Primer set, kit and use for RAA method targeted sequencing analysis of human metapneumovirus genome

Abstract

The present invention belongs to the field of biological detection technology, and specifically, relates to a primer set, a kit and a use for RAA method targeted sequencing analysis of human metapneumovirus genome. The primer set includes 42 pairs of primers with nucleotide sequences as shown in SEQ ID NO:1-SEQ ID NO:84. The primer set provided by the present invention performs capture and library construction sequencing on different samples, and can achieve a whole genome coverage of 100%, with high coverage uniformity. Compared with the metagenomic sequencing method, this method can achieve a whole genome coverage of 100%, and the capture time is shortened by 83.3% compared with the multi-plex PCR method, which greatly improves the sequencing efficiency.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and in particular, relates to a primer set, a kit and uses for RAA method targeted sequencing analysis of human metapneumovirus genome. Background Art

[0002] Human metapneumovirus (HMPV) is one of the main pathogens of acute respiratory infections. It can infect the upper and lower respiratory tracts of all people. After infection, it often causes symptoms such as cough, fever, nasal congestion and shortness of breath. It can cause serious diseases such as bronchitis and pneumonia in susceptible populations such as infants, the elderly and those with weakened immune function. HMPV is mainly transmitted through droplets and close contact, and can also be transmitted through contact with objects contaminated by the virus. After HMPV infection, the body cannot produce effective immune protection and is prone to repeated infection, which seriously threatens human health. However, there are currently no vaccines or specific drugs on the market, and treatment measures are mostly symptomatic supportive treatment.

[0003] HMPV belongs to the family Paramyxoviridae, subfamily Pneumovirinae, genus Metapneumovirus, and is an enveloped single-stranded negative-sense RNA virus. The viral genome is 13.3 kb long and encodes nine proteins in sequence, of which the encoded F protein, attachment glycoprotein (G) and small hydrophobic surface protein (SH) are viral envelope glycoproteins. Other encoded proteins include nucleoprotein (N), phosphoprotein (P), matrix protein (M), transcription elongation factor (M2-1), RNA synthesis regulator (M2-2) and major polymerase subunit (L). Based on its nucleic acid sequence and antigenic variation, HMPV can be divided into two subgroups, A and B. Each subgroup has two subtypes (A1, A2; B1, B2). The four subtypes often circulate at the same time, but there is a dominant subtype every year, and it is usually different every year.

[0004] HMPV infection is prevalent worldwide, with cases reported in America, Europe, Asia, Africa, and Australia. Serological studies have shown that HMPV has infected humans for at least 50 years. The virus is active throughout the year in tropical regions and is prevalent in winter and spring in temperate regions. In the northern hemisphere, it usually occurs in winter, especially from December to April, and its prevalence is usually slightly later than that of respiratory syncytial virus. In Norway, an epidemic of childhood metapneumovirus was reported from November 2002 to April 2003; in Australia, it started at the end of winter and peaked in summer. The subtypes of the virus prevalent in different countries and regions are different. In North America, the subtype that is usually prevalent changes from type B to type A, while the subtype prevalent in Australia usually transitions from type A1 to type A2. According to previous meta-analysis, the global prevalence of HMPV in patients hospitalized for acute respiratory infections is about 6%. HMPV infection can occur in people of all ages, but most of them occur in young children, the elderly, people with underlying diseases or people with weakened immune function.

[0005] At present, the detection methods for human metapneumovirus include virus isolation, antigen detection, antibody detection, nucleic acid detection and gene sequencing. Among them, virus isolation is relatively difficult and has low sensitivity. Antigen and antibody detection have poor specificity and low sensitivity. Nucleic acid detection cannot accurately grasp the virus mutation situation and there is a risk of failure to detect mutant strains. At present, sequencing and typing are mainly carried out on the G and F gene fragments of the virus. Compared with other detection methods, whole genome sequencing can obtain the whole genome sequence of the virus, thereby playing the role of typing identification, tracing and variation analysis. It can more comprehensively understand the evolutionary process and pathobiology of HMPV, identify the variable and conserved regions of the genome, and recognize its genetic diversity to guide the development of diagnostic tools and vaccines. At present, the PCR method is mainly used for HMPV whole genome amplification. Its difficult primer design, poor amplification efficiency, and long amplification time greatly affect the detection of the HMPV genome. Therefore, it is of great significance to develop a primer and method for whole genome sequencing of human metapneumovirus. Summary of the invention

[0006] The purpose of the present invention is to provide a primer set, a kit and a use for RAA method targeted sequencing analysis of the human metapneumovirus genome, so as to achieve sequencing analysis of the whole genome of the human metapneumovirus with high coverage uniformity.

[0007] To this end, the present invention provides the following technical solutions.

[0008] The first aspect of the present invention provides a primer set for RAA method targeted sequencing analysis of human metapneumovirus genome, wherein the primer set comprises 42 pairs of primers with nucleotide sequences shown as SEQ ID NO: 1-SEQ ID NO: 84.

[0009] In a preferred embodiment of the present invention, the primer set further includes a forward primer, and the nucleotide sequence of the forward primer is: 5'-Pd(NNNNNNNN)-3', wherein N is G, A, T or C.

[0010] The second aspect of the present invention provides a kit for analyzing the human metapneumovirus genome by RAA targeted sequencing, wherein the kit comprises a RAA amplification system, and the RAA amplification system comprises the primer set as described above.

[0011] In a preferred embodiment of the present invention, the primer group is divided into Pool A group and Pool B group in the RAA amplification system, the nucleotide sequence of the Pool A group primer pair is shown in SEQ ID NO:1-SEQ ID NO:46, and the nucleotide sequence of the Pool B group primer pair is shown in SEQ ID NO:47-SEQ ID NO:84.

[0012] In a preferred embodiment of the present invention, the RAA amplification system further comprises recombinase T4UvsX, single-strand binding protein gp32, T4 UvsY auxiliary protein, strand displacement Bsu DNA polymerase, creatine kinase, magnesium acetate solution and cDNA / DNA template.

[0013] The strand displacement Bsu DNA polymerase is a DNA isothermal amplification polymerase with strand displacement activity, the recombinase T4 UvsX is a recombinase with pairing and strand transfer activity, the T4 UvsY auxiliary protein can promote the binding of the recombinase to single-stranded DNA, the single-strand binding protein gp32 can stabilize the transiently formed ssDNA region, and the creatine kinase can stimulate the decomposition of ATP and creatine into phosphocreatine and ADP to release energy.

[0014] In a preferred embodiment of the present invention, the kit further comprises a reverse transcription system.

[0015] In a preferred embodiment of the present invention, the reverse transcription system comprises random primers, SSIV RT Buffer, SSIV RT enzyme, ribonuclease inhibitor and DTT.

[0016] In a preferred embodiment of the present invention, the reaction procedure of the kit is: 39° C., 20 min.

[0017] The third aspect of the present invention provides a use of the primer set or kit as described above in preparing a reagent for detecting human metapneumovirus samples.

[0018] In a preferred embodiment of the present invention, the human metapneumovirus is selected from one or more of the four subtypes A1, A2, B1, and B2.

[0019] In a preferred embodiment of the present invention, the sample is selected from one or more of nasopharyngeal swab, throat swab, saliva, sputum, and alveolar lavage fluid.

[0020] A fourth aspect of the present invention provides a detection method of the kit as described above, comprising the following steps:

[0021] (1) extracting RNA from the sample to be tested;

[0022] (2) performing RAA amplification on the RNA to obtain an amplification product;

[0023] (3) Construct the library and sequence the amplified products.

[0024] In a preferred embodiment of the present invention, the step (3) further includes a step of purifying the amplified product with magnetic beads.

[0025] In a preferred embodiment of the present invention, in step (3), the library is selected from a second-generation sequencing library or a third-generation sequencing library.

[0026] In a preferred embodiment of the present invention, the second-generation sequencing library is constructed using Nextera XT DNA library construction kit and Nextera XT tagging kit from Illumina.

[0027] In a preferred embodiment of the present invention, the third-generation sequencing library is constructed using a ligation sequencing kit and a Native labeling kit from Nanopore.

[0028] In a preferred embodiment of the present invention, step (3) further includes a step of quality inspection of the library.

[0029] By means of the above technical solution, the present invention has at least the following advantages:

[0030] The present invention compares the whole genome of human metapneumovirus obtained from the NCBI database, selects the regions with specificity and high conservation, and designs a universal primer set that can cover the whole genome of human metapneumovirus. The present invention adjusts and optimizes the primer set sequence and amplification conditions such as primer concentration, reaction conditions, etc., so that the amplification efficiency of each primer pair is equivalent, and the mutual influence between primers during multiple RAA amplification is reduced, so that more than 98% of the regions of the human metapneumovirus genome can be subjected to multiple RAA (recombinase-mediated isothermal amplification technology, recombinase-aided amplification, referred to as RAA) amplification reactions in two different primer pools, which shortens the capture time by 83.3% compared with the existing multiple PCR method, wherein the multiple PCR method captures 3 hours, while the multiple RAA method only takes 30 minutes. The present invention can realize the whole genome sequencing of human metapneumovirus on different platforms by building a library for the RAA amplification product according to the library construction scheme of the second generation sequencing or the third generation sequencing.

[0031] The primer set provided by the present invention performs capture and library construction sequencing on different samples, which can achieve a 100% coverage of the whole genome and has high coverage uniformity, which is much higher than that of the metagenomic sequencing method, thus greatly improving the sequencing efficiency.

[0032] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the preferred embodiments of the present invention are described in detail as follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The capillary electrophoresis diagram of the products obtained after RAA amplification of RNA from 5 human metapneumovirus-positive samples and 1 human influenza virus-positive sample is shown. A1 (L) represents the marker, B1 represents the human metapneumovirus-positive sample with a Ct value of 26.1, C1 represents the human metapneumovirus-positive sample with a Ct value of 24.65, D1 represents the human metapneumovirus-positive sample with a Ct value of 31.53, E1 represents the human metapneumovirus-positive sample with a Ct value of 35.49, F1 represents the human influenza virus-positive sample with a Ct value of 26.05, G1 represents the human metapneumovirus-positive sample with a Ct value of 28.23, and H1 represents the negative control;

[0034] Figure 2 The whole genome sequence coverage distribution map of the human metapneumovirus sample with a CT value of 24.65 in the second-generation sequencing is shown;

[0035] Figure 3 The whole genome sequence coverage distribution map of the human metapneumovirus sample with a CT value of 31.53 in the second-generation sequencing is shown;

[0036] Figure 4 The whole genome sequence coverage distribution map of the human metapneumovirus sample with a CT value of 24.65 in the third-generation sequencing is shown;

[0037] Figure 5 The whole genome sequence coverage distribution map of the human metapneumovirus sample with a CT value of 31.53 in the third-generation sequencing is shown;

[0038] Figure 6 The whole genome sequence coverage distribution of the human metapneumovirus sample with a CT value of 24.65 in metagenomic sequencing is shown;

[0039] Figure 7 The whole genome sequence coverage distribution of the human metapneumovirus sample with a CT value of 31.53 in metagenomic sequencing is shown. DETAILED DESCRIPTION

[0040] In order to make the technical means, creative features, objectives and effects of the present invention easy to understand, the technical solution of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] Example 1: Sample source and extraction of viral nucleic acid

[0042] (1) Sample source

[0043] Five human metapneumovirus-positive samples and one human influenza virus-positive sample were used, and the sample type was a throat swab. After testing, the CT values ​​of the fluorescent PCR of the five human metapneumovirus samples were 31.53, 24.65, 28.23, and 26.1, respectively. 、 35.49, and the CT value of fluorescent PCR of human influenza virus positive samples was 26.05.

[0044] (2) Extraction of viral nucleic acid

[0045] The nucleic acid in the virus sample was extracted using QIAamp Viral RNA Mini Kit. According to the manufacturer's instructions, the specific steps were as follows: 560 μl of AVL lysate containing 5.6 μg of carrier RNA was added to 140 μl of sample, vortexed for 15 seconds, thoroughly mixed, and incubated at room temperature for 10 minutes. After the incubation, centrifuge briefly, then add 560 μl of anhydrous ethanol to the 700 μl sample solution, vortexed for 15 seconds, and centrifuged briefly. 630 μl of sample solution was added to the column placed in a 2 ml collection tube, centrifuged at 8000 rpm for 1 minute, and the column was placed in a new 2 ml collection tube, and the old tube was discarded. Repeat the previous step and filter the remaining sample solution. Add 500 μl of AW1 solution to the column, centrifuge at 8000 rpm for 1 minute, and place the column in a new 2 ml collection tube, and discard the old tube. Add 500μl of AW2 solution to the column and centrifuge at 13000rpm for 3min. After discarding the liquid in the collection tube, centrifuge the column at 13000rpm for 1min. Place the column in a new 1.5ml centrifuge tube and discard the old tube. Add 40μl of AVE solution to the column, incubate at room temperature for 1min, then centrifuge at 8000rpm for 1min, discard the column, obtain viral nucleic acid, and store at -80℃ for later use. You can also repeat the extraction without discarding the column in the previous step. This process can increase the RNA recovery rate by 20%.

[0046] Example 2: Multiple RAA Primer Design

[0047] The present invention uses a public database (NCBI) to obtain base sequences of various genotypes of human metapneumovirus, and identifies the conserved segments at the head, tail and middle of the genome through bioinformatics analysis. Similar to the multiplex PCR primer design method, this embodiment screens for highly conserved segments of the human metapneumovirus genome, and designs a primer set that can cover the entire genome of human metapneumovirus for this region. The primer set includes 42 pairs of primers and a forward primer, wherein the nucleotide sequence of the forward primer is:

[0048] 5'-Pd(NNNNNNNN)-3', wherein N is G, A, T or C.

[0049] The nucleotide sequences of the 42 primer pairs are shown in Table 1. The primer sets obtained through screening have universality and simplicity of amplification, while increasing the sensitivity and specificity of amplification.

[0050] Table 1 Sequence information of multiple RAA primers

[0051]

[0052]

[0053] Example 3: RAA Amplification of Human Metapneumovirus and Human Influenza Virus

[0054] In this example, a reverse transcription system containing Super Script IV reverse transcriptase and a RAA amplification system containing RAA amplification reagent were used to perform reverse transcription and RAA isothermal amplification on human metapneumovirus nucleic acid extracted from 5 samples and 1 human influenza virus nucleic acid in Example 1 to obtain RAA products corresponding to 6 samples. The specific steps are as follows:

[0055] Mix the viral nucleic acid of each sample in two reaction tubes to obtain a nucleic acid solution. Take 10μl of nucleic acid solution in each tube, add 1μl of forward primer (5'-Pd(NNNNNNNN)-3', where N is G, A, T or C) and 1μl of 10mM dNTPs, flick to mix, and centrifuge instantly. Then add 1μl of 100mM DTT (dithiothreitol), 1μl of SSIV reverse transcriptase, 1μl of ribonuclease inhibitor, 1μl of recombinase T4 UvsX, 1.5μl of single-strand binding protein gp32, 0.5μl of T4 UvsY auxiliary protein, 2μl of strand displacement Bsu DNA polymerase, 0.5μl of creatine kinase, 1.5μl of magnesium acetate solution, and 14μl of Nuclease-Free Water to each tube. After the addition is complete, flick to mix and centrifuge instantly. Then, the primer set shown in the sequence number SEQ ID NO: 1-84 in Example 2 was mixed into two 100 μM primers PoolA: SEQ ID NO: 1-46 and PoolB: SEQ ID NO: 47-84 according to odd and even pairs, and then the two primers PoolA and PoolB were diluted to 10 μM respectively. 4 μl of primer PoolA or primer PoolB was added to each of the two reaction tubes, and the mixture was fully mixed and centrifuged. Then, the RAA amplification reaction was performed, and the reaction procedure was: 39°C, 20 min. After the reaction, the RAA amplification reaction product was obtained. The RAA amplification reaction products of the two reaction tubes were mixed to obtain a mixed RAA product. The mixed RAA product was purified using 1.5 times the volume of Beckman AMpure XP magnetic beads, and then washed with 80% ethanol and eluted with 30 μl of denuclease water to obtain a purified RAA product as a sample to be tested for later use.

[0056] The RAA products of the 6 samples were subjected to capillary electrophoresis. Figure 1 .like Figure 1As shown, among the RAA products of human metapneumovirus positive samples, 4 samples had clear corresponding bands, corresponding to B1, C1, D1, and F1 in the figure, and the fragment size was about 400-1000bp ， However, the band of human metapneumovirus-positive sample E1 with a Ct value of 35.49 was very weak, and the RAA product of human influenza virus-positive sample G1 had no band.

[0057] Example 4: Whole genome next generation sequencing of human metapneumovirus positive samples

[0058] In this example, the purified RAA product obtained in Example 3 was quantified using Qubit 2.0 Fluorometer and Qubit dsDNA HS Assay kit (Thermo Fisher Scientific). The library was constructed using Nextera XT DNA Library Prep Kit V2 and Nextera XT Index Kit V2 from Illumina, and the experiment was performed with a starting input of 5 ng DNA per sample according to the manufacturer's instructions. The fragment integrity test and library quality inspection were performed using the Agilent 4200 TapeStation bioanalyzer and D5000 DNA ScreenTape to verify whether the DNA fragment was within the fragment screening size range. The library was quantified using Qubit 2.0 Fluorometer and Qubit dsDNA HS Assay kit.

[0059] The specific steps include: after constructing the library according to the manufacturer's instructions, the library quality inspection is performed to obtain a library that has passed the quality inspection. The library is mixed at a ratio of 1:10 and diluted to 4nM, then mixed with 0.2nM NaOH in equal proportions, denatured for 5min at room temperature, and a denatured product is obtained. If you are worried that the denaturation is not thorough, the library concentration can be diluted to 12pM, heat denatured at 95°C for 2min, and then placed on ice for more than 2min. Finally, the denatured product is sequenced on the Miseq instrument using the 300-cycle MiSeq sequencing kit (300-cycle MiSeqReagent Kit v2). After sequencing, the fastq file unpacked on the Miseq instrument is uploaded to the CLC Genomics Workbench for analysis. The analysis results show that the coverage of the four samples to be tested is 100%, and the average sequencing depth is 13425.3X to 19732.85X. One sample to be tested was not sequenced because there was no band after RAA amplification in Example 3.

[0060] Figure 2 The whole genome sequence coverage distribution of human metapneumovirus sample with a CT value of 24.65 in the second generation sequencing is shown. Figure 2 As shown, the sequencing results covered the whole genome sequence of HMPV, and the sequencing depth was 106.37X.

[0061] Figure 3 The whole genome sequence coverage distribution diagram of the human metapneumovirus sample with a CT value of 31.53 in the second generation sequencing is shown. Figure 3 As shown, the sequencing results covered the whole genome sequence of HMPV, and the sequencing depth was 106.37X.

[0062] Example 5: Whole genome third generation sequencing of human metapneumovirus positive samples

[0063] In this example, the purified RAA product was quantified using Qubit 2.0 Fluorometer and Qubit dsDNA BR Assay kit (Thermo Fisher Scientific). Ligation sequencing kit (SQK-LSK109) and Native Barcoding Expansion 1-12 (EXP-NBD104) were selected for library construction, and the optimal DNA starting input amount selected according to the length of the amplified fragment was 200 ng. The DNA product or library was quantified using Qubit 2.0 Fluorometer and Qubit dsDNA HS Assay kit. The recommended loading amount for the library is 100-300 ng.

[0064] The specific steps include: After constructing the library according to the manufacturer's instructions, perform library quality inspection to obtain a library that has passed the quality inspection. The resulting library is injected into the R9.4.1 chip, and then the chip is placed in the MinION MK1C for sequencing. Data analysis can be performed using the analysis software of Hangzhou Baiyi Technology Co., Ltd. or other bioanalysis software. The analysis results show that the coverage of the four samples to be tested is 100%, and the average sequencing depth is 1037.41X to 3617.25X.

[0065] Figure 4 The whole genome sequence coverage distribution diagram of the human metapneumovirus sample with a CT value of 24.65 in the third generation sequencing is shown. Figure 4 As shown, the sequencing results covered the whole genome sequence of HMPV, and the sequencing depth was 98.43X.

[0066] Figure 5 The whole genome sequence coverage distribution diagram of the human metapneumovirus sample with a CT value of 31.53 in the third generation sequencing is shown. Figure 5As shown, the sequencing results covered the whole genome sequence of HMPV, and the sequencing depth was 98.43X.

[0067] Example 6: Metagenomic sequencing of human metapneumovirus positive samples

[0068] In this example, RNA was quantified using Qubit 2.0 Fluorometer and Qubit RNA BR Assay kit. 200 ng RNA was taken from the nucleic acid sample obtained in Example 1, and a library was constructed using MGISP-100 and its supporting RNA library construction reagents. The constructed library was quantified using Qubit ssDNA BR Assay kit.

[0069] The specific steps include: After constructing the library according to the manufacturer's instructions, perform library quality inspection to obtain a library that has passed the quality inspection. Take 40fmol of DNA from each library formed by 5 samples and mix them, and use the DNBSEQ DNB preparation kit (SD) to prepare DNB. The obtained DNB is loaded onto the sequencing slide and sequenced on the high-throughput sequencing kit (SE100) MGISEQ-2000RS. Use the analysis software of Hangzhou Boyi Technology Co., Ltd. for analysis. The analysis results show that the coverage of the 5 samples to be tested is 63%.

[0070] Figure 6 The whole genome sequence coverage distribution of human metapneumovirus sample with a CT value of 24.65 in metagenomic sequencing is shown. Figure 6 As shown, the sequencing results can fully cover the whole genome sequence of HMPV, but there are multiple sections where the sequencing depth is lower than 20X, and the sequencing effect is significantly lower than that of Example 4 and Example 5.

[0071] Figure 7 The whole genome sequence coverage distribution of human metapneumovirus sample with a CT value of 31.53 in metagenomic sequencing is shown. Figure 7 As shown, the sequencing results failed to fully cover the HMPV genome sequence, and there were multiple regions with a sequencing depth of 0, and the sequencing effect was significantly lower than that of Example 4 and Example 5.

[0072] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technician familiar with this profession can make some changes or modifications to equivalent embodiments of equivalent changes by using the methods and technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A primer set for RAA method targeted sequencing analysis of human metapneumovirus genome, characterized in that: The primer set includes 42 pairs of primers with nucleotide sequences shown as SEQ ID NO: 1 to SEQ ID NO:

84.

2. The primer set according to claim 1, characterized in that The primer set also includes a forward primer, and the nucleotide sequence of the forward primer is: 5'-Pd(NNNNNNNN)-3', wherein N is G, A, T or C.

3. A kit for analyzing the human metapneumovirus genome by RAA targeted sequencing, characterized in that: The kit comprises a RAA amplification system, and the RAA amplification system comprises the primer set according to claim 1 or 2.

4. The kit according to claim 3, characterized in that The primer group is divided into Pool A group and Pool B group in the RAA amplification system. The nucleotide sequence of the primer pair of the Pool A group is shown in SEQ ID NO:1-SEQ ID NO:46, and the nucleotide sequence of the primer pair of the Pool B group is shown in SEQ ID NO:47-SEQ ID NO:

84.

5. The kit according to claim 3, characterized in that The RAA amplification system also includes recombinase T4UvsX, single-strand binding protein gp32, T4 UvsY auxiliary protein, strand displacement Bsu DNA polymerase, creatine kinase, magnesium acetate solution and cDNA / DNA template.

6. The kit according to any one of claims 3 to 5, characterized in that The kit also includes a reverse transcription system.

7. The kit according to claim 6, characterized in that The reverse transcription system comprises random primers, SSIV RT Buffer, SSIV RT enzyme, ribonuclease inhibitor and DTT.

8. Use of the primer set according to claim 1 or 2, or the kit according to any one of claims 3 to 7 in preparing a reagent for detecting human metapneumovirus samples.

9. The use according to claim 8, characterized in that The human metapneumovirus is selected from one or more of the four subtypes A1, A2, B1, and B2.

10. The use according to claim 8, characterized in that The sample is selected from one or more of nasopharyngeal swab, throat swab, saliva, sputum, and alveolar lavage fluid.

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