A method for detecting the entire genome of the SARS-CoV-2 virus

Abstract

This application provides a method for detecting the entire SARS-CoV-2 virus genome based on nanopore sequencing, a primer combination used in the method, and a kit containing the primer combination. The method provided by the present invention uses a small number of primers in primer design, which greatly saves costs and is economical and reliable. It also utilizes multiplex PCR with the advantages of high throughput and low cost, showing great flexibility.

Description

Technical Field

[0001] The present application belongs to the field of genetic testing, and specifically relates to a method for detecting the whole genome of the SARS-CoV-2 virus based on nanopore sequencing, the primers involved in the method, and a kit containing the aforementioned primers. Background Art

[0002] Whole-genome sequencing of the SARS-CoV-2 virus involves multiple methods, including metatranscriptome sequencing, multiplex PCR amplification sequencing, and hybrid capture sequencing. Metatranscriptome sequencing can investigate all potential pathogens, including the target microorganism, but requires a large amount of sequencing data and has low sensitivity for samples with low viral loads. Hybrid capture sequencing increases the proportion of viral reads, improving detection sensitivity and enabling the detection of base mutations and base frequencies. However, compared to multiplex PCR amplification sequencing, it may miss some mutations, especially when the viral load is low. Multiplex PCR amplification sequencing offers improved sensitivity and is relatively more accurate at low viral loads, especially when the viral load is extremely low. Most commercially available multiplex PCR amplification methods for SARS-CoV-2 virus amplify fragments between 300 and 500 bp, requiring a large number of primers and the simultaneous amplification of two PCR products. Currently, there is a lack of methods that can detect SARS-CoV-2 virus typing, emerging mutations, or variants with high sensitivity and accuracy using fewer primers. Summary of the Invention

[0003] In order to improve the above technical problems, on the one hand, the present invention provides a method for detecting the whole genome of SARS-CoV-2 virus based on nanopore sequencing, which comprises the following steps:

[0004] a. Design and synthesize reverse transcription primers P7R2-Random primers and multiple PCR primers, the nucleic acid sequence of the reverse transcription primer is shown in SEQ ID NO: 34, the multiple PCR primers include Left Primer and Right Primer, the nucleic acid sequence of the Left Primer is shown in SEQ ID NO: 1-32, the nucleic acid sequence of the Right Primer is shown in SEQID NO: 33;

[0005] b. Reverse transcription of the extracted viral RNA was performed using a reverse transcription primer to obtain a cDNA product;

[0006] c. The cDNA product was mixed with the Left Primer pool, Right primer, and Q5® Hot Start HF 2XMaster Mix for multiplex PCR amplification;

[0007] d. DNA purification of multiplex PCR amplification products;

[0008] e. Perform end repair on the purified PCR product and further purify it;

[0009] f. Connecting the end-repaired product to the nanopore sequencing adapter and purifying it;

[0010] g. Sequence the adapter-ligated DNA library using a nanopore sequencer to obtain the complete genome sequence of SARS-CoV-2.

[0011] In one embodiment, the reverse transcription primer P7R2-Random primers in the method comprises a fixed sequence and a random sequence NNNNNN, and the nucleic acid sequence of the fixed sequence is shown in SEQ ID NO:33.

[0012] In one embodiment, the reaction conditions for the multiplex PCR amplification in the method are: pre-denaturation at 98°C for 30 seconds, followed by 35 cycles of amplification, wherein the cycle program is 98°C for 15 seconds, 61°C for 2 minutes, 65°C for 3 minutes, and finally stored at 4°C.

[0013] In one embodiment, the DNA purification, end repair, and adapter ligation steps in the method are all purified using DNAXP magnetic beads, and the target product is eluted using an elution buffer.

[0014] In one embodiment, the sequencing step in step (g) of the method comprises:

[0015] a) Mix the anchor adapter with sequencing buffer and incubate for 5 minutes;

[0016] b) mixing the sample to be tested with sequencing buffer and then flowing into the sequencing system;

[0017] c) Start sequencing.

[0018] In another aspect, the present invention provides a primer combination for detecting the whole genome of SARS-CoV-2, comprising:

[0019] a) a reverse transcription primer, the nucleotide sequence of which is shown in SEQ ID NO: 34);

[0020] b) a multiplex PCR primer pool comprising a Left Primer and a Right Primer, wherein the nucleic acid sequence of the Left Primer is shown in SEQ ID NO: 1-32, and the nucleic acid sequence of the Right Primer is shown in SEQ ID NO: 33.

[0021] In another aspect, the present invention provides a kit for detecting the whole genome of SARS-CoV-2, comprising:

[0022] a) the aforementioned primer combination;

[0023] b) Q5® Hot Start HF 2X Master Mix;

[0024] c) DNA XP magnetic beads;

[0025] d) nanopore sequencing adapter;

[0026] e) Sequencing buffer.

[0027] In one embodiment, the kit is used to detect the whole genome of SARS-CoV-2 in low viral load samples by nanopore sequencing technology.

[0028] Beneficial effects

[0029] The multiplex PCR used in the present invention only requires the PCR reaction to be completed in the same reaction tube, avoiding errors and saving time and reagents; the present invention uses a small number of primers in primer design, greatly saving costs and being economical and reliable; the multiplex PCR used in the present invention has the advantages of high throughput and low cost, showing great flexibility; the multiplex PCR amplification products used in the present invention are of different lengths, which can give full play to the advantage of nanopore sequencing that is not limited to fragment length. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The operational steps for whole genome sequencing of the SARS-CoV-2 virus provided in this application are shown.

[0031] Figure 2 The results of 1% agarose gel electrophoresis detection of multiplex PCR products are shown.

[0032] Figure 3 The distribution diagram of each fragment of SARS-CoV-2 virus sample is shown.

[0033] Figure 4 The sequencing depth distribution of each fragment of SARS-CoV-2 virus sample is shown.

[0034] Figure 5 The base sequencing depth distribution of each fragment of the SARS-CoV-2 virus sample is shown.

[0035] Figure 6 The average sequencing depth of each fragment of SARS-CoV-2 virus samples was calculated. DETAILED DESCRIPTION

[0036] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0037] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0038] Example 1 Detection of the entire SARS-CoV-2 virus genome based on nanopore sequencing

[0039] Primers were designed and synthesized based on the complete SARS-CoV-2 genome. The reverse transcription primer, P7R2-Randomprimers, has a sequence of 5'-CCTTGGCACCCGAGAATTCCANNNNNN (its nucleotide sequence is shown in SEQ ID NO:34). The LEFT primer pool (its nucleotide sequence is shown in SEQ ID NOs:1-32) divides the SARS-CoV-2 viral sequence into 29 1.2 kb segments. Multiple specific primers were designed to evenly divide the SARS-CoV-2 viral genome into multiple small segments for amplification, ensuring that each segment is fully amplified. This achieves uniform coverage of the viral genome, reduces coverage bias caused by insufficient amplification of certain regions, ensures complete genomic information, and facilitates accurate monitoring of viral mutations. The right primer is the P7R2 primer, with a sequence of 5'-CCTTGGCACCCGAGAATTCCA (its nucleotide sequence is shown in SEQ ID NO:33). Primers were prepared by Shanghai Sangon Biotechnology Co., Ltd. and purified by HPLC and identified by mass spectrometry. The steps for detecting the full genome of SARS-CoV-2 virus based on nanopore sequencing are as follows:

[0040] 1. For 1×10 5 Nucleic acid was extracted from SARS-CoV-2 virus at a concentration of 10 copies / mL. The extracted RNA was reverse transcribed using P7R2-Random primers according to the instructions for Thermo MaximaH Minus Reverse Transcriptase. These specific primers accurately recognize and bind to specific regions of the SARS-CoV-2 viral genome, avoiding nonspecific binding to the host genome or other non-target nucleic acid sequences. This ensures that the amplified product is the target gene fragment, improving the accuracy of the sequencing results.

[0041] Prepare the following reaction system in a nuclease-free PCR tube as shown in Table 1:

[0042] Table 1

[0043]

[0044] After gentle mixing, place the sample in a PCR instrument at 65°C for 5 min and place on ice.

[0045] Then add the following reaction system as shown in Table 2:

[0046] Table 2

[0047]

[0048] Mix gently and run the reaction according to the following program shown in Table 3:

[0049] Table 3

[0050]

[0051] The final cDNA product was stored at 4°C overnight or at -80°C for long-term storage.

[0052] 2. Multiplex PCR

[0053] Prepare the following reaction system in a PCR tube as shown in Table 4:

[0054] Table 4

[0055]

[0056] Mix thoroughly and perform PCR amplification according to the following procedure as shown in Table 5.

[0057] Table 5

[0058]

[0059] 3. After PCR, 1% agarose gel electrophoresis was used to detect the amplified bands. A diffuse band was observed around 750 bp. Figure 2 , indicating that the amplification was successful.

[0060] 4. Purify the amplicon product.

[0061] 5. End Repair and Purification

[0062] (1) Melt the end repair reagents on ice, mix gently and centrifuge briefly to the bottom of the tube. Prepare the following reaction system as shown in Table 6 in a sterile PCR tube:

[0063] Table 6

[0064]

[0065] (2) After thorough mixing, place the PCR tube into the PCR instrument and set the reaction program to: 20℃ 5 min; 65℃ 5 min; 4℃ ∞; the PCR instrument heat cover is 75℃.

[0066] (3) Take out the DNA purification magnetic beads half an hour in advance, equilibrate to room temperature and mix thoroughly; transfer the end repair product to a 1.5mL centrifuge tube, add 60μL of resuspended magnetic beads and mix well, and incubate at room temperature for 5 minutes.

[0067] (4) Prepare freshly prepared 80% ethanol and place the centrifuge tube on a magnetic rack to separate the magnetic beads for about 5 minutes. After the solution is clear, carefully remove the supernatant and discard it. Keep the centrifuge tube on the magnetic rack and add 200 μL of freshly prepared 80% ethanol to incubate for 30 seconds. Carefully remove the supernatant and discard it. Repeat this step once.

[0068] (5) Open the centrifuge tube cap and let it air dry for 3-5 minutes until there is no obvious water mark on the surface of the magnetic beads; remove the centrifuge tube from the magnetic rack, add 63 μL of nuclease-free water to thoroughly mix the magnetic beads, and incubate at room temperature for 5 minutes; place the centrifuge tube on the magnetic rack to separate the magnetic beads for about 2-3 minutes. After the solution is clear, carefully aspirate the supernatant and transfer it to a new centrifuge tube, and take 2 μL to measure the concentration.

[0069] 6. Ligation of Adaptor Complex and Purification

[0070] (1) Briefly centrifuge the adapter complex and DNA ligase and place on ice. Thaw the ligation buffer on ice and mix thoroughly by pipetting. Thaw the fragment recovery buffer and elution buffer at room temperature, mix thoroughly, and immediately place on ice. Prepare the following reaction system as shown in Table 7 in a 1.5 mL centrifuge tube:

[0071] Table 7

[0072]

[0073] (2) After thorough mixing, place the 1.5 mL centrifuge tube in a 20°C metal bath and incubate for 15 minutes.

[0074] (3) Take out the DNA purification magnetic beads half an hour in advance and equilibrate them to room temperature and mix them thoroughly; transfer the product to a 1.5mL centrifuge tube, add 0.8 times the volume of the resuspended magnetic beads and mix them, and incubate at room temperature for 10 minutes; place the centrifuge tube on a magnetic rack to separate the magnetic beads for about 5 minutes. After the solution is clear, carefully remove the supernatant and discard it.

[0075] (4) Remove the centrifuge tube and add 200 μL of fragment recovery buffer to resuspend the magnetic beads (use long fragment recovery buffer when recovering DNA fragments >3Kb; use long and short fragment recovery buffer when recovering DNA fragments of various lengths). Then place the centrifuge tube on the magnetic rack to separate the magnetic beads. After the solution is clarified, carefully remove the supernatant and discard it. Repeat this step once; aspirate as much residual liquid as possible in the tube.

[0076] (5) Remove the centrifuge tube from the magnetic stand, add 20 μL of elution buffer to thoroughly mix the magnetic beads, and incubate at 37°C for 10 minutes; place the centrifuge tube on the magnetic stand to separate the magnetic beads for about 2-3 minutes. After the solution is clear, carefully transfer the supernatant to a new centrifuge tube and take 2 μL to measure the concentration; sequencing library quality control uses the Thermo Fisher Qubit fluorescence quantitative instrument to perform nucleic acid quantitative determination on each purified sample in the library preparation. Take 1 μL of the purified sample and add 199 μL of Qubit™ 1X dsDNAHS Working Solution to quantitatively detect the sample concentration.

[0077] 7. Sequencing

[0078] The nanopore sequencing method was used to sequence the cells using the Polyseq One ​​nanopore sequencer developed by Beijing Puyi Biotechnology Co., Ltd. The sequencing steps were as follows:

[0079] (1) Mix 5 μL of anchor adapter, 95 μL of enzyme-free water, and 100 μL of sequencing buffer into the system and incubate for 5 minutes;

[0080] (2) Take 200 ng of the sample to be tested, fill it up to 50 μL with enzyme-free water, then add 50 μL of sequencing buffer and mix, and flow into the system;

[0081] (3) Start sequencing.

[0082] 8. Viral genome alignment

[0083] (1) The viral reference genome is SARS-CoV-2 MN908947.3. The sequencing data after quality control is aligned with the reference sequence to obtain a bam file of the alignment between the sequencing data and the reference sequence.

[0084] (2) Calculate the coverage, where the 1× sequencing coverage value is 98.83%, the 10× sequencing coverage value is 97.67%, the 50× sequencing coverage value is 94.57%, the 100× sequencing coverage value is 93.94%, and the 1000× sequencing coverage value is 72.48%. Figure 3 The distribution of each fragment of SARS-CoV-2 sample is shown. Figure 4 The total base sequencing depth distribution is shown. Figure 5 The base sequencing depth distribution of 29 fragments is shown. Figure 6 The average sequencing depth of 29 fragments was calculated (the lowest average sequencing depth was 1306.25 for cov7 and the highest average sequencing depth was 41125.7 for cov29). The sequencing data met the requirements for SARS-CoV-2 whole-genome sequencing and typing analysis. The SARS-CoV-2 genome coverage exceeded 93%, providing a sequencing solution for sequencing samples with low viral loads. Furthermore, nanopore whole-genome sequencing technology reduces the sequencing and analysis time of SARS-CoV-2 samples. Through real-time base calling and bioinformatics analysis, it rapidly identifies pathogenic mutations, improving the efficiency and sensitivity of SARS-CoV-2 sequencing.

[0085] The above describes the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

[0086] The primer sequences involved in this application are shown in Table 8.

[0087] Table 8

[0088] .

Claims

1. A method for detecting the whole genome of SARS-CoV-2 virus based on nanopore sequencing for non-diagnostic purposes, comprising the following steps: a. Design and synthesize reverse transcription primers P7R2-Random primers and multiple PCR primers, the nucleic acid sequence of the reverse transcription primer is shown in SEQ ID NO: 34, the multiple PCR primers include Left Primer and Right Primer, the nucleic acid sequence of the Left Primer is shown in SEQ ID NO: 1-32, and the nucleic acid sequence of the Right Primer is shown in SEQ ID NO: 33; b. Reverse transcription of the extracted viral RNA was performed using a reverse transcription primer to obtain a cDNA product; c. The cDNA product was mixed with the Left Primer pool, Right primer, and Q5® Hot Start HF 2X MasterMix for multiplex PCR amplification; d. DNA purification of multiplex PCR amplification products; e. Perform end repair on the purified PCR product and further purify it; f. Connecting the end-repaired product to the nanopore sequencing adapter and purifying it; g. Sequence the adapter-ligated DNA library using a nanopore sequencer to obtain the complete genome sequence of SARS-CoV-2. The reverse transcription primer P7R2-Random primers comprises a fixed sequence and a random sequence NNNNNN, and the nucleic acid sequence of the fixed sequence is shown in SEQ ID NO:

33.

2. The method according to claim 1, wherein the reaction conditions for the multiplex PCR amplification are: initial denaturation at 98°C for 30 seconds, followed by 35 cycles of amplification, wherein the cycling program is 98°C for 15 seconds, 61°C for 2 minutes, and 65°C for 3 minutes, and finally stored at 4°C.

3. The method according to claim 1, wherein the DNA purification, end repair and adapter ligation steps are all performed using DNA XP magnetic beads, and the target product is eluted using elution buffer.

4. The method according to claim 1, wherein the sequencing step in step (g) comprises: a) Mix the anchor adapter with sequencing buffer and incubate for 5 minutes; b) mixing the sample to be tested with sequencing buffer and then flowing into the sequencing system; c) Start sequencing.

5. A primer combination for detecting the whole genome of SARS-CoV-2, comprising: a) a reverse transcription primer, the nucleotide sequence of which is shown in SEQ ID NO: 34; b) a multiplex PCR primer pool comprising a Left Primer and a Right Primer, wherein the nucleic acid sequence of the Left Primer is shown in SEQ ID NO: 1-32, and the nucleic acid sequence of the Right Primer is shown in SEQ ID NO:

33.

6. A kit for detecting the whole genome of SARS-CoV-2, comprising: a) the primer combination according to claim 5; b) Q5® Hot Start HF 2X Master Mix; c) DNA XP magnetic beads; d) nanopore sequencing adapter; e) Sequencing buffer.

7. The kit according to claim 6, which can be used to detect the whole genome of SARS-CoV-2 in low viral load samples by nanopore sequencing technology.

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