sequence. According to the detection method developed by the invention, a whole genome sequence can be directly obtained from a clinical sample, the problem that the chlamydia is difficult to detect is solved, the detection efficiency and typing accuracy of the chlamydia are greatly improved, and the method has an important application prospect in clinical detection.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a method for detecting the whole genome of Chlamydia and an application thereof. Background Art

[0002] Chlamydia are tiny, round to oval microorganisms, intermediate in size between bacteria and viruses. They are obligate intracellular organisms with a unique life cycle and are Gram-negative. Currently, there are three known chlamydiae that can cause human disease: Chlamydia psittaci, Chlamydia trachomatis, and Chlamydia pneumoniae. The incubation period for Chlamydia pneumoniae infection is 10-65 days, with a lack of specific manifestations. Latent infection and mild symptoms are common. Chlamydia trachomatis infection manifests clinically in a variety of ways, with varying incubation periods.

[0003] Pneumonia caused by Chlamydia pneumoniae has clinically similar symptoms to most other pneumonias, including Mycoplasma pneumoniae, viral pneumonia, and Legionella pneumonia, making differential diagnosis challenging. Conventional testing for Chlamydia includes smear and culture methods. The former is simple and rapid, suitable for rapid screening of high-risk populations, but its sensitivity is easily affected by the prevalence of infection in the population, and the determination of a positive result is subjective. The latter is time-consuming and requires advanced laboratory skills, making it less commonly used.

[0004] Therefore, developing a new method for whole genome detection of Chlamydia and obtaining whole genome sequences directly from clinical samples has important application value for the detection and typing of Chlamydia. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a method for whole-genome detection of Chlamydia and its application. Based on next-generation sequencing (NGS) technology, the present invention develops a method for whole-genome detection of Chlamydia. This method can directly obtain whole-genome sequences from clinical samples, addressing the difficulty of Chlamydia detection and significantly improving detection efficiency and typing accuracy. This method holds great promise for clinical applications.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a probe for capturing the whole genome of Chlamydia, wherein the probe is designed based on the Chlamydia genome sequence as a reference sequence, and the specific reference genome is shown in the following table;

[0008]

[0009]

[0010] The probes are designed in a shingled manner with 3-10 layers of probes, for example, 3, 4, 5, 6, 7, 8, 9 or 9, and each probe is 90-120nt long, for example, 90nt, 95nt, 100nt, 105nt, 110nt, 115nt or 120nt, etc., and a total of 50,000-200,000 probes are designed.

[0011] The present invention designs capture probes using all Chlamydia genomes as reference sequences. The designed capture probes have good compatibility and can meet the needs of whole-genome detection of different Chlamydia.

[0012] In the present invention, the probe design method includes:

[0013] (1) Genome download: Search NCBI (https: / / www.ncbi.nlm.nih.gov / ) for the reference genes of Chlamydia listed in the download table.

[0014] (2) Probe sequence design: The downloaded reference genome is slid according to a fixed window size (90-120bp), and the sliding range is dynamically adjusted according to the GC content. If the GC content is ≥50%, the probe is encrypted 1X, that is, the sliding range is the designed probe length of 90-120bp; if the GC content is between 40% and 50%, the probe is encrypted 2X, that is, the sliding range is 1 / 2 of the designed probe length, 45-60bp; if the GC content is between 30% and 40%, the probe is encrypted 4X, that is, the sliding range is 1 / 4 of the designed probe length, 22bp-30bp; if the GC content is <30%, the probe is encrypted 8X, that is, the sliding range is 10bp-15bp, and the candidate probe set is obtained.

[0015] The length of the probe designed to be 90-120 bp can be, for example, 90 nt, 95 nt, 100 nt, 105 nt, 110 nt, 115 nt or 120 nt; the GC content can be 40% to 50%, for example, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49 or 50%; the length of the probe designed to be 45-60 bp can be, for example, 45 bp, 47 bp, 49 bp, 50 bp, 51 bp, 53 bp, 55 bp, 57 bp, 59 bp or 60bp, etc.; the GC content is 30% to 40%, for example, it can be 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39% or 40%, etc.; the length of the probe design is 22bp-30bp, for example, it can be 22bp, 23bp, 24bp, 25bp, 26bp, 27bp, 28bp, 29bp or 30bp; the length of the probe design is 10bp-15bp, for example, it can be 10bp, 11bp, 12bp, 13bp, 14bp or 15bp.

[0016] (3) Probe selection:

[0017] (3.1) Filtering probes in the plasmid region (adjustable according to actual needs). The plasmid library is from: https: / / ccb-microbe.cs.uni-saarland.de / plsdb / plasmids / download / . The probes are aligned to the plasmid genome and filtered according to the alignment consistency of the probe and plasmid of 90%;

[0018] (3.2) Filtering non-specific probes caused by common host assembly contamination. Probes were mapped to human, bird, and poultry genomes. The genomes were obtained from NCBI (https: / / www.ncbi.nlm.nih.gov / ). Filtering was performed based on a 90% consistency between the probe and the host genome.

[0019] (3.3) Probe homology filtering: The candidate probe sets filtered in 3.2 were subjected to homology filtering with a 95% consistency rate.

[0020] In a second aspect, the present invention provides a kit for detecting Chlamydia, comprising the probe for capturing the whole genome of Chlamydia according to the first aspect.

[0021] Preferably, the kit further comprises: any one or a combination of at least two of a DNA library construction reagent, a hybridization capture reagent or an auxiliary reagent.

[0022] Preferably, the DNA library construction reagents include: any one or a combination of at least two of: fragmentation enzyme, ligase, ligation buffer, amplification reaction solution, amplification primers, adapters or purification magnetic beads.

[0023] In the present invention, the DNA library construction reagent is used for DNA amplification. In a specific embodiment of the present invention, the DNA library construction reagent is a sequencing library preparation kit from Guangzhou Jinqirui Biotechnology Co., Ltd., with the product number KS130-CAPJK-48.

[0024] Preferably, the hybrid capture reagent comprises: any one or a combination of at least two of: sample diluent, enrichment enhancer, amplification primer, amplification solution, enrichment buffer, eluent, enrichment magnetic beads or purification magnetic beads.

[0025] In the present invention, the hybrid capture reagent is used to capture the target nucleic acid. In a specific embodiment of the present invention, the hybrid capture reagent is MetaCAP hybrid capture reaction solution from Guangzhou Jinqirui Biotechnology Co., Ltd., with the product number KS129-CAPBH-6.

[0026] Preferably, the auxiliary reagents include: nucleic acid extraction reagents and / or high-throughput sequencing reagents.

[0027] In the present invention, the nucleic acid extraction reagent and high-throughput sequencing reagent are universal kits for sequencing reactions.

[0028] In a third aspect, the present invention provides use of the probe for capturing the whole genome of Chlamydia according to the first aspect and / or the kit for detecting Chlamydia according to the second aspect in the preparation of a product for detecting Chlamydia.

[0029] In a fourth aspect, the present invention provides a method for detecting the whole genome of Chlamydia, which uses the probe for capturing the whole genome of Chlamydia described in the first aspect to perform hybridization capture and high-throughput sequencing analysis on the sample to be tested to obtain the whole genome sequence.

[0030] Preferably, the method comprises:

[0031] (1) extracting nucleic acid from the sample to be tested;

[0032] (2) The extracted nucleic acid is sequentially fragmented, end-repaired, and ligated with adapters, and then amplified to obtain a Chlamydia whole genome library;

[0033] (3) hybridizing and incubating the obtained Chlamydia whole genome library with the probe for capturing the Chlamydia whole genome described in the first aspect, and then sequentially performing magnetic bead capture and amplification to obtain a high-throughput sequencing sample;

[0034] (4) The obtained high-throughput sequencing samples are sequentially subjected to library dilution, library denaturation, sequencing and data analysis.

[0035] In the present invention, when detecting the whole genome of Chlamydia, the method can directly detect the sample without relying on cultivation, which solves the problem of difficulty in obtaining the whole genome sequence due to the difficulty in culturing Chlamydia; compared with the conventional method that the whole genome must be cultured, the method significantly improves the success rate of whole genome detection of Chlamydia in clinical samples, greatly shortens the detection cycle, and is conducive to the traceability, variation tracking and monitoring of Chlamydia; the method has good compatibility and is compatible with highly mutated sequences, while meeting the requirements for whole genome detection of common Chlamydia such as Chlamydia pneumoniae and Chlamydia psittaci.

[0036] Preferably, in step (2), a purification step is further included after linking the adapter and / or after amplification.

[0037] In the present invention, purification magnetic beads are used to purify the sample.

[0038] Preferably, in step (3), the amount of the library and probe hybridization incubation is 0.05-2 fmol of probe for every 200-500 ng of library.

[0039] Among them, the 200-500ng library can be, for example, 200ng, 250ng, 300ng, 350ng, 400ng, 450ng or 500ng, etc.; the 0.05-2fmol probe can be, for example, 0.05fmol, 0.1fmol, 0.25fmol, 0.5fmol, 0.75fmol, 1fmol, 1.25fmol, 1.5fmol, 1.75fmol or 2fmol, etc.

[0040] In this invention, the library and probe dosages are optimized after testing. Too little library dosage can lead to missed detections, while too much can result in a high number of invalid samples or decreased performance. Too little library and probe dosage can lead to missed detections, while too much can increase costs.

[0041] Preferably, in step (3), the conditions for hybridization incubation of the library and the probe are as follows:

[0042] Incubate hybridization at 58-62°C for 28-32 min; incubate capture at 63-67°C for 28-32 min; wash at least twice at 66-70°C; and wash at least three times at 46-50°C.

[0043] Among them, 58-62℃ can be, for example, 58℃, 59℃, 60℃, 61℃ or 62℃; 28-32min can be, for example, 28min, 29min, 30min, 31min or 32min; 63-67℃ can be, for example, 63℃, 64℃, 65℃, 66℃ or 67℃; 66-70℃ can be, for example, 66℃, 67℃, 68℃, 69℃ or 70℃; 46-50℃ can be, for example, 46℃, 47℃, 48℃, 49℃ or 50℃.

[0044] As a preferred technical solution of the present invention, the conditions for hybridization incubation of the library and the probe are as follows: hybridization incubation at 60°C for 30 minutes; capture incubation at 65°C for 30 minutes; washing at 68°C for 2 times; and washing at 48°C for 3 times.

[0045] Preferably, in step (4), the on-machine sequencing uses second-generation sequencing technology to obtain the whole genome sequence from the sample.

[0046] In a fifth aspect, the present invention provides use of the method for detecting the whole genome of Chlamydia in a culture sample according to the fourth aspect in detecting Chlamydia.

[0047] The numerical range described in the present invention includes not only the point values ​​listed above, but also any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] (1) The whole genome detection of Chlamydia does not rely on culture, which can simplify the detection process, shorten the detection time, and quickly generate result reports during clinical use, thereby improving the detection efficiency of Chlamydia and facilitating the rapid confirmation of Chlamydia typing.

[0050] (2) The nucleic acid content in non-culture enriched samples is low, and the capture and enrichment can improve the success rate of detection, avoid detection errors and non-detection situations; and significantly improve the success rate of Chlamydia whole genome detection in non-culture enriched samples.

[0051] (3) The present invention uses all chlamydia genome sequences as reference sequences, and the designed capture probes have good compatibility and can meet the needs of whole genome detection of different chlamydia. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 This is a schematic diagram of library loading.

[0053] Figure 2 It is a schematic diagram of the sequencing interface.

[0054] Figure 3 This is a schematic diagram of sequencing parameter settings.

[0055] Figure 4 This is a schematic diagram of running the sequencing program.

[0056] Figure 5 This is a schematic diagram of the self-test before sequencing.

[0057] Figure 6 It is a schematic diagram of the sequencing interface.

[0058] Figure 7 It is a detection flow chart. DETAILED DESCRIPTION

[0059] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0060] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0061] Example 1

[0062] This embodiment provides a probe for capturing the entire genome of Chlamydia. The probe is designed based on the entire genome sequence of Chlamydia as a reference sequence. The probe is a shingled design of 3-10 layers of probes, each probe is 120 nt long, and a total of 50,000 to 200,000 probes are designed.

[0063] The probe design method comprises:

[0064] (1) Genome download: Search and download all Chlamydia reference genes (Table 1) on NCBI (https: / / www.ncbi.nlm.nih.gov / ).

[0065] Table 1

[0066]

[0067]

[0068] (2) Probe sequence design: The downloaded reference genome is slid according to a fixed window size of 120 bp. The sliding range is dynamically adjusted according to the GC content. If the GC content is ≥50%, the probe is encrypted 1X, that is, it slides 120 bp; if the GC content is between 40% and 50%, the probe is encrypted 2X, that is, it slides 60 bp; if the GC content is between 30% and 40%, the probe is encrypted 4X, that is, it slides 30 bp; if the GC content is <30%, the probe is encrypted 8X, that is, it slides 15 bp, and the candidate probe set is obtained.

[0069] (3) Probe selection:

[0070] (3.1) Filter probes in the plasmid region (adjustable according to project needs). The plasmid library is from: https: / / ccb-microbe.cs.uni-saarland.de / plsdb / plasmids / download / . The probes are aligned to the plasmid genome and filtered according to the alignment consistency of the probe and plasmid of 90%;

[0071] (3.2) Filtering non-specific probes caused by common host assembly contamination. Probes were mapped to human, bird, and poultry genomes. The genomes were obtained from NCBI (https: / / www.ncbi.nlm.nih.gov / ). Filtering was performed based on a 90% consistency between the probe and the host genome.

[0072] (3.3) Probe homology filtering: The candidate probe sets filtered in 3.2 were subjected to homology filtering with a 95% consistency rate.

[0073] Among them, some targets and their corresponding probe sequences are shown below. The following sequences are some probes designed with reference to the above design method (Table 2).

[0074] Table 2

[0075]

[0076]

[0077] Example 2

[0078] The present invention provides a kit for detecting Chlamydia, which comprises: the probe for capturing the whole genome of Chlamydia described in Example 1, a DNA library construction reagent, a hybridization capture reagent and an auxiliary reagent. The DNA library construction reagent comprises: a fragmentation enzyme, a ligase, a ligation buffer, an amplification reaction solution, an amplification primer, a linker and purification magnetic beads. The hybridization capture reagent comprises: an enrichment tube, a sample diluent, an enrichment enhancer, an amplification primer, an amplification solution, an enrichment buffer, an eluent, an enrichment magnetic bead and a purification magnetic bead. The auxiliary reagents include a nucleic acid extraction reagent and a high-throughput sequencing reagent, which is a universal kit for sequencing reactions (Guangzhou Medical Equipment No. 20180841).

[0079] Example 3

[0080] This example provides a method for detecting the entire genome of Chlamydia in culture samples, using whole genome sequencing (WGS) as a comparison. The instruments and reagents used for the test are shown in Table 3 below. The samples were obtained from the Chinese Center for Disease Control and Prevention. CPS-YE, CPN1, and CPS-LAN were culture-enriched samples, while BYSY46, BYSY49, XU, LAN, SUN, PAN, and BAO were not culture-enriched samples. The experimental steps are as follows:

[0081] Table 3

[0082]

[0083] 1. Nucleic acid extraction

[0084] 1.1. Pipette 100 μL of sample into a 1.5 mL EP tube.

[0085] 1.2. Add 10 μL proteinase K and 100 μL buffer AL and vortex for 15 seconds to mix.

[0086] 1.3. Incubate at 56°C for 10 min.

[0087] 1.4. After centrifugation for 5 seconds, add 50 μL of anhydrous ethanol, invert 10 times to mix, and let stand at room temperature for 3 minutes.

[0088] 1.5. Centrifuge for 5 seconds, transfer the reaction solution to a MinElute column, centrifuge at 6000g for 1 minute, and discard the filtrate.

[0089] 1.6. Add 500 μL of buffer AW1 to the elution column, centrifuge at 6000 g for 1 min, and discard the filtrate.

[0090] 1.7. Add 500 μL of buffer AW2 to the elution column, centrifuge at 6000 g for 1 min, and discard the filtrate.

[0091] 1.8. Centrifuge at 20000g for 3 minutes.

[0092] 1.9. Transfer the eluted column to a new 1.5 mL EP tube.

[0093] 1.10. Add 50 μL of Buffer AE to the elution column and let it stand at room temperature for 1 min.

[0094] 1.11. Centrifuge at 20000g for 1 min and collect the filtrate.

[0095] 2. Library construction

[0096] 2.1. Prepare the end repair system in a PCR tube according to Table 4 below.

[0097] Table 4

[0098]

[0099]

[0100] 2.2. Gently shake the reagent to mix well and centrifuge for 5 seconds.

[0101] 2.3. Place the reagents on a PCR instrument and perform the reaction according to the reaction conditions in Table 5 below.

[0102] Table 5

[0103] temperature time Number of cycles 4℃ 1min 1 30℃ 5min 1 72℃ 20min 1 4℃ Keep ∞

[0104] 2.4. Prepare the linker ligation reaction system according to Table 6 below and add it to the product of step 2.3 and mix well.

[0105] Table 6

[0106] Components Volume per portion (μL) Ligation buffer 25 Ligase 3 connector 2

[0107] 2.5. Place the reagents from step 2.4 on a PCR instrument and perform the reaction according to the reaction conditions in Table 7 below.

[0108] Table 7

[0109] temperature time Number of cycles 20℃ 15min 1 4℃ Keep 1

[0110] 2.6. Remove the nucleic acid purification magnetic beads and place them at 25°C for 30 minutes.

[0111] 2.7. After thorough vortexing, add 60 μL of B1 purified magnetic beads to the product from step 2.5, pipette up and down to mix thoroughly, and place at 25°C for 5 minutes.

[0112] 2.8. Place the sample from step 2.7 on a magnetic stand and let it stand for 2 minutes. Once the solution is clear, discard the supernatant.

[0113] 2.9. Add 200 μL of 80% ethanol to the magnetic beads in step 2.8. Rotate the EP tube twice. After the solution is clarified, discard the supernatant.

[0114] 2.10. Repeat step 2.9 once.

[0115] 2.11. Place at 25°C for 5 minutes until the surface of the beads turns matte.

[0116] Resuspend the beads in 22 μL of nuclease-free water, pipette to mix, and incubate at 25°C for 3 min. Place on a magnetic stand for 2 min until the solution clears. Carefully transfer 20 μL of the supernatant to a new PCR tube for later use.

[0117] Prepare the PCR reaction mixture according to Table 8 below and add it to the product from step 2.12 and mix thoroughly.

[0118] Table 8

[0119] Components Volume per portion (μL) PCR reaction solution 25 PCR primers 5

[0120] 2.14. Place the reagents from step 2.13 on a PCR instrument and perform the reaction according to the reaction conditions in Table 9 below.

[0121] Table 9

[0122]

[0123] After thorough vortexing, add 75 μL of B1 purified magnetic beads to the product from step 2.14. Pipette up and down to mix thoroughly and incubate at 25°C for 5 min.

[0124] Place the sample from step 2.15 on a magnetic rack for 2 min. Once the solution is clear, discard the supernatant.

[0125] Add 200 μL of 80% ethanol to the magnetic beads from step 2.16. Swirl the EP tube twice to clarify the solution and discard the supernatant.

[0126] 2.18. Repeat step 2.17 once.

[0127] 2.19. Incubate at 25°C for 5 minutes until the surface of the beads turns matte.

[0128] Resuspend the beads in 22 μL of nuclease-free water, pipette to mix, and incubate at 25°C for 3 min. Place on a magnetic stand for 2 min until the solution clears. Carefully transfer 20 μL of the supernatant to a new PCR tube for later use.

[0129] 2.21. Take 1 μL of the product from step 2.20 and Follow the instructions of the dsDNA HS Assay Kit to determine its concentration and record the results.

[0130] Note: The WGS library skips step 3 and goes directly to step 4 for sequencing. The library of the present invention is then subjected to hybridization capture in step 4.3 and then sequenced in step 4.4.

[0131] 3. Hybridization capture

[0132] 3.1. Pool 250 ng of each library from 2.21 into a 0.2 mL PCR tube and add 1 fmol of Chlamydia probe.

[0133] 3.2. Take out the B3 purification magnetic beads in advance and equilibrate them to room temperature (30 minutes), then vortex and mix.

[0134] 3.3. Based on the total volume of the pooling library, add 1.8 times the volume of B3 purification magnetic beads (if 1.8 times the volume of magnetic beads is less than 50 μL, add 50 μL) to the pooling library, vortex to mix, and incubate at room temperature for 5 minutes.

[0135] 3.4. Briefly centrifuge the PCR tube and place it on a magnetic rack. Once the solution is clear, carefully remove the supernatant.

[0136] 3.5. After brief centrifugation, carefully remove all remaining alcohol at the bottom of the tube using a 10 μL pipette.

[0137] 3.6. Add 6 μL C2 hybridization buffer and 15 μL C3 hybridization enhancer equilibrated at room temperature, resuspend the magnetic beads, shake thoroughly and centrifuge for 3 seconds. At this time, the solution is separated and the magnetic bead suspension in the PCR tube is centrifuged for use.

[0138] 3.7. Set up the PCR hybridization capture program according to Table 10 (heated lid 85°C, volume 100 μL) and start the run for preheating.

[0139] Table 10

[0140]

[0141] 3.8. Transfer all the concentrated product from step 3.4 (a total of 21 μL, separated into layers, with the lower layer being the magnetic bead suspension) to the C1 enrichment tube. After removing all bubbles from the tube, place the PCR tube on the PCR instrument from step 3.7.

[0142] 3. After incubating at 98°C for 1 minute, remove the PCR tube, shake for 20 seconds, and centrifuge for 3 seconds. (At this point, the solution in the tube will separate into layers, with the magnetic bead suspension at the bottom.)

[0143] 3.10. Use the SKIP function to end the 68°C∞ program. When the PCR instrument reaches 95°C, place the PCR tube from step 2.3 back into the PCR instrument.

[0144] 3.11. Use the SKIP function to end the 95℃∞ program and enter the 95℃5min program.

[0145] 3.1 After 2.5 minutes, the PCR instrument enters the 60°C∞ program to begin the hybridization reaction. This step must be maintained for at least 30 minutes.

[0146] 3.13.B2 After the enrichment magnetic beads are equilibrated at room temperature for 30 minutes, vortex and shake for 5 seconds to mix thoroughly and set aside.

[0147] For each reaction, pipette 25 μL of B2 enrichment magnetic beads into a new PCR tube, place on a magnetic stand for 15 seconds, and discard the supernatant.

[0148] Add 100 μL of C6 enrichment buffer, pipette to mix the beads, centrifuge for 1 second, place on a magnetic stand for 15 seconds, and discard the supernatant.

[0149] 3.16. Repeat step 3.15 twice, for a total of three washes.

[0150] 3.17. Add 20 μL of C6 enrichment buffer, pipette and mix the beads, and store at room temperature.

[0151] 3.18. After step 3.12 has run for 30 min, open the incubation tube on the PCR instrument, add 80 μL of C6 enrichment buffer, vortex to mix, centrifuge briefly for 1 second, and place on a magnetic rack. Once the solution has clarified, transfer the entire supernatant to 20 μL of B2 enrichment magnetic bead suspension from step 3.17, vortex to mix thoroughly, and centrifuge briefly for 1 second.

[0152] 3.19. Use the SKIP function to end the 60°C∞ program and enter the 65°C∞ program. Place the PCR tube from step 3.18 on the PCR instrument and incubate for 30 minutes. Pipet and mix every 5 minutes to resuspend the enriched magnetic beads, ensuring that there are no magnetic beads clumping at the bottom and no magnetic beads remaining on the tube cap.

[0153] During 3.20, preheat C7 Wash Solution 1 (320 μL / each reaction) and C8 Wash Solution 2 (480 μL / each reaction) in a PCR instrument.

[0154] 3.21. After the reaction in step 3.19 is completed, remove the samples from the PCR instrument (C7 Wash 1 and C8 Wash 2 must remain on the PCR instrument), use the SKIP function to end the 65°C∞ program, and enter the 68°C∞ program.

[0155] Centrifuge the sample for 1 second, place on a magnetic rack, and discard the supernatant after the solution has clarified.

[0156] Add 150 μL of preheated C7 Wash Buffer 1, pipette to mix, and incubate at 68°C for 5 min. Ensure that there are no beads clumping at the bottom of the tube and no beads remaining on the cap.

[0157] Place the beads on the magnetic rack for approximately 2-5 seconds. Discard the supernatant immediately after clarification.

[0158] Resuspend the beads by adding 150 μL of preheated C7 Wash Buffer 1. Transfer the entire suspension to a new PCR tube and incubate at 68°C for 5 min. Ensure that there are no beads clumping at the bottom and no beads remaining on the tube cap.

[0159] Place on the magnetic stand for approximately 2-5 seconds. After the beads have been clarified, discard the supernatant immediately.

[0160] 3.27. Use the SKIP function to end the 68°C∞ program and enter the 48°C∞ program.

[0161] Add 150 μL of preheated C8 Wash Buffer 2, pipette to mix, and incubate at 48°C for 5 min to ensure that there are no beads clumping at the bottom of the tube and no beads remaining on the cap.

[0162] Place on the magnetic stand for about 2-5 seconds. After the beads are clarified, discard the supernatant immediately.

[0163] 3.30. Repeat steps 3.28-3.29 twice for a total of three washes.

[0164] Centrifuge the magnetic beads from step 3.30 for 3 seconds. Place them on a magnetic rack for approximately 5 seconds and then carefully remove any remaining liquid from the bottom of the tube using a 10 μL pipette.

[0165] Resuspend the beads in 22 μL of NF-Water, pipette to mix, and use 11 μL for the downstream amplification step (store the remaining 11 μL at -20°C for later use).

[0166] Prepare the PCR system according to Table 11:

[0167] Table 11

[0168] Reaction system (25 μL) Single dosage (μL) Magnetic bead suspension 11 C4 amplification primer 1.5 C5 Amplification Buffer 12.5

[0169] 3.34. Mix the prepared PCR system and perform the reaction according to the procedure in Table 12:

[0170] Table 12

[0171]

[0172]

[0173] 3.35.B3 After equilibration of the purified magnetic beads at room temperature for 30 min, vortex and mix thoroughly.

[0174] 3.36. Add 38 μL of B3 purification magnetic beads to the product from step 3.34, pipette to mix, and incubate at room temperature for 5 min.

[0175] 3.37. Briefly centrifuge the PCR tube for 3 seconds and place it on a magnetic rack. After the solution is clear, aspirate and discard the supernatant.

[0176] Add 200 μL of freshly prepared 80% ethanol and transfer the PCR tube twice on a magnetic rack to thoroughly wash the beads. Once the solution is clear, aspirate and discard the supernatant.

[0177] 3.39. Repeat step 3.38 once, for a total of two washes.

[0178] Centrifuge the magnetic beads from step 3.39 for 5 seconds. Place them on a magnetic rack for approximately 30 seconds and then carefully remove any remaining liquid from the bottom of the tube using a 10 μL pipette.

[0179] After the beads have dried, add 22 μL of nuclease-free water, pipette to mix, and let stand at room temperature for 3 min.

[0180] Briefly centrifuge the PCR tube for 3 seconds, place on a magnetic rack, and after the solution has clarified, transfer 20 μL of the supernatant to a new PCR tube and store at -20°C until further use.

[0181] 3.43. Determine the concentration of the product from step 3.42 using a Qubit 3.0 / 4.0 assay according to the instructions for the Qubit dsDNA HS Assay Kit (Invitrogen) and record the concentration. A concentration ≥ 1 ng / μL is considered acceptable.

[0182] 4. High-throughput sequencing

[0183] Use a 1 mL pipette tip to pierce the tin foil of well 16 (labeled "Load Library Here"), replace the tip with a new 1 mL pipette tip, take 500 μL of the sequencing library from step 3.43, and press Figure 1 Add to well 16. Click "Sequence" on the software interface to enter the sequencing parameter setting interface ( Figure 2 ). In the "Run Mode" interface, select Manual and click "Next". Figure 3 Set the sequencing run parameters as shown (the Run Name can be personalized) and click "Next". Follow the instrument prompts to load the sequencing chip into the chip compartment, the reagent cartridge into the reagent compartment, and the empty waste bottle into the waste compartment. Close the chip compartment door and the reagent compartment door, and click "Next". Figure 4 After entering the Check interface, the instrument will perform a self-check. After all options have passed the self-check, click Start to run the sequencing program ( Figure 5 and Figure 6 After sequencing, the data were subjected to bioinformatics analysis.

[0184] 5. Results: In three cultures, the 1x coverage of WGS was 94.42%, 55.45%, and 99.95%, respectively, while the 1x coverage of the present invention was 94.60%, 91.33%, and 99.94%, respectively. In seven uncultured samples, the 1x coverage of WGS was 0% to 0.21%, while the 1x coverage of the present invention was 3.84% to 99.74%. The number of WGS samples with an assembly completeness greater than 90% was 2 / 10, while that of the present invention was 5 / 10. Specific results are shown in Table 13 below.

[0185] Table 13

[0186]

[0187] The above results show that the detection method of the present invention is more suitable for the detection of non-culture enriched samples than WGS, and significantly improves the success rate of whole genome detection of Chlamydia in non-culture enriched samples.

[0188] In summary, the present invention provides a method for whole genome detection of Chlamydia, based on NGS technology, to design whole genome probes for Chlamydia. Figure 7 As shown, the nucleic acid library of the clinical sample is first constructed, and then the nucleic acid library of Chlamydia is obtained by hybridization capture using designed probes. Finally, the library is sequenced and analyzed by high-throughput sequencing to obtain the whole genome information of Chlamydia.

[0189] The applicant declares that the above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the scope of protection and disclosure of the present invention.

Claims

1. A probe for capturing the whole genome of Chlamydia, characterized in that The probes are designed based on the Chlamydia genome sequence as a reference sequence, and the specific reference genome is shown in the following table; The probes are designed in a shingled manner with 3-10 layers of probes, each probe is 90-120 nt long, and a total of 50,000-200,000 probes are designed.

2. A kit for detecting Chlamydia, characterized in that: The kit comprises the probe for capturing the whole genome of Chlamydia according to claim 1.

3. The kit for detecting Chlamydia according to claim 2, characterized in that The kit further comprises: any one or a combination of at least two of a DNA library construction reagent, a hybridization capture reagent or an auxiliary reagent; Preferably, the DNA library construction reagents include: any one or a combination of at least two of fragmentation enzymes, ligases, ligation buffers, amplification reaction solutions, amplification primers, adapters, or purification magnetic beads; Preferably, the hybrid capture reagent comprises: any one or a combination of at least two of: a sample diluent, an enrichment enhancer, an amplification primer, an amplification solution, an enrichment buffer, an eluent, enrichment magnetic beads or purification magnetic beads; Preferably, the auxiliary reagents include: nucleic acid extraction reagents and / or high-throughput sequencing reagents.

4. Use of the probe for capturing the whole genome of Chlamydia according to claim 1 and / or the kit for detecting Chlamydia according to claim 2 or 3 in the preparation of a product for detecting Chlamydia.

5. A method for detecting the whole genome of Chlamydia, characterized in that: The probe for capturing the whole genome of Chlamydia according to claim 1 is used to perform hybridization capture and high-throughput sequencing analysis on the sample to be tested to obtain the whole genome sequence.

6. The method for detecting the whole genome of Chlamydia according to claim 5, characterized in that: The method comprises: (1) extracting nucleic acid from the sample to be tested; (2) The extracted nucleic acid is sequentially fragmented, end-repaired, and ligated with adapters, and then amplified to obtain a Chlamydia whole genome library; (3) hybridizing and incubating the obtained Chlamydia whole genome library with the probe for capturing the Chlamydia whole genome according to claim 1, and then sequentially performing magnetic bead capture and amplification to obtain a high-throughput sequencing sample; (4) The obtained high-throughput sequencing samples are sequentially subjected to library dilution, library denaturation, sequencing and data analysis.

7. The method for detecting the whole genome of Chlamydia according to claim 6, characterized in that: In step (2), a purification step is also included after connecting the linker and / or after amplification.

8. The method for detecting the whole genome of Chlamydia according to claim 6 or 7, characterized in that: In step (3), the amount of the library and probe hybridization incubation is 0.05-2 fmol of probe for every 200-500 ng of library; Preferably, in step (3), the conditions for hybridization incubation of the library and the probe are as follows: Incubate hybridization at 58-62°C for 28-32 min; incubate capture at 63-67°C for 28-32 min; wash at least twice at 66-70°C; and wash at least three times at 46-50°C.

9. The method for detecting the whole genome of Chlamydia according to any one of claims 6 to 8, characterized in that: In step (4), the on-machine sequencing uses second-generation sequencing technology to obtain the whole genome sequence from the sample.

10. Use of the Chlamydia whole genome detection method according to any one of claims 6 to 9 in detecting Chlamydia.

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