A PCR primer set for detecting African swine fever virus based on whole-genome and a nanopore sequencing method based on single-molecule sequencing

Abstract

The present invention discloses a PCR primer set for detecting African swine fever virus based on whole-genome and a nanopore sequencing method based on single-molecule sequencing. The ultra-multiplex PCR primer set for detecting African swine fever virus provided by the present invention can achieve 106-plex PCR detection, and is used for rapidly detecting and analyzing the genotypes of African swine fever virus. The ultra-multiplex PCR detection of the present invention has strong specificity and high sensitivity. By simultaneously detecting 106 segments, rapid detection is achieved, which is of great significance for the rapid screening and early prevention and control of African swine fever virus.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a PCR primer set for detecting African swine fever virus based on whole genome and a nanopore sequencing method based on single molecule sequencing. Background Art

[0002] The prevalence of African swine fever virus will cause a huge impact on pig farming. In the natural environment, African swine fever virus is still constantly mutating, resulting in various genotypes and deletion strains.

[0003] However, the lethality of African swine fever virus after gene mutation is relatively low, while wild strains with high lethality almost all cause acute or subacute symptoms and are difficult to be detected in advance. Therefore, the prevention and control of ASF is difficult.

[0004] Therefore, the rapid detection of the ASF genome is of great significance in the detection of African swine fever virus types and the prevention and control of African swine fever. Summary of the Invention

[0005] The purpose of this part is to outline some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments.

[0006] As one aspect of the present invention, the present invention provides a...

[0007] A PCR primer set for detecting African swine fever virus based on whole genome, the PCR primer set consists of 106 pairs of primers, and the sequences of the PCR primer set are shown as SEQ ID NO: 1-212. The PCR primer set is used for super-multiplex PCR detection.

[0008] The present invention also provides the nanopore sequencing method based on single molecule sequencing as described above, which includes the following steps:

[0009] (1) Collect samples and extract sample DNA;

[0010] (2) Perform super-multiplex PCR amplification using the 106 pairs of PCR primer sets;

[0011] (3) Construct a nanopore sequencing library;

[0012] (4) Perform bioinformatics analysis by comparing with the African swine fever virus genome sequence.

[0013] Wherein, the samples include various types such as pig nasal swabs, throat swabs, blood samples, tissue samples, oral fluids, saliva, etc.

[0014] Among them, the reaction program of the multiplex PCR amplification is: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 10 s, extension at 65°C for 3 min, and the number of reaction cycles is 40.

[0015] Among them, the construction of the nanopore sequencing library is carried out according to the connection kit to construct the nanopore sequencing library, including PCR product purification, tag ligation, and adapter ligation.

[0016] Among them, the bioinformatics analysis includes sample data merging, distinguishing host (pig) sequences and non-host (virus) sequences, whole-genome sequence identification, whole-genome strain typing, and virulence identification.

[0017] Advantages of the present invention: The multiplex PCR primer set for detecting African swine fever virus based on whole-genome provided by the present invention can achieve 106-plex PCR detection, and is used for rapid detection and analysis of the genotype of African swine fever virus. The multiplex PCR detection of the present invention has strong specificity and high sensitivity, and can detect 106 fragments simultaneously, realizing rapid detection, which is of great significance for the rapid screening and early prevention and control of African swine fever virus. Brief Description of the Drawings

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for description in the embodiments will be briefly introduced below, where:

[0019] Figure 1 It is a flow chart of the nanopore sequencing method. Detailed Embodiments

[0020] To make the above objects, features, and advantages of the present invention more obvious and understandable, the following detailed description of the specific embodiments of the present invention will be given with reference to specific embodiments.

[0021] Example 1:

[0022] Design and synthesis of 106 pairs of primers for targeted amplification of African swine fever virus (genomic sequence reference NCBI accession number MK333180):

[0023] Table 1

[0024]

[0025]

[0026]

[0027]

[0028]

[0029]

[0030]

[0031]

[0032] PCR conditions:

[0033] 20 μL reaction system, with the enzyme being biotechrabbit multiplex PCR enzyme:

[0034] ① 10 μL of 4×CAPITAL qPCR Probe Master Mix

[0035] ② 1 μL of 20×RTase with RNase lnhibitor

[0036] ③ 5 μL of primer set (100 μmol / L)

[0037] ④ 1 μL of deionized water

[0038] ⑤ 5 μL of nucleic acid of the sample to be tested

[0039] The reaction procedure is as follows:

[0040] ① Pre-denaturation at 95°C for 3 min

[0041] ② Denaturation at 95°C for 10 s

[0042] ③ Extension at 65°C for 3 min, with 40 reaction cycles

[0043] The brand of the PCR instrument tested is Tianlong 96R.

[0044] Dilution of primers and positive control plasmid:

[0045] 106 pairs of primers are diluted to 100 μmol / L with TE Bμffer as recommended; the dilution method of the positive control plasmid is as follows:

[0046] (1) Take 10 μl of African swine fever virus plasmid solution into a 1.5 ml centrifuge tube, add 990 μl of TE Bμffer, and the concentration of African swine fever virus plasmid is 1.134X 10 9 copies / μl, labeled as "ASF positive plasmid 10 9 "; The copy number calculation formula: copy number = (6.02×10 14 )a / 660b (a: stock solution concentration, b: plasmid length).

[0047] (2) Dilute the ASF positive plasmid according to the following steps (10 9 ~10n copies / μl):

[0048] Take 100 μl of 10 9 copies / μl of the African swine fever positive plasmid into a 1.5 ml centrifuge tube, add 900 μl of TE Buffer, with a concentration of 10 8 copies / μl, labeled as "African swine fever positive plasmid 10 8 ". Vortex for 5 min and centrifuge for 30 s.

[0049] Take 100 μl of 10 8 copies / μl of the African swine fever positive plasmid into a 1.5 ml centrifuge tube, add 900 μl of TE Buffer, with a concentration of 10 7 copies / μl, labeled as "African swine fever positive plasmid 10 7 ". Vortex for 5 min and centrifuge for 30 s.

[0050] Repeat the dilution method to obtain "African swine fever positive plasmid 10 9 " to "African swine fever positive plasmid 10 n ".

[0051] Verification of the amplification effect and specificity of PCR primers:

[0052] First, verify the single primers. Design 106 segments of plasmids as DNA templates, and all 106 segments correspond to single pairs of primers. In the first step, mix 106 pairs of primers and perform amplification without adding a DNA template. After amplification, perform DNA agarose gel electrophoresis on 106 different amplification products, and no bands are found, indicating that no dimers are formed between the 106 pairs of primers.

[0053] In the second step, add single pairs of primers and the corresponding positive plasmids (plasmid concentration selected as 10000 copies / mL) to each system, for a total of 106 reaction systems. After single - plex ordinary PCR amplification, perform DNA agarose gel electrophoresis on 106 different amplification products, observe the electrophoresis pattern, and evaluate the amplification effect of all single pairs of primers. After verification, all 106 pairs of primers can show obvious bands during gel running.

[0054] In the third step, add single pairs of primers and a mixture of 105 pairs of plasmids that have no amplification relationship with the primer (final plasmid concentration is 100000 copies / mL) to each system, for a total of 106 reaction systems, to verify the specificity of each pair of primers. After verification, no bands appear for all 106 pairs of primers during gel running, indicating good specificity of primer design.

[0055] Ultra - multiplex PCR sensitivity experiment:

[0056] Sensitivity tests were performed on the primer sets using 106 segments of the above-mentioned African swine fever positive plasmids diluted to different gradients. The sensitivity results are shown in Table 2:

[0057] Table 2

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068] In the table, ASFV-1-L represents the first upstream primer among the 106 pairs of primers, and ASFV-1-R represents the first downstream primer among the 106 pairs of primers.

[0069] Ultra-multiplex PCR stability experiment:

[0070] Select 10 5 copies / mL, 10 6 copies / mL, 10 7 copies / mL, 10 8 copies / mL dilution of 4 different positive plasmid standards were used for repeated experiments (taking 5 μL of three dilutions of the standard as templates), with 10 replicates for each. After amplification, nanopore sequencing was performed (the specific sequencing process is described below), and the number of amplified products was counted (each sample ran 100 M B of data). The coefficient of variation was calculated using the coefficient of variation calculation formula. The results showed that the coefficients of variation for the three gradients were all less than 2%, indicating that the method has good repeatability. The specific data are shown in the following table:

[0071] Table 3

[0072]

[0073]

[0074] Clinical manifestation verification:

[0075] Send the primers and a mixed sample of 100 sera, oral fluids, and tissues with determined specific information to the third-party testing company, Sangon Biotech. Among them, 70 samples are African swine fever positive samples of various types, and 30 are samples of other common swine diseases, such as porcine reproductive and respiratory syndrome, foot-and-mouth disease, pseudorabies, etc. Sequence the clinical samples using the second-generation Illumina platform and the third-generation nanopore sequencing method, and finally compare the detected sequences with the African swine fever virus database to verify the performance of this method clinically.

[0076] The detection results of the second-generation high-throughput sequencing are as follows: The African swine fever typing determined after comparing the 70 African swine fever samples with the database is consistent with the information of the positive samples, while none of the 30 other pathogen samples are matched with the African swine fever database, and the results show negative. The results of the third-generation sequencing are consistent with those of the second-generation.

[0077] After the primer set of the present invention is used by a third party, the clinical test results are consistent with the true information of the samples, indicating that the ultra-multiplex PCR of this patent can be used for the detection of clinical samples and has reliable sensitivity and accuracy.

[0078] Specificity verification of 106-plex ultra-multiplex PCR:

[0079] Prepare 20 samples: 15 African swine fever positive plasmids with random fragments and 5 negative ones. Experimenter 1 numbers them from 1 to 20, with samples 1 - 15 being positive samples and samples 16 - 20 being negative samples. Rearrange the numbers from 1 to 20 and re-number them as A - T and hand them over to Experimenter 2. After nanopore sequencing, conduct a comparison. Check whether there are false positives and false negatives, and detect the accuracy of the experiment. The detection results show no false positives and false negatives, and after base comparison using the nanopore sequencing method, the results show that each amplified product corresponds to the primer amplification fragment. This indicates that the 106-plex ultra-multiplex PCR has high specificity and accuracy.

[0080] Nanopore sequencing library construction:

[0081] Library construction process: After the PCR targeted enrichment of the African swine fever whole genome is completed, construct the sequencing library according to the SQK-NBD114.24 ligation kit (Oxford Nanopore Technologies). The operation is as follows:

[0082] Purification of PCR products:

[0083] Prepare reagents and consumables:

[0084] ① Magnetic beads, ② Washing solution (note to add 24 mL of absolute ethanol before use), ③ Elution solution, ④ 1.5 mL centrifuge tube, ⑤ Qubit TMQuantitative tube (Invitrogen, catalog number: Q32856), ⑥ Qμbit dsDNA HS Assay Kit (ThermoFisher, catalog number: Q32851).

[0085] Take out the barcode (NB01 - 24) according to the experimental requirements, mix well, centrifuge, and each sample or quality control requires a separate label number; prepare the following reactions in the tube and incubate at room temperature for 20 min;

[0086] Reagent Name Dosage Final Modified Product 7.5ul Barcode NB01-24 2.5ul Label Enzyme Premix 10ul Total Volume 20 μL

[0087] After incubation, add 2 μL of EDTA (ONT ligation sequencing kit) to each tube, pipette to mix well, and centrifuge briefly;

[0088] Transfer all ligation products to a 1.5 mL centrifuge tube and mix well. Add 0.4 times the total volume of magnetic beads (e.g., if the mixed product is 100 μL, add 40 μL of magnetic beads) for purification; pipette thoroughly to mix well and incubate at room temperature for 10 min;

[0089] Remove the centrifuge tube, centrifuge briefly, place it on the magnetic stand until clear, and aspirate and discard the supernatant;

[0090] While maintaining the magnetic state, add 700 μL of washing solution for washing. Note that the magnetic beads should not be disturbed during washing, and aspirate and discard the supernatant. Repeat twice;

[0091] Centrifuge briefly, place the centrifuge tube back on the magnetic stand, remove the residual washing solution with a pipette, and air dry for 30 seconds;

[0092] Remove the centrifuge tube, add 35 μL of elution buffer, flick the bottom of the tube to resuspend, centrifuge briefly, and incubate at 37 °C for 10 min;

[0093] Centrifuge briefly, place it on the magnetic stand to magnetize until clear, and transfer 35 μL of the supernatant to a clean 1.5 mL centrifuge tube. This is the labeled ligation product.

[0094] Adapter ligation:

[0095] Prepare reagents and consumables: ① Magnetic beads, ② Ligation enzyme premix, ③ Ligation, ④ Ligation sequencing kit (ONT, catalog number: SQK - NBD114.24), ⑤ Centrifuge tubes (0.2 mL, 1.5 mL), ⑥ Qμbit TM Quantitative tube (Invitrogen, catalog number: Q32856), ⑦ Qμbit dsDNA HS Assay Kit (ThermoFisher, catalog number: Q32851).

[0096] Thaw the required reagents, flick to mix well, centrifuge briefly, and place on ice;

[0097] In a 1.5 mL centrifuge tube, mix the following reagents in the order shown in the table below. After adding each reagent, mix well, centrifuge briefly, and incubate at room temperature for 20 minutes;

[0098] Reagent Name Dosage Label Ligation Product 30 uL Sequencing Adapter (NA) 5 μL Ligase Premix 10 uL Ligase 5 μL Total Volume 50 uL

[0099] Add 20 μL of the well-mixed magnetic beads, flick to mix well, and incubate at room temperature for 10 min;

[0100] Remove the centrifuge tube, centrifuge briefly, place it on a magnetic stand, and magnetize until the supernatant is clear. Aspirate and discard the supernatant;

[0101] Remove the centrifuge tube from the magnetic stand, add 125 μL of SFB, flick to resuspend, and centrifuge briefly. Place it on the magnetic stand and magnetize until the supernatant is clear. Aspirate and discard the supernatant. Repeat this twice;

[0102] Centrifuge briefly, place the centrifuge tube back on the magnetic stand, use a pipette to aspirate the residual liquid, remove the centrifuge tube, add 15 μL of EB, flick the bottom of the tube to resuspend, centrifuge briefly, and incubate at 37 °C for 10 min;

[0103] Centrifuge briefly, place it on the magnetic stand and magnetize until the supernatant is clear. Transfer 15 μL of the supernatant to a clean 1.5 mL centrifuge tube. This is the prepared nucleic acid library;

[0104] Take 1 μL of the prepared library for Qμbit quantification. The ideal loading amount for the library on the machine is 3.3 ng - 6.7 ng. If the library concentration is too high, it can be diluted with EB;

[0105] Chip loading:

[0106] Prepare reagents and consumables:

[0107] ① RNase-free water, ② Auxiliary buffer, ③ Flow Cell Flush (FCF) (ONT, product number: SQK-NBD114.24), ④ Flow Cell Tether (FCT) (ONT, product number: SQK-NBD114.24), ⑤ Library Solution (LIS) (ONT, product number: SQK-NBD114.24), ⑥ Library Beads (LIB) (ONT, product number: SQK-NBD114.24), ⑦ Sequencing Buffer (SB) (ONT, product number: SQK-NBD114.24).

[0108] Sequencing chip SpotON (ONT, product number: FLO-MIN106D)

[0109] Thaw the required reagents at room temperature in advance, mix well and centrifuge, and equilibrate the chip at room temperature for about 10 min and perform quality inspection.

[0110] Prepare the Priming reagent FB in a 1.5 mL centrifuge tube according to the following table:

[0111] Reagent Name Dosage Flow Cell Flush (FCF) 1170 μL Flow Cell Tether (FCT) 30 μL Auxiliary Buffer 5 μL Total Volume 1205 μL

[0112] Prepare the sequencing chip and remove the air bubbles inside the chip;

[0113] Aspirate 800 μL of FB and slowly push it into the pipeline from the Priming port. Note that a small amount of liquid should be left at the tip of the pipette. Equilibrate at room temperature for 5 min;

[0114] Prepare the library for loading according to the following table. If the prepared nucleic acid library is less than 12 μL, add EB to make it up to 12 μL;

[0115] Reagent Name Dosage Sequencing Buffer (SB) 37.5 μl Library Beads (LIB) 25.5 μl Nucleic Acid Library 12ul Total Volume 75ul

[0116] After the chip equilibration is completed, gently push outwards to open the SpotON;

[0117] Aspirate 200 μL of FB and slowly push it into the pipeline from the Priming port.

[0118] Vortex the prepared library for loading several times, and take 75 μL and add it drop by drop to the SpotON sample port.

[0119] First, close the SpotON sample cover, then rotate counterclockwise to close the Priming port, and finally aspirate the liquid in the pipeline from Wasteport 1 and place it on the sequencer to set up the experiment.

[0120] Sequencing on the machine: For the steps of sequencing on the machine and parameter settings, please refer to the official instruction manual of the ONT sequencer for operation.

[0121] Chip cleaning: For the chip cleaning steps, please refer to the official instruction manual of the ONT sequencing chip (product number EXP-WSH004) for operation.

[0122] Data analysis is carried out according to the conventional bioinformatics process (the database contains the whole genome of African swine fever). Use the shell language to build a bioinformatics data analysis process for parallel processing of multiple samples; this process can interact with the sequencing platform to obtain sequencing data and perform the following analysis:

[0123] 1. Merge the sample data, and merge multiple sequencing fastq files with the same barcode into one;

[0124] 2. Distinguish between host sequences and non-host sequences

[0125] a. Align the fastq to the porcine reference genome. The reads that are aligned are the porcine genomic sequences, and the reads that are not aligned are the viral sequences.

[0126] b. Count the number of reads and the total length of reads in the fastq file

[0127] 3. Whole-genome sequence identification and whole-genome strain typing

[0128] a. Align the fastq sequences after removing the host to the genome to obtain a sam file

[0129] b. Generate a consensus sequence for the primary bam

[0130] c. Call variants on the consensus sequence file

[0131] d. Replace the bases at the corresponding positions in the genome with the mutated bases to generate the assembled genome sequence

[0132] e. Perform depth statistics and calculate the coverage: cov = number of positions with depth > 30 / total length of the genome

[0133] 4. Virulence identification

[0134] a. Use blastn to align the virulence gene sequences to the assembled genome. If a virulence gene sequence aligns to multiple positions in the genome, they are merged

[0135] b. According to the alignment positions in the blastn alignment results, intercept the corresponding sequences on the assembled genome and perform protein translation on the sequences to obtain 6 protein sequences

[0136] c. Align the translated proteins to the corresponding virulence gene protein sequences and select the best-aligned protein sequences

[0137] d. When the similarity of both the base alignment and the protein alignment is > 85%, it is determined that the virulence gene exists (not missing), otherwise it is temporarily determined that the virulence gene is missing. Similarity calculation: similarity in the blastn results * length of alignment / total length of the gene (protein) * 100%

[0138] e. Perform primer product verification on the virulence genes determined to be missing in the previous step: Align the off-machine data to the primer product sequences of the virulence genes. For each virulence gene primer product sequence, if there are reads aligned, it is determined that the virulence gene is indeed missing, otherwise it is determined that the virulence gene exists

[0139] f. According to the virulence level determination rules of different strains, judge the virulence level of the strain.

[0140] The flowchart is asFigure 1 as shown

[0141] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

Claims

1. A PCR primer set for detecting African swine fever virus based on the whole genome, characterized in that: The PCR primer set consists of 106 pairs of primers, and the sequences of the PCR primer set are shown in SEQ ID NOs: 1-212.

Images