A primer and kit for enriching the whole genome of influenza A virus suitable for sequencing analysis

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to a whole genome enrichment primer and a kit for influenza A virus suitable for sequencing analysis. The present invention provides a pair of specially designed primers, and the primer sequence consists of an artificially designed special sequence and a target binding sequence. The primer pair greatly improves the coverage of different influenza A types, and at the same time enhances the amplification ability of the primers for targets of different lengths. The 8-segment sequence of the whole gene of influenza A can be uniformly amplified, which not only improves the coverage of the whole sequence, but also has a strong tolerance for samples of different qualities. Samples with lower concentrations or samples that have been degraded to a certain extent can achieve high uniformity full sequence amplification. The method has strong sensitivity and uniformity.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to influenza A virus whole genome enrichment primers and a kit suitable for sequencing analysis. Background Art

[0002] Influenza A virus belongs to the family Orthomyxoviridae and is a single-stranded segmented negative-strand RNA virus. The viral genome consists of 8 segments, of which hemagglutinin (HA) and neuraminidase (NA) are two important surface glycoproteins with high genetic diversity. Currently, 18 HA subtypes and 11 NA subtypes have been discovered, some of which are highly pathogenic. Influenza A virus is the most common influenza virus and is highly pathogenic to humans. Among influenza A viruses, the avian influenza virus subtypes that can directly infect humans are: A / H1N1, H5N1, H7N1, H7N2, H7N3, H7N7, H7N9, H9N2 and H10N8, of which H1, H5 and H7 subtypes are highly pathogenic. Influenza A virus is also the most prone to mutation, and avian influenza (abbreviated as human-avian influenza) is an acute respiratory infectious disease caused by some strains of certain subtypes of avian influenza A virus. After the virus gene mutates, it can infect humans. The symptoms after infection are mainly high fever, cough, runny nose, myalgia, etc., most of which are accompanied by severe pneumonia. In severe cases, multiple organ failure such as heart and kidney leads to death. The mortality rate is very high. Therefore, monitoring the mutation of influenza virus is crucial to assessing changes in the influenza situation.

[0003] There are many ways to monitor influenza, including nucleic acid testing, virus isolation and culture, sequencing and analysis, etc. Nucleic acid testing and virus isolation and culture can only perform basic typing of the virus, and cannot obtain more sequence variation information. Therefore, whole genome sequencing analysis of influenza A virus is currently the most effective means of tracking virus mutation analysis, new mutation types, and changes in epidemic situation. Although this technology has been developed by some departments, there are still many technical problems that are difficult to overcome. The mutation rate of influenza A is extremely high, and both human influenza and avian influenza can cause explosive infections. Therefore, it is difficult to cover all subtypes and unknown new influenza pathogens in the process of whole genome capture. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a whole genome enrichment primer and kit for influenza A virus, which can not only completely cover all subtypes of human and avian influenza, but also complete the entire reverse transcription and amplification process in one step with high uniformity.

[0005] In one aspect, the present invention provides a primer for amplifying the whole genome of influenza A virus, wherein the primer sequences are shown in SEQ ID NO.1-SEQ ID NO.2.

[0006] In yet another aspect, the present invention provides a kit for amplifying the whole genome of influenza A virus, the kit comprising the aforementioned primers.

[0007] Specifically, the kit also includes RNA extraction reagent, RNA reverse transcription reagent and PCR amplification reagent.

[0008] Preferably, the kit further comprises an RNA extraction reagent and a one-step RT-qPCR reaction reagent.

[0009] More specifically, the RNA extraction reagent includes, but is not limited to: cell disruption buffer, hemolysis buffer, protease, RNase inhibitor, chloroform / isopropanol and ethanol.

[0010] The one-step RT-qPCR reaction reagents include, but are not limited to, primers, dNTPs, reverse transcriptase, high-fidelity enzyme, Taq enzyme, and multiple amplification reaction solution.

[0011] More specifically, the reaction procedure of the PCR amplification reagent is:

[0012]

[0013] Preferably, the reaction procedure of the PCR amplification reagent is:

[0014]

[0015]

[0016] Specifically, the kit also includes library construction reagents for library construction and library enrichment.

[0017] The library construction reagents described more specifically include but are not limited to: fragmentation enzyme, magnetic beads, fragmentation reaction solution, fragmentation termination reaction solution, Taq enzyme, Taq enzyme reaction solution, etc.

[0018] In another aspect, the present invention provides a method for amplifying the whole genome of influenza A virus, which specifically comprises the following steps:

[0019] (1) Extracting influenza A virus RNA from samples;

[0020] (2) performing one-step RT-qPCR using the primers described in claim 1 to obtain an amplified product;

[0021] (3) constructing a library and sequencing the amplified product obtained in step (2).

[0022] Specifically, the sample in step (1) can be a nasal swab, a throat swab, a nasal aspirate, a nasal wash or a tracheal aspirate.

[0023] Specifically, the one-step RT-qPCR reaction system described in step (2) consists of the following components:

[0024]

[0025] Preferably, the one-step RT-qPCR reaction system in step (2) consists of the following components:

[0026]

[0027]

[0028] Specifically, the reaction procedure of the reaction system is:

[0029]

[0030] Preferably, the reaction procedure of the reaction system is:

[0031]

[0032] In yet another aspect, the present invention provides use of the aforementioned primers or kit in whole genome sequencing of influenza A virus.

[0033] Specifically, the whole genome sequencing of influenza A virus includes second generation sequencing or third generation sequencing.

[0034] Specifically, the influenza A virus includes but is not limited to: H1N1, H2N2, H3N2, H5N1, H7N1, H7N2, H7N3, H7N7, H7N9, H9N2 and H10N8.

[0035] The technical effect achieved by the present invention is that the influenza A virus genome-wide enrichment primers and kit of the present invention have a coverage of ≥99.9% for each subtype, and the amplification efficiency is uniform. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the full genome coverage map of the H1N1 subtype samples.

[0037] Figure 2 This is the full genome coverage map of the H3N2 subtype samples.

[0038] Figure 3 This is the full genome coverage map of the H7N9 subtype samples.

[0039] Figure 4This is the whole genome coverage map of primer pair 1 of comparative example 1.

[0040] Figure 5 This is the whole genome coverage map of primer pair 2 of comparative example 2. DETAILED DESCRIPTION

[0041] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0042] Example 1 Full gene amplification and library construction

[0043] Samples: Throat swab samples from patients with influenza A confirmed to be infected with H1N1, H3N2, H7N9, and H5N1, respectively, stored at -20°C. Nucleic acid was extracted from the samples using a nucleic acid extraction reagent (CN8043, Zhuocheng Huisheng). The Ct value of the sample nucleic acid was detected using the influenza A virus nucleic acid real-time fluorescence PCR detection kit (A3011, Zhuocheng Huisheng).

[0044] The present invention adopts a high-efficiency one-step reverse transcription and a high-fidelity Taq enzyme amplification system to amplify the sample. The system adopted is SuperScript III One-Step RT-PCR Platinum Taq HiFi (invitrogen / 12574-035) and the amplification primer sequences are shown in SEQ ID NO.1-SEQ ID NO.2.

[0045] FluA-F(SEQ ID NO.1):CGCGTTGTAG AGCRAAAGCAG ;

[0046] FluA-R(SEQ ID NO.2):CGCGTTGTAG AGTAGAAACAAG ;

[0047] Among them, the underlined part is the matching part, and the non-underlined part is the artificially designed sequence.

[0048] The reaction system is shown in Table 1 below:

[0049] Table 1

[0050]

[0051]

[0052] The reaction procedure is shown in Table 2 below:

[0053] Table 2

[0054]

[0055] The library was constructed using Illumina's Nextera XT Library Preparation Kit (Cat. No.: FC-131-1024). The specific process is as follows:

[0056] (1) Add the above amplified products to a total volume of 100 μL, and then purify with 180 μL of purification magnetic beads;

[0057] (2) adding the purified products from the previous round to ATM and TD components for fragmentation;

[0058] (3) The fragmented sequences were further amplified using the Nextera XT Index Kit (FC-131-1001, Illumina) to complete library construction;

[0059] (4) Perform library quality inspection: The dsDNA HS Assay Kit (Invitrogen / Q33230) was used for library quantification. The yield of a qualified library should be above 50 ng.

[0060] Example 2 Bioinformatics Analysis Assembly

[0061] Bioinformatics methods were used to assemble the reference genomes of influenza A viruses of various types. The full length of the genome was 113432bp. The coverage depth of each genomic site (i.e., how many sequencing fragments covered each site) was calculated. The number of genomic sites with a sequencing depth of more than 30× was counted as x bp, and the coverage = x / 113432×100%. The coverage of each subtype was ≥99.9%, and the full sequence coverage greater than 20% of the average depth could reach more than 88%, with uniform amplification efficiency. Among them, the 100X depth of Ct32 samples could reach a coverage of more than 99%, indicating that the technical method has good detection sensitivity and accuracy. The results are shown in Table 3 below:

[0062] Table 3

[0063]

[0064] Comparative Example

[0065] A comparative example was set up with reference to the methods of Example 1 and Example 2. The difference between the comparative example and Example 1 is that the primers are different. The samples used in the comparative example are the same as those in the example (throat swab samples of patients with influenza A confirmed to be infected with H1N1 clinically). The primer sequences of the comparative example are shown in Table 4 below:

[0066] Table 4

[0067]

[0068] The whole genome sequencing results of the comparative primer amplified samples are shown in Table 5:

[0069] Table 5

[0070]

[0071] From the data of Comparative Examples 1-2, it can be seen that different designed primer sequences cannot obtain the whole genome sequence of the sample with high uniformity, and amplification unevenness is prone to occur, resulting in the inability to obtain a complete whole genome sequence after the final sequencing. Some sequences are lost or the depth is extremely low, resulting in unreliable accuracy of the results.

Claims

1. A primer for amplifying the whole genome of influenza A virus, characterized in that: The primer sequences are shown in SEQ ID NO.1-SEQ ID NO.2; the primers complete the entire reverse transcription and amplification process in one step.

2. A kit for amplifying the whole genome of influenza A virus, characterized in that: The kit comprises the primer according to claim 1.

3. The kit according to claim 2, characterized in that The kit also includes an RNA extraction reagent, an RNA reverse transcription reagent and a PCR amplification reagent.

4. The kit according to claim 2, characterized in that The kit also includes an RNA extraction reagent and a one-step RT-qPCR reaction reagent.

5. The kit according to claim 3, characterized in that The kit also includes library construction reagents for library construction and library enrichment.

6. The kit according to claim 5, characterized in that The reaction procedure of the PCR amplification reagent is:

7. A method for amplifying the whole genome of influenza A virus, characterized in that: The following steps are involved: (1) Extract influenza A virus RNA from samples; (2) performing one-step RT-qPCR using the primers described in claim 1 to obtain an amplified product; (3) Construct a library and sequence the amplified product obtained in step (2).

8. The amplification method according to claim 7, characterized in that The sample described in step (1) includes a nasal swab, a throat swab, a nasal aspirate, a nasal wash and / or a tracheal aspirate.

9. The amplification method according to claim 7, characterized in that: The reaction procedure of the one-step RT-qPCR described in step (2) is:

10. Use of the primers according to claim 1 or the kit according to any one of claims 2 to 6 in whole genome sequencing of influenza A virus; the use is for non-diagnostic purposes.

11. The use according to claim 10, characterized in that: The whole genome sequencing of influenza A virus includes second generation sequencing or third generation sequencing.

12. The use according to claim 10 or 11, characterized in that: The influenza A viruses include H1N1, H2N2, H3N2, H5N1, H7N1, H7N2, H7N3, H7N7, H7N9, H9N2 and H10N8.

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