Primer set, library construction method and application for detecting african swine fever virus based on whole genome

Abstract

The application provides a primer set, a library construction method and application based on whole genome sequencing of African swine fever virus, the primer set comprises 182 pairs of primers, and the sequences of the upstream primers and the downstream primers of the 182 pairs of primers are shown in SEQ ID NO. 1-SEQ ID NO. 364. The primer set has good specificity and sensitivity, and has a good application prospect in the whole genome detection of African swine fever virus and the preparation of the required reagent or kit for detection. The library construction method comprises the following steps: using the primer set to perform multiplex PCR amplification; the library construction method is suitable for a second-generation sequencing platform and a nanopore sequencing platform, and has a high degree of automation; even in a sample with a low virus content of 10 copies / μL, more than 95% of the African swine fever virus sequences can be obtained, and the sequencing depth can reach more than 1000X.

Description

TECHNICAL FIELD

[0001] The application relates to the field of high-throughput technology of viruses, in particular to a primer set for detecting African swine fever virus based on whole genome, a library construction method and application. BACKGROUND

[0002] African swine fever virus (ASFV) is a large, icosahedral, linear, double-stranded DNA virus and the only member of the Asfarviridae family. African Swine Fever Virus, abbreviated ASFV The genome of ASFV is 170-193 kb in length and contains 150-167 open reading frames. The central part of the genome is a conserved region of about 125 kb in length, and the two ends are variable regions containing five multigene families. The MGF (mast cell growth factor) gene region often has multiple copies of genes deleted or inserted, with a length of up to 20 kb, which is related to viral antigen variation and is beneficial to escape from the host immune system. The division of ASFV genotypes and subtypes is based on the slight variation in the central variable region (CVR) of the central conserved region of the genome. So far, according to the partial nucleotide sequence of the p72 gene, ASFV can be divided into 24 genotypes. The division of subtypes is based on the tandem repeat sequence of the CVR region of the B620L gene and the intergenic sequence between the 173R-1329L genes on the right side of the genome. Rapid differential diagnosis of samples infected with ASFV and obtaining the whole genome information of the virus can provide a basis for early detection, early control and tracing of the virus, and minimize the economic losses to the pig industry.

[0003] Currently, the nucleic acid detection methods in virus detection mainly include fluorescent quantitative PCR (qPCR), Sanger sequencing (first-generation sequencing) and high-throughput sequencing (second-generation sequencing and nanopore sequencing). qPCR can monitor the change of fluorescent signal in the PCR amplification process in real time, realize the quantitative analysis of target nucleic acid, and can quickly identify ASFV infection, and is suitable for accurate determination of virus content. However, qPCR usually only detects specific African swine fever virus (ASFV) gene fragments (such as MGF gene, B620L gene, etc.), and cannot provide the whole genome information of African swine fever virus. The first-generation sequencing adopts the method of sequencing by synthesis, and determines the DNA sequence by capturing the special markers (usually fluorescent molecular markers) carried by the newly added bases in the DNA replication process. It is suitable for small fragment sequence analysis due to its high accuracy and long read length advantage, and its combination with fluorescent quantitative PCR can realize the qualitative and typing of ASFV. With the continuous deepening of genomics research, the demand for sequencing throughput and speed is increasing, and the fluorescent quantitative PCR and first-generation sequencing technology gradually cannot meet the demand of large-scale genome sequencing, so the high-throughput sequencing technology emerges as the times require. The high-throughput sequencing rapidly becomes the mainstream technology in genomics research due to its characteristics of high throughput, low cost and fast speed. Therefore, it is of great significance to provide a primer set suitable for whole genome sequencing of African swine fever virus by high-throughput sequencing technology for rapid diagnosis and typing of African swine fever virus and whole genome research. SUMMARY

[0004] In view of the deficiencies of the prior art, the purpose of the present application is to provide a primer set for detecting African swine fever virus (ASFV) based on whole genome, a library construction method and application. The primer set of the present application has good specificity and sensitivity, and has good application prospect in African swine fever virus whole genome detection and preparation of detection reagents or kits. The library construction method of the present application is suitable for second-generation sequencing platform and nanopore sequencing platform, has high automation degree, and can obtain more than 95% of African swine fever virus sequences even in samples with low virus content of 10 copies / μL, and the sequencing depth can reach more than 1000X; and when sequencing on the second-generation sequencing platform, the sequence number of each sample is only 5MB; and when sequencing on the nanopore sequencing platform, the data amount of each sample is only 500MB.

[0005] The purpose of the present application is realized by the following technical solutions:

[0006] In a first aspect, the present application provides a primer set for sequencing African swine fever virus based on whole genome, which comprises 182 pairs of primers, and the sequences of the upstream primers and downstream primers of the 182 pairs of primers are shown in SEQ ID NO. 1-SEQ ID NO. 364.

[0007] In some embodiments of the present application, "the sequences of the upstream primers and the downstream primers of the 182 pairs of primers are as shown in SEQ ID NO. 1-SEQ ID NO. 364" means that the sequences of the upstream primers and the downstream primers of the 182 pairs of primers are as shown in SEQ ID NO. 1-SEQ ID NO. 364 in turn. Among them, the sequences of the upstream primers and the downstream primers in each pair of primers can be exchanged, for example, the sequences of the upstream primer and the downstream primer of the first pair of primers are SEQ ID NO. 1 and SEQ ID NO. 2, when SEQ ID NO. 1 represents the sequence of the upstream primer of the first pair of primers, SEQ ID NO. 2 represents the sequence of the downstream primer of the first pair of primers; when SEQ ID NO. 1 represents the sequence of the downstream primer of the first pair of primers, SEQ ID NO. 2 represents the sequence of the upstream primer of the first pair of primers. The sequence numbers of the primer pairs are only for distinction and grouping, and there is no difference in sequence. Each pair of primers is independent.

[0008] In some embodiments of the present application, the 182 pairs of primers with sequences as shown in SEQ ID NO. 1-SEQ ID NO. 364 are divided into primer group 1 and primer group 2 at intervals, as shown in Table 4.

[0009] In the second aspect, the present application provides a reagent for whole genome amplification of African swine fever virus, which comprises the primer group of the first aspect.

[0010] In some embodiments of the present application, the reagent comprises two groups of reagents, wherein one group of reagents comprises the primer group 1 of the first aspect, and the other group of reagents comprises the primer group 2 of the first aspect.

[0011] In the third aspect, the present application provides a kit for whole genome amplification of African swine fever virus, which comprises the primer group of the first aspect or the reagent of the second aspect.

[0012] In the fourth aspect, the present application provides the use of the primer group of the first aspect or the reagent of the second aspect or the kit of the third aspect in whole genome amplification of African swine fever virus.

[0013] In the fifth aspect, the present application provides a whole genome library construction method of African swine fever virus, which comprises multiplex PCR amplification using the primer group of the first aspect or the reagent of the second aspect or the kit of the third aspect.

[0014] In some embodiments of the present application, the method comprises the following steps:

[0015] (1) Collecting samples and extracting nucleic acids from the samples;

[0016] (2) using the primer set of the first aspect or the reagent of the second aspect or the kit of the third aspect to perform multiplex PCR amplification on the nucleic acid extracted in step (1);

[0017] (3) constructing a sequencing library;

[0018] (4) sequencing;

[0019] (5) bioinformatics analysis.

[0020] In some embodiments of the present application, the sample of step (1) comprises one or more of a pig nasal swab, a throat swab, a blood sample, a tissue sample, etc.

[0021] In some embodiments of the present application, the method of nucleic acid extraction of step (1) comprises a magnetic bead method.

[0022] In some embodiments of the present application, the length of the amplification product obtained in step (2) is between 1000-1400 bp, preferably 1200 bp.

[0023] In some embodiments of the present application, the amplification program of step (2) is: 98℃ pre-denaturation for 30s; 98℃ denaturation for 10s, 60℃ denaturation for 30s, 72℃ denaturation for 90s, for a total of 30 cycles; 72℃ extension for 5min.

[0024] In some embodiments of the present application, the reaction reagent for amplification of step (2) comprises VAHTS Pathogen DNA Multiplex PCR Mix, gDNA, ddH2O, and the primer set of the first aspect.

[0025] In some embodiments of the present application, the amplification of step (2) is performed in two groups, i.e., using primer set 1 of the first aspect and primer set 2 of the first aspect to perform multiplex PCR amplification on the nucleic acid extracted in step (1), and then combining the amplification products.

[0026] In some embodiments of the present application, one reaction reagent for amplification of step (2) comprises VAHTS Pathogen DNA Multiplex PCR Mix 12.5μL, cDNA 2.5μL, ddH2O 5μL, and primer set 1 of the first aspect 5μL per 25μL reaction reagent; another reaction reagent comprises VAHTS Pathogen DNA Multiplex PCR Mix 12.5μL, cDNA 2.5μL, ddH2O 5μL, and primer set 2 of the first aspect 5μL per 25μL reaction reagent.

[0027] In some embodiments of the present application, the step (2) further comprises purifying the amplified product obtained by amplification.

[0028] In some embodiments of the present application, the bioinformatics analysis in the step (5) comprises sequence alignment, mutation detection and evolutionary analysis.

[0029] In some embodiments of the present application, the step (3) of constructing a sequencing library is performed on a second-generation sequencing platform; preferably, the step of constructing a sequencing library specifically comprises the following steps: fragment breaking, end repair & dA addition, adapter ligation, library amplification, single-stranded circularization, enzyme digestion, DNB preparation; more preferably, the step of constructing a sequencing library further comprises purifying the product obtained in the step of adapter ligation and / or purifying the product obtained in the step of enzyme digestion.

[0030] In some embodiments of the present application, the purification described above comprises using purified magnetic beads for purification; preferably, the purification comprises:

[0031] 1) mixing the product to be purified with magnetic beads, and discarding the supernatant after the magnetic beads are completely adsorbed;

[0032] 2) adding freshly prepared 80% ethanol to rinse the magnetic beads, and carefully removing the supernatant;

[0033] 3) drying the magnetic beads at room temperature for 5-10 min, adding ddH2O to resuspend the dried magnetic beads, and recovering the supernatant after the magnetic beads are completely adsorbed, to obtain the purified product.

[0034] In some embodiments of the present application, the volume ratio of the product to be purified to the magnetic beads in step 1) is (0.5-3): 1.

[0035] In some embodiments of the present application, step 2) is repeated 1-3 times.

[0036] In some embodiments of the present application, the fragment size obtained after the fragment breaking step is in the range of 150-350 bp.

[0037] In some embodiments of the present application, the step (3) of constructing a sequencing library is performed on a nanopore sequencing platform; preferably, the step of constructing a sequencing library specifically comprises the following steps: end repair & dA addition, barcode ligation, mixing, adapter ligation; more preferably, the step of constructing a sequencing library further comprises purifying the product obtained in the step of end repair & dA addition and / or purifying the product obtained in the step of barcode ligation and / or purifying the product obtained in the step of adapter ligation.

[0038] In some embodiments of the present application, the mixing of samples refers to mixing different samples (especially the samples with barcodes attached obtained from different samples after the foregoing steps); preferably, the mixing of different samples is mixing by equal mass or mixing by a specific ratio, and the total amount of DNA in the sample after mixing is between 1-2 μg. Mixing the samples with barcodes attached before detecting the samples can reduce the number of sample detections in the subsequent steps of the library construction method, thereby shortening the library construction time.

[0039] In some embodiments of the present application, the purification method in the purification of the product obtained in the end repair & dA addition step and / or the purification of the product obtained in the barcode ligation step comprises using purified magnetic beads for purification; preferably, the purification method comprises the following steps:

[0040] 1) mixing the product to be purified with magnetic beads, and discarding the supernatant after the magnetic beads are completely adsorbed;

[0041] 2) adding freshly prepared 80% ethanol to rinse the magnetic beads, and carefully removing the supernatant;

[0042] 3) drying the magnetic beads at room temperature for 5-10 min, adding ddH2O to resuspend the dried magnetic beads, and recovering the supernatant after the magnetic beads are completely adsorbed, thereby obtaining the purified product.

[0043] In some embodiments of the present application, the volume ratio of the product to be purified to the magnetic beads in step 1) is (0.5-3): 1.

[0044] In some embodiments of the present application, step 2) is repeated 1-3 times.

[0045] In some embodiments of the present application, the purification method in the purification of the product obtained in the linker ligation step comprises using purified magnetic beads for purification; preferably, the purification method comprises the following steps:

[0046] Ⅰ) mixing the product to be purified with magnetic beads, and discarding the supernatant after the magnetic beads are completely adsorbed;

[0047] Ⅱ) adding Wash Buffer to rinse the magnetic beads and discarding the supernatant;

[0048] Ⅲ) adding elution buffer to resuspend the magnetic beads, removing the magnetic stand, incubating at room temperature for 5-10 min, then transferring the reaction tube to the magnetic stand, and recovering the supernatant after the magnetic beads are completely adsorbed, thereby obtaining the purified product.

[0049] In some embodiments of the present application, the volume ratio of the product to be purified to the magnetic beads in step I) is (0.5-3): 1.

[0050] In some embodiments of the present application, step II) is repeated 1-3 times.

[0051] In some embodiments of the present application, the Wash Buffer of step II) is a Wash Buffer matched with the purified magnetic beads.

[0052] In some embodiments of the present application, the elution buffer of step III) is an elution buffer matched with the purified magnetic beads.

[0053] In some embodiments of the present application, when the purified magnetic beads are used for purification, the container containing the product to be purified and the magnetic beads is placed on a magnetic stand during the purification process so that the magnetic beads can be adsorbed by magnetic force during the purification process.

[0054] In some embodiments of the present application, the bioinformatics analysis in step (5) includes sequence alignment, mutation detection and evolutionary analysis.

[0055] The beneficial effects of the present application are as follows:

[0056] The present application provides a primer set for detecting African swine fever virus based on whole genome, which is designed by the imbricated primer design method, and has an overlapping region of 100-200 bp between adjacent primer pairs, which can ensure that the primer set can cover the entire genome. In addition, adjacent primer pairs are placed in different primer sets, and then two different primer sets are used for amplification and the amplification products are combined, which can effectively avoid non-specific binding of primers and improve amplification efficiency. The primer set of the present application has good specificity and sensitivity, and has good application prospect in African swine fever virus whole genome amplification and preparation of reagents or kits required for amplification.

[0057] The library construction method of the present application is suitable for second-generation sequencing platform and nanopore sequencing platform, and has high automation. Even in a sample with a virus content as low as 10 copies / μL, more than 95% of African swine fever virus sequences can be obtained, and the sequencing depth can reach more than 1000X. When sequencing on the second-generation sequencing platform, the sequence number of each sample only needs 5MB; when sequencing on the nanopore sequencing platform, the data amount of each sample only needs 500MB. Further, the present application sets the amplicon size of each primer pair to 1000-1400 bp, which can meet the requirements of the second-generation sequencing platform and the nanopore sequencing platform for fragment size, thereby improving the adaptability of the library construction method to the sequencing platform. BRIEF DESCRIPTION OF DRAWINGS

[0058] Figure 1 The present application shows the design schematic of ASFV whole genome targeting panel.

[0059] Figure 2The technical route of the primer set of the application in the whole genome sequencing of African swine fever virus on the second-generation sequencing platform and the nanopore sequencing platform is shown.

[0060] Figure 3 The whole genome coverage obtained from Example 1 of the application is shown. DETAILED DESCRIPTION

[0061] The technology of the application is further illustrated by the following examples. These examples are an illustration and an example of the application, and do not limit the scope of the application in any form.

[0062] Some experimental reagents used in the examples of the application are shown in Table 1:

[0063] Table 1 Experimental reagents

[0064]

[0065] Some experimental instruments and equipment used in the examples of the application are shown in Table 2:

[0066] Table 2 Experimental instruments and equipment

[0067]

[0068] The experimental samples used in the examples of the application are shown in Table 3:

[0069] Table 3 Experimental samples

[0070]

[0071] In Table 3, the sample source corresponding to ZKJY-A1 is the pure culture of HR / 20 strain, and the reference genome is MW656282.1; the sample source corresponding to ZKJY-A2 is the pure culture of SD / 21 strain, and the reference genome is MZ945537.1; the sample source corresponding to ZKJY-A3 is the pure culture of HLJ / 18 strain, and the reference genome is MK333180.1. The viruses corresponding to ZKJY-A1, ZKJY-A2 and ZKJY-A3 are all virus standard samples provided by the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences.

[0072] The primer set used in the examples of the application is shown in Table 4:

[0073] Table 4 ASFV whole genome primer set

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082] Note: In the primer name of Table 4, ASFV_1 represents the first pair of primers, ASFV_2 represents the second pair of primers, and so on, LEFT represents the upstream primer, RIGHT represents the downstream primer, ASFV_1_LEFT represents the upstream primer of the first pair of primers, ASFV_1_RIGHT represents the downstream primer of the first pair of primers, and so on.

[0083] The primer group in Table 4 refers to the primer group to which the primer belongs, and the purpose is to group all primers into primer group 1 and primer group 2; in addition, it can also clearly indicate that the 182 pairs of primers are spaced into primer group 1 and primer group 2, that is, adjacent primer pairs are placed in different primer groups.

[0084] The sequence number of the primer in Table 4 corresponds to the primer sequence number in SEQ ID NO. 1-SEQ ID NO. 364 and the sequence listing.

[0085] The design idea of the primer group in Table 4 above: Referring to relevant literature at home and abroad, all known ASFV whole genome sequences are summarized, repeated sequences are removed through multiple sequence alignment and sequence clustering, and representative sequences are selected as reference genomes for primer design. Then, the primer is designed by the shingled primer design method (adjacent primer pairs have an overlapping region of 100-200 bp) (the primer design schematic diagram is shown as Figure 1 ), and finally 182 pairs of primers covering the whole genome of ASFV are obtained. The technical route for using the primer group of the present application to perform whole genome sequencing of African swine fever virus (ASFV) on a second-generation sequencing platform and a nanopore sequencing platform is shown as Figure 2 , and the specific process is described in the following Example 1 and Example 2.

[0086] Example 1 (second-generation sequencing)

[0087] The experimental samples of the present embodiment are: three nucleic acid samples extracted from three African swine fever virus standards provided by the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, respectively: ZKJY-A1, ZKJY-A2, ZKJY-A3, the nucleic acid concentration in the samples is: 10000 copies / μL; ZKJY-A4, ZKJY-A5, ZKJY-A6, ZKJY-A7 are samples obtained by diluting ZKJY-A3, the nucleic acid concentrations in the samples are: 1000 copies / μL, 100 copies / μL, 10 copies / μL, 1 copies / μL, respectively; a total of 7 samples, each sample is set with 3 replicates, a total of 21 samples. The acquisition method of ZKJY-A1, ZKJY-A2, ZKJY-A3 is: take 100 μL of pure virus culture of the strain to extract nucleic acid by magnetic bead method using the nucleic acid automatic extractor KINGFISHER FLEX of Thermo Fisher Scientific and MagMAX CORE Nucleic Acid Purification Kit, and use 90 μL Elution Buffer to dissolve the nucleic acid, obtain 90 μL nucleic acid solution; then take 5 μL of nucleic acid solution to perform qPCR reaction by PCR instrument (Applied Biosystems real-time fluorescent quantitative PCR instrument QuantStudio 1Plus, same below), calculate the corresponding copy number according to the CT value obtained; and adjust the copy number to 10000 copies / μL. The library construction experiment process is as follows:

[0088] 1. ASFV whole genome amplification

[0089] The reagents for amplification are prepared according to the reagents and amounts in Table 5 using MiniAmp Plus Thermal Cycle PCR instrument, VAHTS Pathogen DNA Multiplex PCR Mix reagent kit and primer set in Table 4, shaken and mixed, and centrifuged briefly, and placed in the PCR instrument for ASFV whole genome multiplex PCR amplification according to the program in Table 6. Among them, the reagents in Table 5 are two different sets of amplification reagents, one set of amplification reagents contains VAHTS Pathogen DNA Multiplex PCR Mix 12.5 μL, cDNA 2.5 μL, ddH2O 5 μL, primer set 1 5 μL; the other set of amplification reagents contains VAHTS Pathogen DNA Multiplex PCR Mix 12.5 μL, cDNA 2.5 μL, ddH2O 5 μL, primer set 2 5 μL. Both sets of amplification reagents are subjected to ASFV whole genome multiplex PCR amplification according to the program in Table 6.

[0090] Table 5 Reagents and amounts for ASFV whole genome amplification

[0091]

[0092] Table 6 PCR reaction program for ASFV whole genome amplification

[0093]

[0094] 2. PCR reaction product purification

[0095] After the PCR reaction program is completed, 25 μL of the amplification products of primer group 1 and primer group 2 are combined into one tube, 50 μL of VAHTS DNA Clean Beads is added, incubated at room temperature for 5 min, then transferred to a magnetic stand and stand for 5 min, and after the magnetic beads are completely adsorbed, the supernatant is aspirated; then 200 μL of freshly prepared 80% ethanol is added and stand for 30 s, then the supernatant is aspirated, 200 μL of freshly prepared 80% ethanol is added to the reaction tube and stand for 30 s, then the supernatant is aspirated; then the magnetic beads are dried at room temperature for 8 min, 40 μL of ddH2O is added to resuspend the magnetic beads, the magnetic stand is removed and incubated at room temperature for 5 min, then transferred to a magnetic stand and stand for 5 min, after the magnetic beads are completely adsorbed, the supernatant is aspirated and transferred to a 0.2 mL centrifuge tube, then the supernatant is measured for nucleic acid concentration and purity using Nanodrop 2000, and temporarily stored in a -20°C refrigerator for standby.

[0096] 3. Library construction

[0097] The MGIEasy Fast enzyme digestion library preparation kit is used, and 150 ng of the amplification product purified in step 2 is used for library construction. According to the MGIEasy Fast enzyme digestion library preparation kit instructions, the reagents required for library construction are prepared in advance. Then the amplification product is broken and end-repaired & dA-added according to the required fragment size, followed by adapter ligation and product purification, library amplification steps to obtain double-stranded DNA library. Then use Equalbit 1 × dsDNA HS Assay Kit to quantify the double-stranded DNA library, adjust the concentration of the double-stranded DNA library to more than 10 ng / μL, then perform single-stranded circularization, enzyme digestion and product purification to obtain single-stranded DNA library. Again use Equalbit 1 × ssDNA HS Assay Kit to quantify the single-stranded DNA library to confirm the library construction results (library concentration > 1 ng / μL is qualified). The specific steps for constructing the sequencing library are as follows:

[0098] 3.1. Fragment breaking and end-repair & dA addition

[0099] The purified amplification product (dsDNA) obtained in step 2 was mixed with other components in the amounts shown in Table 7 (where the amount of dsDNA was 150 ng, and the volume was calculated based on the concentration determined in step 2), shaken to mix and centrifuged briefly, and placed in a PCR instrument for reaction according to the procedure in Table 8.

[0100] Table 7 Reagents and amounts for fragment breaking and end repair & dA addition

[0101]

[0102] Table 8 PCR reaction procedure for fragment breaking and end repair & dA addition

[0103]

[0104] 3.2. Adapter ligation

[0105] The reagents were prepared according to Table 9, and 25 μL of the prepared reagents were taken and added to the reaction tube after the reaction in step 3.1, shaken to mix and centrifuged briefly, and placed in a PCR instrument for reaction according to the procedure in Table 10.

[0106] Table 9 Reagents and amounts for adapter ligation

[0107]

[0108] Table 10 PCR reaction procedure for adapter ligation

[0109]

[0110] After the PCR reaction procedure was completed, 100 μL of VAHTS DNA Clean Beads was added to the reaction tube, incubated at room temperature for 5 min, and then the reaction tube was transferred to a magnetic stand and left to stand for 5 min, after which the supernatant was aspirated after the magnetic beads were completely adsorbed; then 200 μL of freshly prepared 80% ethanol was added to the reaction tube and left to stand for 30 s, after which the supernatant was aspirated, and then 200 μL of freshly prepared 80% ethanol was added to the reaction tube and left to stand for 30 s, after which the supernatant was aspirated; then the magnetic beads were dried at room temperature for 8 min, 50 μL of ddH2O was added to resuspend the dried magnetic beads, the magnetic stand was removed, and the reaction tube was incubated at room temperature for 5 min, and then the reaction tube was transferred to the magnetic stand and left to stand for 5 min, after which the supernatant was aspirated and transferred to a new tube, and finally 48 μL of the ligation product was obtained.

[0111] 3.3. Library amplification

[0112] The adapter ligation product obtained in step 3.2 was placed in a PCR instrument and subjected to reaction according to the procedure in Table 11.

[0113] Table 11 PCR reaction procedure for library amplification

[0114]

[0115] 3.4. Single-strand circularization

[0116] Prepare reagents according to Table 12, and pipette 12 μL of the prepared reagent into the reaction tube after step 3.3, shake and mix briefly, and centrifuge briefly. Place the reaction tube in a PCR instrument, and perform the reaction according to the program in Table 13.

[0117] Table 12 Reagents and amounts for single-strand circularization

[0118]

[0119] Table 13 PCR reaction program for single-strand circularization

[0120]

[0121] 3.5. Enzymatic digestion

[0122] Prepare reagents according to Table 14, and pipette 4 μL of the prepared reagent into the reaction tube after step 3.4, shake and mix briefly, and centrifuge briefly. Place the reaction tube in a PCR instrument, and perform the reaction according to the program in Table 15.

[0123] Table 14 Reagents and amounts for enzymatic digestion

[0124]

[0125] Table 15 PCR reaction program for enzymatic digestion

[0126]

[0127] After the PCR reaction program is completed, add 3 μL of Exo Stop Buffer to the reaction tube, and vortex 3 times for 3 s each time using a vortex shaker VORTEX-5, and then centrifuge briefly to collect the reaction product to the bottom of the tube. Then add 100 μL of VAHTS DNA Clean Beads to the reaction tube, and incubate at room temperature for 5 min. Then transfer the reaction tube to a magnetic stand and stand for 5 min, and then discard the supernatant after the magnetic beads are completely adsorbed. Then add 200 μL of 80% ethanol to the reaction tube, stand for 30 s, and then discard the supernatant. Then add 200 μL of 80% ethanol to the reaction tube, stand for 30 s, and then discard the supernatant. Then dry the magnetic beads at room temperature for 8 min with the cover open, add 20 μL of ddH2O to resuspend the dried magnetic beads, remove the magnetic stand, and incubate at room temperature for 5 min. Then transfer the reaction tube to the magnetic stand and stand for 5 min, and then pipette the supernatant to a new tube after the magnetic beads are completely adsorbed. Finally, 20 μL of single-strand library is obtained.

[0128] 4. Preparation of DNB and sequencing

[0129] DNB was prepared according to the required ssDNA library volume calculated by the MGISEQ-G99RS high-throughput sequencing reagent kit (PE150) requirements. The required ssDNA library amount for each DNB preparation system was 20 fmol. Then the DNB was quantified using Equalbit 1x ssDNA HS Assay Kit, and the DNB concentration was confirmed to be ≥8 ng / μL. The final total amount of DNB required for sequencing was 40 fmol. Sequencing was performed using the Huada DNBSEQ-G99RS gene sequencer, and the sequence number of each sample was 5MB. The sequencing time was 6-12h.

[0130] 4.1. Denaturation

[0131] The single-stranded library obtained in step 3.5 was mixed with other components according to the amount in Table 16, shaken and mixed, and centrifuged briefly. It was placed in a PCR instrument for reaction according to the program in Table 17.

[0132] Table 16 Reagents and amounts for denaturation

[0133]

[0134] Table 17 PCR reaction program for denaturation

[0135]

[0136] 4.2. DNB preparation

[0137] The reagents were prepared according to Table 18, and 22 μL of the prepared reagents were added to the reaction tube after step 4.1 reaction. Shake and mix, and centrifuge briefly. Place in a PCR instrument for reaction according to the program in Table 19. When the temperature of the PCR instrument reaches 4℃, immediately add 10 μL of DNB stop buffer, and mix slowly with a wide-bore pipette for 6 times. It can be placed at 4℃ before use.

[0138] Table 18 Reagents and amounts for DNB preparation

[0139]

[0140] Table 19 PCR reaction program for DNB preparation

[0141]

[0142] 5. DNB loading system preparation and sequencing

[0143] The DNB loading system was prepared according to Table 20, and the sequencing experiment was performed according to the instructions of the Huada DNBSEQ-G99RS gene sequencer.

[0144] Table 20 Reagents and amounts used in the preparation of DNB loading system

[0145]

[0146] 6. Bioinformatics analysis

[0147] The data was quality controlled using Fastpc software, aligned to the reference genome using BWA software, converted file format using samtools software, counted the alignment results, generated bed files using bedtools software, and calculated coverage, sequencing depth and other indicators using bamdst.

[0148] The results obtained by the above-described method are shown in Table 21. From the results in Table 21, it can be seen that the primer set of the present application has good specificity, good sensitivity, and a genome coverage of greater than 95% (of which the whole genome coverage graph is shown in Figure 3 The minimum detection limit at a depth of 1000X can reach 10 copies / μL.

[0149] Table 21 Sequencing results

[0150]

[0151] In Table 21, PK15 is a negative control. After targeted amplification, the DNA concentration was very low, indicating that the targeted amplification failed, and the library could not be built and sequenced.

[0152] Example 2 (nanopore sequencing)

[0153] The experimental samples of the present embodiment are: three nucleic acid samples extracted from three African swine fever virus standards provided by the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences: ZKJY-A1, ZKJY-A2, ZKJY-A3, the nucleic acid concentration in the samples is: 10000 copies / μL; ZKJY-A4, ZKJY-A5, ZKJY-A6, ZKJY-A7 are samples obtained by diluting ZKJY-A3, the nucleic acid concentrations in the samples are: 1000 copies / μL, 100 copies / μL, 10 copies / μL, 1 copies / μL; a total of 7 samples, each sample is set up 3 replicates, a total of 21 samples. The acquisition method of ZKJY-A1, ZKJY-A2, ZKJY-A3 is: take 100 μL of pure virus culture of the strain, use the nucleic acid automatic extractor KINGFISHER FLEX of Thermo Fisher Scientific Co., Ltd. and MagMAX CORE Nucleic Acid Purification Kit to extract nucleic acid by magnetic bead method, use 90 μL Elution Buffer to dissolve nucleic acid, get 90 μL nucleic acid solution; then take 5 μL nucleic acid solution for qPCR reaction, calculate the corresponding copy number according to the CT value obtained; and adjust its copy number to 10000 copies / μL. The library construction process is as follows:

[0154] First, the amplification product is obtained according to the method described in steps 1-2 of Example 1, then the obtained amplification product is used to construct a library according to the Cyclone SEQ library construction kit instructions, the library construction steps specifically include end repair & dA addition and product purification, barcode connection and product purification, sample mixing, adapter ligation and product purification, the final library recovery rate > 10% is normal, the library quality > 1 μg, which meets the requirements of the machine, and the Cyclone SEQ sequencing kit and Huada Zhi Zao Cyclone SEQ-WT02 sequencer are used for machine sequencing, and the sequencing time is 12 h. The experimental process is as follows:

[0155] 1.1. End repair & dA addition

[0156] The components of Table 22 are shaken and mixed, and centrifuged briefly, and placed in a PCR instrument for reaction according to the program in Table 23. Among them, the volume of the amplification product (double-stranded DNA) is calculated according to the concentration of double-stranded DNA in the amplification product.

[0157] Table 22 Reagents and amounts for end repair & dA addition

[0158]

[0159] Table 23 PCR reaction program for end repair & dA addition

[0160]

[0161] After the PCR reaction program is completed, 60 μL of VAHTS DNA Clean Beads is added to the reaction tube, incubated at room temperature for 5 min, and then transferred to a magnetic stand for 5 min. After the magnetic beads are completely adsorbed, the supernatant is discarded. Then 200 μL of freshly prepared 80% ethanol is added to the reaction tube and incubated for 30 s, and then the supernatant is discarded. Then 200 μL of freshly prepared 80% ethanol is added to the reaction tube and incubated for 30 s, and then the supernatant is discarded. Then the magnetic beads are dried at room temperature for 8 min, 27 μL of ddH2O is added to resuspend the dried magnetic beads, and the reaction tube is removed from the magnetic stand and incubated at room temperature for 5 min. Then the reaction tube is transferred to the magnetic stand and incubated for 5 min. After the magnetic beads are completely adsorbed, the supernatant is transferred to a new tube, and 25 μL of supernatant is obtained.

[0162] 1.2. Barcode connection

[0163] The supernatant (dsDNA after end repair) obtained in step 1.1 is prepared with the reagents in Table 24, shaken and centrifuged, and then placed in a PCR instrument for reaction according to the program in Table 25.

[0164] Table 24 Reagents and amounts for barcode connection

[0165]

[0166] Table 25 PCR reaction program for barcode connection

[0167]

[0168] After the PCR reaction program is completed, 40 μL of VAHTS DNA Clean Beads is added to the reaction tube, incubated at room temperature for 5 min, and then transferred to a magnetic stand for 5 min. After the magnetic beads are completely adsorbed, the supernatant is discarded. Then 200 μL of freshly prepared 80% ethanol is added to the reaction tube and incubated for 30 s, and then the supernatant is discarded. Then 200 μL of freshly prepared 80% ethanol is added to the reaction tube and incubated for 30 s, and then the supernatant is discarded. Then the magnetic beads are dried at room temperature for 8 min, 17 μL of ddH2O is added to resuspend the dried magnetic beads, and the reaction tube is removed from the magnetic stand and incubated at room temperature for 5 min. Then the reaction tube is transferred to the magnetic stand and incubated for 5 min. After the magnetic beads are completely adsorbed, the supernatant is transferred to a new tube, and 15 μL of supernatant is obtained, and the DNA concentration in the supernatant is determined using Equalbit 1×dsDNA HS Assay Kit.

[0169] 1.3. Mixing

[0170] According to the total DNA input amount is 1.0 μg, the product obtained by ZKJY-A1, ZKJY-A2, ZKJY-A1, ZKJY-A4, ZKJY-A5, ZKJY-A6 and ZKJY-A7 seven samples after end repair & add dA and product purification, barcode connection and product purification steps are mixed with equal quality (the volume of each sample required for mixing is calculated according to the sample concentration determined in step 1.2), mix each sample needed for mixing to 1.5 mL low adsorption tube, and add water to a total volume of 125 μL after mixing.

[0171] 1.4. Adapter ligation

[0172] The product obtained by mixing in step 1.3 is prepared with reagents according to Table 26, shaken and mixed, and centrifuged briefly, then placed in a PCR instrument for reaction according to the program in Table 27.

[0173] Table 26 Reagents and amounts for adapter ligation

[0174]

[0175] Table 27 PCR reaction program for adapter ligation

[0176]

[0177] After the PCR reaction program is completed, 80 μL of VAHTS DNA Clean Beads is added to the reaction tube, incubated at room temperature for 5 min, then the reaction tube is transferred to the magnetic stand and placed for 5 min, and the supernatant is aspirated after the magnetic beads are completely adsorbed; then 300 μL of Wash Buffer 1 (i.e. Wash Buffer 1 provided by Nanjing Novozyme Bio-tech Co., Ltd. for use with VAHTS DNA Clean Beads, the same below) is added to the reaction tube for elution, and the supernatant is aspirated; 300 μL of Wash Buffer 1 for use with magnetic beads is added to the reaction tube for elution, and the supernatant is aspirated; then 42 μL of elution buffer for use with magnetic beads (i.e. elution buffer provided by Nanjing Novozyme Bio-tech Co., Ltd. for use with VAHTS DNA Clean Beads) is added to resuspend the magnetic beads, the magnetic stand is removed and incubated at room temperature for 7 min, then the reaction tube is transferred to the magnetic stand and placed for 5 min, and 40 μL of supernatant is aspirated and transferred to a new 1.5 mL centrifuge tube, and the DNA concentration in the supernatant is measured using Equalbit 1xdsDNA HS Assay Kit.

[0178] 1.5. Sequencing

[0179] The product obtained in step 1.4 was sequenced by using Cyclone SEQ Sequencing Kit and Huada Cyclone SEQ-WT02 gene sequencer according to the Cyclone SEQ Sequencing Kit instruction.

[0180] 2. Bioinformatics analysis

[0181] The data obtained was quality controlled by using Nanofilt software, aligned to the reference genome by using minimap software, converted file format by using samtools software, counted the alignment results, generated bed files by using bedtools software, and counted coverage, sequencing depth and other indicators by using bamdst.

[0182] The results obtained by the above-described method are shown in Table 28. From the results in Table 28, it can be seen that the primer set of the application has good specificity, good sensitivity, and a genome coverage of greater than 95%, and the minimum detection limit of 1000X depth can reach 10 copies / μL.

[0183] Table 28 Nanopore sequencing results

[0184]

[0185] In Table 28: PK15 is a negative control product. After targeted amplification, the DNA concentration is very low, indicating that the targeted amplification fails, and the library cannot be built and sequenced.

[0186] It should be noted that the above-described embodiments are only used to explain the present application and do not constitute any limitation on the present application. The present application is described by referring to the embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than limiting words. The present application can be modified within the scope of the claims, and the present application can be revised without departing from the scope and spirit of the present application. Although the present application described therein relates to specific methods, materials and embodiments, it does not mean that the present application is limited to the specific examples disclosed therein, on the contrary, the present application can be extended to all other methods and applications with the same function.

Claims

1. A primer set for detecting African swine fever virus based on whole genome sequencing, characterized by, The primer set comprises 182 pairs of primers, and sequences of upstream primers and downstream primers of the 182 pairs of primers are shown in SEQ ID NO. 1-SEQ ID NO.

364.

2. A reagent for whole genome amplification of African swine fever virus, characterized by, The reagent comprises the primer set of claim 1.

3. The agent of claim 2, wherein The 182 pairs of primers with sequences shown in SEQ ID NO. 1-SEQ ID NO. 364 are sequentially and intervally divided into primer group 1 and primer group 2, and the reagent comprises two groups of reagents, one of which comprises the primer group 1 and the other of which comprises the primer group 2.

4. A kit for whole genome amplification of African swine fever virus, characterized by, The kit comprises the primer set of claim 1 or the reagent of claim 2 or 3.

5. Application of the primer set of claim 1 or the reagent of claim 2 or 3 or the kit of claim 4 in whole genome amplification of African swine fever virus, which is for non-diagnostic purposes.

6. A method of whole genome sequencing of African swine fever virus for non-diagnostic purposes, characterized by, The method comprises multiplex PCR amplification using the primer set of claim 1 or the reagent of claim 2 or 3 or the kit of claim 4.

7. The method of claim 6, wherein, The method comprises the following steps: (1) collecting samples and extracting nucleic acids from the samples; (2) performing multiplex PCR amplification on the nucleic acids extracted in step (1) using the primer set of claim 1 or the reagent of claim 2 or 3 or the kit of claim 4; (3) constructing a sequencing library; (4) sequencing on a machine; (5) bioinformatics analysis.

8. The method of claim 7, wherein, The samples in step (1) comprise one or more of pig nasal swabs, throat swabs, blood samples and tissue samples; And / or, the method for extracting nucleic acids in step (1) comprises a magnetic bead method; And / or, the length of the amplification product obtained in step (2) is between 1000-1400 bp; And / or, the amplification program in step (2) is: 98℃ pre-denaturation for 30s; 98℃ denaturation for 10s, 60℃ denaturation for 30s, 72℃ denaturation for 90s, a total of 30 cycles; 72℃ extension for 5min; And / or, the reaction reagent for amplification in step (2) comprises VAHTS Pathogen DNA Multiplex PCR Mix, gDNA, ddH2O and the primer set of claim 1; And / or, step (2) further comprises purifying the amplification product obtained by amplification; And / or, the construction of the sequencing library in step (3) is performed on a second-generation sequencing platform or a nanopore sequencing platform; And / or, the bioinformatics analysis in step (5) comprises sequence alignment, variation detection and evolution analysis.

9. The method according to claim 7 or 8, characterized in that, The 182 pairs of primers with sequences shown in SEQ ID NO. 1-SEQ ID NO. 364 are sequentially and intervally divided into primer group 1 and primer group 2, and the amplification in step (2) is performed in two groups, respectively using the primer group 1 and the primer group 2 to perform multiplex PCR amplification on the nucleic acids extracted in step (1), and then the amplification products are combined.

10. The method of claim 8, wherein, The length of the amplification product obtained in step (2) is 1200 bp.

11. The method of claim 8, wherein, The step (3) of constructing a sequencing library is performed on a second-generation sequencing platform, and the constructing of the sequencing library specifically comprises the following steps: fragment breaking, end repair & dA addition, adapter ligation, library amplification, single-strand circularization, enzyme digestion, and DNB preparation. Alternatively, the step (3) of constructing a sequencing library is performed on a nanopore sequencing platform, and the constructing of the sequencing library specifically comprises the following steps: end repair & dA addition, barcode ligation, sample mixing, and adapter ligation.

12. The method of claim 11, wherein, When the step (3) of constructing a sequencing library is performed on a second-generation sequencing platform, the step of constructing a sequencing library further comprises purifying the product obtained in the adapter ligation step and / or purifying the product obtained in the enzyme digestion step. When the step (3) of constructing a sequencing library is performed on a nanopore sequencing platform, the step of constructing a sequencing library further comprises purifying the product obtained in the end repair & dA addition step and / or purifying the product obtained in the barcode ligation step and / or purifying the product obtained in the adapter ligation step.

13. The method of any one of claims 8, 10, 11, 12, wherein, The purification comprises purifying using purified magnetic beads.

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