Method for detecting SARS-CoV-2 virus whole genome

Abstract

The invention provides a method for detecting an SARS-CoV-2 virus whole genome based on nanopore sequencing, a primer combination used in the method and a kit containing the primer combination. According to the method provided by the invention, the number of primers used in primer design is small, the cost is greatly saved, the method is economical and reliable, and by utilizing the advantages of high throughput and low cost of multiple PCR, the method shows 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 SARS-CoV-2 virus based on nanopore sequencing, the primers involved in the method and a kit containing the aforementioned primers. Background Art

[0002] The whole genome sequencing of SARS-CoV-2 virus involves a variety of methods, including macrotranscriptome sequencing, multiplex PCR amplification sequencing, and hybrid capture sequencing. Macrotranscriptome sequencing can study all possible pathogenic microorganisms including target microorganisms, requires a large amount of sequencing data, and has low detection sensitivity for samples with low viral load. Hybrid capture sequencing can increase the proportion of viral reads, improve detection sensitivity, and detect base mutations and base frequencies. Compared with multiplex PCR amplification sequencing, some mutation sites may be missed, especially when the sample viral load is low. Multiplex PCR amplification sequencing has better detection sensitivity and is relatively more accurate under low viral load conditions, especially when the sample viral load is extremely low. Most of the amplified fragments of SARS-CoV-2 virus multiplex PCR on the market are 300-500bp, requiring a large number of primers, and requiring the amplification of 2 tubes of PCR products at the same time. At present, there is a lack of methods that use fewer primers to detect the typing, new mutations or variant strains of SARS-CoV-2 virus with higher sensitivity and accuracy. 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: a. Designing and synthesizing reverse transcription primers P7R2-Random primers and multiple PCR primers, wherein 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 SEQID NO: 33; b. using reverse transcription primers to reverse transcribe the extracted RNA virus to obtain a cDNA product; c. Mixing the cDNA product with the Left Primer pool, Right primer and Q5® Hot Start HF 2XMaster Mix 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. Use a nanopore sequencer to sequence the adapter-ligated DNA library to obtain the complete genome sequence of SARS-CoV-2.

[0004] 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.

[0005] 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.

[0006] 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.

[0007] In one embodiment, the sequencing step in step (g) of the method comprises: a) Mix the anchor adapter with sequencing buffer and incubate for 5 minutes; b) mixing the sample to be tested with the sequencing buffer and then flowing into the sequencing system; c) Start sequencing.

[0008] On the other hand, the present invention provides 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.

[0009] On the other hand, the present invention provides a kit for detecting the whole genome of SARS-CoV-2, comprising: a) the aforementioned primer combination; b) Q5® Hot Start HF 2X Master Mix; c) DNA XP magnetic beads; d) Nanopore sequencing adapter; e) Sequencing buffer.

[0010] 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.

[0011] Beneficial Effects The multiplex PCR adopted in the present invention only needs to complete the PCR reaction in the same reaction tube, thus 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 adopted in the present invention has the advantages of high throughput and low cost, and shows great flexibility; the multiplex PCR amplification products adopted in the present invention are of different lengths, which can give full play to the advantage of nanopore sequencing that is not limited to the length of fragments. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The operational steps for whole genome sequencing of the SARS-CoV-2 virus provided in this application are shown.

[0013] Figure 2 The results of 1% agarose gel electrophoresis for detecting multiplex PCR products are shown.

[0014] Figure 3 The distribution diagram of each fragment of SARS-CoV-2 virus sample is shown.

[0015] Figure 4 The sequencing depth distribution of each fragment of SARS-CoV-2 virus sample is shown.

[0016] Figure 5 The base sequencing depth distribution map of each fragment of the SARS-CoV-2 virus sample is shown.

[0017] Figure 6 The average sequencing depth of each fragment of SARS-CoV-2 virus samples was counted. DETAILED DESCRIPTION

[0018] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions 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 included in the scope that the present invention is intended to protect.

[0019] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0020] Example 1 Detection of the entire genome of SARS-CoV-2 virus based on nanopore sequencing Primers were designed and synthesized based on the whole genome of SARS-CoV-2 virus. The reverse transcription primer P7R2-Randomprimers sequence was 5'-CCTTGGCACCCGAGAATTCCANNNNNN (its nucleic acid sequence is shown in SEQ ID NO:34). The sequence of the LEFT primer pool (its nucleic acid sequence is shown in SEQ ID NO:1-32) is a sequence that divides the SARS-CoV-2 virus sequence into 29 segments of 1.2 Kb. Through the design of multiple specific primers, the whole genome of SARS-CoV-2 virus is evenly divided into multiple small fragments for amplification, so that each fragment can be fully amplified, thereby achieving uniform coverage of the whole genome of the virus, reducing coverage deviation caused by insufficient amplification of certain regions, ensuring the acquisition of complete genome information, and helping to accurately monitor the mutation of the virus. The right primer is P7R2 primers, and the primer sequence is 5'-CCTTGGCACCCGAGAATTCCA (its nucleic acid sequence is shown in SEQ ID NO:33). The primers were completed by Shanghai Shenggong Biotechnology Co., Ltd., and HPLC purification and mass spectrometry identification were performed. The steps for detecting the whole genome of SARS-CoV-2 virus based on nanopore sequencing are as follows: 1. For 1×10 5 The nucleic acid of SARS-CoV-2 virus with 100 copies / mL was extracted, and the extracted RNA was reverse transcribed. The extracted RNA virus was reverse transcribed using P7R2-Random primers random primers, and the operation was performed according to the instructions of Thermo MaximaH Minus reverse transcriptase. This specific primer can accurately identify and bind to specific regions of the SARS-CoV-2 viral genome, avoiding non-specific binding with the host genome or other non-target nucleic acid sequences, thereby ensuring that the amplified product is the target gene fragment and improving the accuracy of the sequencing results.

[0021] Prepare the following reaction system in a nuclease-free PCR tube as shown in Table 1: Table 1

[0022] After gentle mixing, place the sample in a PCR instrument at 65°C for 5 min and place on ice when finished.

[0023] Then add the following reaction system as shown in Table 2: Table 2

[0024] Mix gently and run the reaction according to the following procedure as shown in Table 3: Table 3

[0025] The final cDNA product was stored at 4°C overnight or at -80°C for long-term storage.

[0026] 2. Multiplex PCR Prepare the following reaction system in a PCR tube as shown in Table 4: Table 4

[0027] Mix thoroughly and perform PCR amplification according to the following procedure as shown in Table 5.

[0028] Table 5

[0029] 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.

[0030] 4. Purify the amplicon product.

[0031] 5. End Repair and Purification (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: Table 6

[0032] (2) After thorough mixing, place the PCR tube into the PCR instrument and set the reaction program as follows: 20℃ 5 min; 65℃ 5min; 4℃ ∞; PCR instrument heat cover to 75℃.

[0033] (3) Take out the DNA purification magnetic beads half an hour in advance, equilibrate them to room temperature and mix them thoroughly; transfer the end repair product to a 1.5 mL centrifuge tube, add 60 μL of the resuspended magnetic beads and mix them well, and incubate at room temperature for 5 minutes.

[0034] (4) Prepare freshly prepared 80% ethanol and place the centrifuge tube on the 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, add 200 μL of freshly prepared 80% ethanol and incubate for 30 seconds. Carefully remove the supernatant and discard it. Repeat this step once.

[0035] (5) Open the centrifuge tube lid 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.

[0036] 6. Ligation of Adaptor Complex and Purification (1) Briefly centrifuge the adapter complex and DNA ligase and place on ice. Melt the ligation buffer on ice and mix well by pipetting. Melt the fragment recovery buffer and elution buffer at room temperature, mix well and immediately place on ice. Prepare the following reaction system as shown in Table 7 in a 1.5 mL centrifuge tube: Table 7

[0037] (2) After thorough mixing, place the 1.5 mL centrifuge tube in a 20°C metal bath and incubate for 15 min.

[0038] (3) Take out the DNA purification magnetic beads half an hour in advance, equilibrate to room temperature and mix thoroughly; transfer the product to a 1.5mL centrifuge tube, add 0.8× volume of the resuspended magnetic beads and mix well, 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.

[0039] (4) Remove the centrifuge tube and add 200 μL of fragment recovery buffer to resuspend the magnetic beads (use long fragment recovery buffer to recover DNA fragments >3 Kb; use long and short fragment recovery buffer to recover DNA fragments of various lengths). Then place the centrifuge tube on a 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.

[0040] (5) Remove the centrifuge tube from the magnetic stand, add 20 μL of elution buffer to mix the magnetic beads thoroughly, 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 clarified, carefully transfer the supernatant to a new centrifuge tube and take 2 μL to measure the concentration; The sequencing library quality control uses the Thermo Fisher Qubit fluorescence quantification 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 Qubit™ 1X dsDNAHS Working Solution to quantitatively detect the sample concentration.

[0041] 7. Sequencing The nanopore sequencing method was used to perform sequencing using the nanopore sequencer Polyseq One ​​developed by Beijing Puyi Biotechnology Co., Ltd. The sequencing steps are as follows: (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; (2) Take 200 ng of the sample to be tested, make up to 50 μL with enzyme-free water, then add 50 μL of sequencing buffer and mix, and flow into the system; (3) Start sequencing.

[0042] 8. Viral genome alignment (1) The viral reference genome is SARS-CoV-2 MN908947.3. The sequencing data after quality control is compared with the reference sequence to obtain a bam file of the comparison between the sequencing data and the reference sequence.

[0043] (2) Calculate the coverage rate: 1× sequencing coverage rate is 98.83%, 10× sequencing coverage rate is 97.67%, 50× sequencing coverage rate is 94.57%, 100× sequencing coverage rate is 93.94%, and 1000× sequencing coverage rate is 72.48%. Figure 3 The distribution map of each fragment of SARS-CoV-2 sample is shown. Figure 4 The total base sequencing depth distribution diagram is shown. Figure 5 The base sequencing depth distribution diagram of 29 fragments is shown. Figure 6 The average sequencing depth of 29 fragments was counted (the lowest average sequencing depth cov7 was 1306.25, and the highest average sequencing depth cov29 was 41125.7. The sequencing data met the requirements for SARS-CoV-2 whole genome sequencing and typing analysis). The SARS-CoV-2 genome coverage reached more than 93%, providing a sequencing solution for sequencing low viral load samples. At the same time, nanopore whole genome sequencing technology reduces the sequencing and analysis time of SARS-CoV-2 samples, and quickly identifies pathogenic mutations through real-time base recognition and bioinformatics analysis, improving the efficiency and sensitivity of SARS-CoV-2 sequencing.

[0044] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

[0045] The primer sequences involved in this application are shown in Table 8.

[0046] Table 8

[0047] .

Claims

1. A method for detecting the whole genome of SARS-CoV-2 virus based on nanopore sequencing, comprising the following steps: a. Design and synthesize reverse transcription primer P7R2-Random primers and multiple PCR primers, wherein 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. using reverse transcription primers to reverse transcribe the extracted RNA virus to obtain a cDNA product; c. Mixing the cDNA product 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. Use a nanopore sequencer to sequence the adapter-ligated DNA library to obtain the complete genome sequence of SARS-CoV-2.

2. The method according to claim 1, wherein 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.

3. The method according to claim 1, wherein the reaction conditions of the multiplex PCR amplification 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.

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

5. 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 the sequencing buffer and then flowing into the sequencing system; c) Start sequencing.

6. 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.

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

8. The kit according to claim 7 is used to detect the whole genome of SARS-CoV-2 in low viral load samples by nanopore sequencing technology.

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