Primer group and method for human metapneumovirus whole genome sequencing and application

Abstract

The invention discloses a primer group and method for human metapneumovirus whole genome sequencing and application. Two groups of specific primer pools are designed for the metapneumovirus, totally comprise 21 primers, can effectively amplify the metapneumovirus type A and the metapneumovirus type B, are high in sensitivity and suitable for multi-platform detection, and provide a new direction for detection, typing, traceability and evolutionary analysis of the metapneumovirus.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a primer set, method, and application for whole-genome sequencing of human metapneumovirus. Background Technology

[0002] Human metapneumovirus (hMPV) is a common human respiratory pathogen. Under electron microscopy, its particles exhibit pleomorphism, spherical shape, and fibrous appearance. hMPV belongs to the Pneumoviridae family, genus Metapneumovirus, and is an enveloped, single-stranded, negative-sense RNA virus. hMPV has two genotypes, A and B, containing 8 genes and 9 open reading frames. The proteins it encodes mainly include nucleoprotein (N), phosphoprotein (P), matrix protein (M), fusion protein (F), and transcription elongation factor (M2). 1) RNA synthesis regulator (M2) 2) Genes of small hydrophobic surface proteins, adhesion proteins (G), and polymerase (L) subunits.

[0003] There are currently three commonly used methods for detecting hMPV: antigen-antibody detection, viral cell culture, and reverse transcription polymerase chain reaction (RT-PCR). PCR). Viral cell culture technology requires advanced techniques and is time-consuming, and cytopathic effects are difficult to identify, resulting in low sensitivity. Clinically, HMPV antigen detection commonly uses immunofluorescence, immunochromatography, and enzyme immunoassay. However, these techniques can only identify the pathogen and cannot perform whole-genome sequencing, thus hindering in-depth tracking and tracing of viral variations.

[0004] Therefore, it is crucial to provide a method for amplifying and sequencing the entire genome of human metapneumovirus. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, the present invention aims to provide a high-sensitivity, high-throughput primer and sequencing method for metapneumovirus whole genome amplification that is applicable to multiple platforms.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of this invention provides a primer combination for enriching the whole genome of metapneumovirus.

[0007] Furthermore, the primer combination includes primer pool A and primer pool B.

[0008] Furthermore, the nucleotide sequence of primer pool A is shown in SEQ ID NO:1-9.

[0009] Furthermore, the nucleotide sequence of primer pool B is shown in SEQ ID NO:10-21.

[0010] Furthermore, the primer combination also includes nucleotide sequences as shown in SEQ ID NO:74-75 or SEQ ID NO:76-139.

[0011] In this invention, primer pool A and Pool A can be used interchangeably; similarly, primer pool B and Pool B can also be used interchangeably.

[0012] In some embodiments, the primer assemblies further include primer sequences having at least 90% sequence identity with the sequences shown in SEQ ID NO:1-21. Identity / similarity refers to the similarity or identity between two or more nucleic acid sequences, or two or more amino acid sequences, expressed according to the identity or similarity between sequences. Sequence identity can be measured by percentage identity; the higher the percentage, the more consistent the sequences. Homologous or orthologous nucleic acid or amino acid sequences have a relatively high level of sequence identity / similarity when aligned using standard methods. The primers disclosed in this application are not limited to the exact sequences shown, and as those skilled in the art will recognize, the sequences can be altered if necessary without significantly affecting the primers' ability to function. Those skilled in the art will recognize that these provided sequence identity ranges are for guidance purposes only, and primers outside these ranges may also be used.

[0013] Furthermore, the primer combination includes degenerate bases.

[0014] Furthermore, the degenerate position includes R, W, H or Y, wherein R includes A or G, W includes A or T, H includes A, C or T, and Y includes C or T.

[0015] In some embodiments, the degenerate positions of the degenerate sequence include R, W, M, Y, K, B, D, H, N, S, or V, wherein R includes A or G, W includes A or T, M includes A or C, Y includes C or T, K includes G or T, B includes A, G, or T, D includes A, G, or T, H includes A, C, or T, N includes A, G, C, or T, S includes G or C, and V includes A, C, or G.

[0016] In some embodiments, at least one of the primers may be labeled with a labeling substance. The labeling substance includes fluorescent labels, radioactive isotopes, chemiluminescent molecules, and paramagnetic ions.

[0017] In some embodiments, the primer set includes at least one modified nucleotide.

[0018] In some embodiments, the modified nucleotides include, but are not limited to, 2'-modified nucleotides or 5-methylcytosine. Specifically, the 2'-modified nucleotides include, but are not limited to, 2'-O-methyl modified nucleotides and 2'-fluorine modified oligonucleotides; the 5-methylcytosine includes, but is not limited to, 5-methyl-deoxycytosine; and the 5-methyl-deoxycytosine includes, but is not limited to, 5-Me-dC and 5-methyl-2'-deoxycytosine. In some embodiments, the primers may include two or more modified nucleotides. The two or more modified nucleotides may have the same or different modifications. In some embodiments, the primer set may include one or more 5-methylcytosines. The primer set may have 0, 1, 2, 3, 4, 5, 6, 7, 8 or more 2'-O-methyl modified nucleotides, 2'-fluorine modified oligonucleotides, 5-methylcytosine, or combinations thereof.

[0019] A second aspect of the present invention provides a method for amplifying the whole genome sequence of metapneumovirus.

[0020] Furthermore, the amplification method includes performing an amplification reaction using the primer combination described in the first aspect of the present invention.

[0021] Furthermore, the amplification method includes: performing a first round of amplification using primer pool A and primer pool B respectively with metapneumovirus cDNA as a template; and performing a second round of amplification using the primers shown in SEQ ID NO:74-75 or SEQ ID NO:76-139 with the first round amplification product as a template.

[0022] Furthermore, the first round of amplification products is a mixture of amplification products from primer pool A and primer pool B.

[0023] Furthermore, the amplification method also includes extracting metapneumovirus nucleic acid from the sample to be tested.

[0024] Furthermore, the amplification method also includes reverse transcription of viral nucleic acid to obtain cDNA.

[0025] Furthermore, the amplification method also includes steps for purifying and quantifying the amplification products.

[0026] Furthermore, the amplification method also includes the step of constructing a library using the second-round amplification products for sequencing.

[0027] Furthermore, the amplification reaction is performed using methods including LCR, NASBA, SDA, TMA, bDNA, and PCR.

[0028] Furthermore, the amplification reaction is performed using PCR.

[0029] In some implementations, the PCR includes, but is not limited to, real-time PCR, multiplex PCR, assembly PCR, sequence-specific PCR, reverse PCR, ligation-mediated PCR, digital PCR, nested PCR, and overlap extension PCR.

[0030] Furthermore, the system for the first round of amplification consisted of 25 μl PrimeSTAR Max, 20 μl primer pool A or primer pool B, and 5 μl cDNA template.

[0031] Furthermore, the system for the second round of amplification consists of 25 μl PrimeSTAR Max, 5 μl primers shown in SEQ ID NO:74-75 or SEQ ID NO:76-77, 5 μl of the first round amplification product, and 15 μl ddH2O.

[0032] Furthermore, the PCR amplification reaction conditions are as follows: (98°C 10s→50°C 15s→72°C 40s) 5 cycles, (98°C 10s→55°C 5s→72°C 20s) 30 cycles, 72°C 5min 1 cycle, 4°C Hold.

[0033] In some implementations, amplification conditions involve a temperature and / or incubation time suitable for obtaining a detectable amount of the target. Therefore, similar amplification conditions mean that, if desired, each target can be assayed at a similar temperature. Similar amplification conditions also mean that, if desired, each target can be assayed at a similar incubation time. In some cases, similar amplification conditions also involve the number of amplification cycles. However, it is well known in the art that the number of cycles is not always strict. For example, some samples may be removed or left for additional amplification cycles before other samples. In other cases, similar amplification conditions also involve the nature of the buffer and amplification reagents (enzymes, nucleotides, salts, etc.) used. Similar amplification conditions also mean that conditions (e.g., time, buffer, number of cycles, temperature, etc.) can be slightly varied or can be the same.

[0034] Furthermore, the samples to be tested include nasopharyngeal swabs, throat swabs, saliva, sputum, and bronchoalveolar lavage fluid.

[0035] Furthermore, the purification is performed using magnetic beads.

[0036] Furthermore, the sequencing includes second-generation sequencing and third-generation sequencing.

[0037] In this invention, whole-genome sequencing refers to the laboratory process of determining the DNA sequence of each DNA strand in a sample. The resulting sequences may be referred to as "raw sequencing data" or "readings." A "library" refers to a mixture of DNA fragments containing whole-genome DNA from a single organism, which is used for sequencing.

[0038] In this invention, second-generation sequencing, also known as high-throughput sequencing, has advantages over first-generation sequencing technologies, such as Sanger sequencing, including high throughput, high yield, high accuracy, and automated analysis. Second-generation sequencing typically refers to the parallel synthesis or ligation sequencing platforms currently used by companies like Illumina, Life Technologies, and Roche.

[0039] In this invention, third-generation sequencing, also known as "de novo sequencing," refers to single-molecule sequencing technology. DNA sequencing eliminates the need for PCR amplification, enabling the individual sequencing of each DNA molecule. It is mainly divided into two major technological camps: the first is single-molecule fluorescence sequencing, represented by technologies such as Helicos' SMS technology and Pacific Bioscience's SMRT technology. The second is nanopore sequencing, represented by Oxford Nanopore Technologies.

[0040] A third aspect of the present invention provides a product for detecting metapneumovirus or amplifying the whole genome sequence of metapneumovirus.

[0041] Furthermore, the product includes the primer combination described in the first aspect of the present invention.

[0042] Furthermore, the products include chips, nucleic acid membrane strips, and reagent kits.

[0043] Furthermore, the kit also includes acceptable adjuvants.

[0044] Furthermore, the acceptable aids include PCR amplification buffer and amplification enzyme.

[0045] Furthermore, the amplification enzyme includes DNA polymerase or RNA polymerase.

[0046] Furthermore, the DNA polymerase includes PrimeSTAR Max DNA polymerase, Taq DNA polymerase, Pfu DNA polymerase, KOD DNA polymerase, Q5 high-fidelity DNA polymerase, Q5 hot-start ultra-fidelity DNA polymerase, Phanta high-fidelity DNA polymerase, and VAHTS HiFi DNA polymerase.

[0047] In this invention, amplification enzyme refers to an enzyme that can catalyze the production of polynucleotides or nucleic acids from existing DNA or RNA templates. In a specific embodiment of this invention, the amplification enzyme is selected from PrimeSTAR Max DNA polymerase.

[0048] Furthermore, the acceptable auxiliaries also include reverse transcription reaction systems.

[0049] Furthermore, the reverse transcription reaction system includes primers, reverse transcription buffer, reverse transcriptase, ribonuclease inhibitor, DTT, and dNTPs.

[0050] Furthermore, the primers are random primers.

[0051] Furthermore, the reverse transcription buffer includes M-MLV reverse transcription buffer, Hifair® III Reverse Transcriptase Buffer, Hifair® V Buffer, and SSIV Buffer.

[0052] Furthermore, the reverse transcriptase includes M-MLV RTase, HIV RTase, ASLV RTase, RSV RTase, AMV RTase, REV-A RTase, RAV RTase, MAV RTase, and SSIV RTase.

[0053] In this invention, reverse transcriptase refers to any enzyme that exhibits reverse transcription activity as determined by the methods described in this invention or by methods known in the art. In a specific embodiment of this invention, the reverse transcriptase is selected from SSIV RT enzymes.

[0054] Furthermore, the kit also includes instructions.

[0055] In some implementations, the kit may contain viral genomic cDNA as a positive control for PCR and sterile water as a negative control.

[0056] In some embodiments, a suitable amount of primers is provided in a container or immobilized on a matrix. The primers may be provided as a suspension in an aqueous solution or, for example, as a lyophilized or freeze-dried powder. The container providing the nucleic acid can be any conventional container capable of containing the provided form, such as a microcentrifuge tube, ampoule, or bottle. The kit may contain labeled or unlabeled probes for pneumovirus nucleic acid sequences.

[0057] In some implementations, primers can be provided in pre-measured single-use amounts in separate, typically disposable tubes or equivalent containers. Using such a setup, samples for testing the presence of metapneumovirus can be added to separate tubes for direct amplification.

[0058] In some implementations, the amount of nucleic acid primers provided in the kit can be any suitable amount, depending on the target market for the product. For example, if the kit is intended for research or clinical applications, the amount of each nucleic acid primer provided can be sufficient to initiate several PCR amplification reactions. The kit may contain more than two primers to facilitate PCR amplification of larger quantities of pneumovirus nucleic acid sequences.

[0059] The fourth aspect of the present invention provides the use of the primer combination described in the first aspect of the present invention in the preparation of products for detecting metapneumovirus or in the preparation of products for metapneumovirus whole genome amplification and / or sequencing.

[0060] Furthermore, the metapneumovirus includes metapneumovirus type A and / or metapneumovirus type B.

[0061] The fifth aspect of this invention provides the application of the primer combinations described in the first aspect of this invention or the products described in the third aspect of this invention in metapneumovirus typing, tracing, or evolutionary analysis.

[0062] Furthermore, the metapneumovirus includes metapneumovirus A and / or metapneumovirus B.

[0063] Advantages and beneficial effects of the present invention: This invention designs two sets of specific primer pools for metapneumovirus, containing a total of 21 primers, which can effectively amplify metapneumovirus type A and metapneumovirus type B. The primers are highly sensitive and suitable for multi-platform detection. This invention provides a new direction for the detection, typing, tracing or evolutionary analysis of metapneumovirus. Attached Figure Description

[0064] Figure 1 This is a flowchart illustrating the amplification process of the present invention; Figure 2 Example image of next-generation sequencing results for 1 hMPV-A serotype using primer set; Figure 3 Example image of next-generation sequencing results for primer set 2 hMPV-A serotype; Figure 4 Example image of next-generation sequencing results for 3 hMPV-A serological type primer set; Figure 5 Example image of next-generation sequencing results for 4 hMPV-A serological type primer set; Figure 6 Example image of next-generation sequencing results for 5 hMPV-A serological type primer set; Figure 7 Example image of next-generation sequencing results for 1 hMPV-B serological type primer set; Figure 8 Example image of next-generation sequencing results for primer set 2 hMPV-B serological type; Figure 9 Example image of next-generation sequencing results for 3 hMPV-B serological type primer set; Figure 10 Example image of next-generation sequencing results for 4 hMPV-B serological type primer set; Figure 11 Example image of next-generation sequencing results for 5 hMPV-B serological type primer set; Figure 12 Example image of third-generation sequencing results for primer set 1 hMPV-A serotype; Figure 13 Example image of third-generation sequencing results for primer set 2 hMPV-A serotype; Figure 14 Example image of third-generation sequencing results for primer set 3 hMPV-A serological type; Figure 15 Example image of third-generation sequencing results for 4 hMPV-A serotype primer set; Figure 16 Example image of third-generation sequencing results for primer set 5 hMPV-A serotype; Figure 17 Example image of third-generation sequencing results for primer set 1 hMPV-B serotype; Figure 18 Example image of third-generation sequencing results for primer set 2 hMPV-B serotype; Figure 19 Example image of third-generation sequencing results for primer set 3 hMPV-B serological type; Figure 20 Example image of third-generation sequencing results for primer set 4 hMPV-B serological type; Figure 21 This is an example image of the third-generation sequencing results for the primer set 5 hMPV-B serological type. Detailed Implementation

[0065] To make the technical means, creative features, and achieved objectives and effects of this invention easier to understand, the invention is further illustrated below with specific embodiments. However, the following embodiments are merely preferred embodiments of this invention and not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the protection scope of this invention. Unless otherwise specified, the operating methods and equipment used in the following embodiments are conventional operating methods, and the materials and equipment used in each embodiment are the same.

[0066] Example 1: Design and Synthesis of Primer Sets This invention designed a total of 5 primer sets (primer sets 1-5). The 5' end of all specific primers is pre-ligated with a universal M13 sequence to form an "M13-tail" structure. The sequence information of primer sets 1-4 is shown in Table 1, and the sequence information of primer set 5 is shown in Table 2.

[0067] Table 1. hMPV Primer Set 1-4: Specific Primers

[0068] Table 2 hMPV Primer Set 5 Specific Primers

[0069] Note: Lowercase letters without bold indicate the M13 primer sequence; uppercase letters in bold indicate RSV-specific primers, where red underlines represent degenerate bases.

[0070] Example 2: Clinical Sample Testing The overall experimental strategy of this invention is as follows: A two-round amplification method is used. In the first round of amplification, the virus-specific primers of Example 1 are used to amplify the product to obtain a (3000±500bp fragment) + M13 adapter. In the second round of amplification, the M13 + pacbioBarcode primers are used to amplify the product of the first round of amplification. After purification, the amplified product is used for pacbio sequencing or next-generation sequencing to construct a library for sequencing.

[0071] 1. Sample collection With the informed consent of the individuals being tested, 94 positive human metapneumovirus samples were used, and the sample type was pharyngeal swabs.

[0072] 2. Viral nucleic acid extraction Nucleic acid was extracted from the samples using a viral RNA extraction kit (QIAamp Viral RNA Mini Kit, Qiagen, 52904 / 52906). The extraction method was performed in accordance with the kit instructions.

[0073] 3. Reverse transcription: Viral RNA was converted into cDNA using SuperScript IV Reverse Transcriptase (Invitrogen 18091050): (1) Prepare reaction system mix1 according to Table 3, incubate at 65℃ for 5 min, and then incubate on ice for 1 min; Table 3 Reaction system mix1

[0074] (2) Prepare reaction system mix2 according to Table 4, mix mix1 and mix2, centrifuge briefly, place in PCR instrument, and perform the following in sequence: 23℃ for 10 min, 50℃ for 10 min, 80℃ for 10 min; Table 4 Reaction system mix2

[0075] (3) Add 1 μl of Rnase H to the above reaction system, incubate at 37°C for 20 min, and keep at 4°C for later use.

[0076] 4. First round of amplification: Each sample was amplified in the first round using primers from Pool A and Pool B, with two reactions (Pool A and Pool B). Each reaction used 2.5 μl of cDNA, and each sample required a total of 2.5 μl × 2 = 5 μl of cDNA. The amplification products from Pool A and Pool B were then mixed for the second round of amplification.

[0077] (1) First round of primer preparation: Calculate the required primer amount based on 100 samples, 100 × 10 μl primer / sample = 1000 μl. Take 5-15 μl of each of the 100 μM primer groups in Pool A and Pool B, and prepare primer pools A and B with a concentration of 0.5-1.5 μM. The specific preparation information for primer group 5 is shown in Table 5. The preparation of other primer groups is the same as that for primer group 5.

[0078] Table 5 Primer preparation for Pool A and Pool B

[0079] (2) Prepare the first round of PCR reaction system according to Table 6 and run the reaction program according to Table 7.

[0080] Table 6 First-round PCR reaction system

[0081] Table 7 Reaction Procedure

[0082] 5. Second round of amplification: The mixed products from pool A and pool B of the first round were amplified using the second-round amplification primers (M13 + barcode combination) (third-generation sequencing). The specific primer sequences are shown in Table 8. If second-generation sequencing is performed, only the M13 primer is needed for the second-round amplification. The forward primer M13 sequence is: GTAAAACGACGGCCAGT (SEQ ID NO:74); the reverse primer M13 sequence is: CAGGAAACAGCTATGAC (SEQ ID NO:75).

[0083] Table 8 Primer sequences for the second round of amplification in third-generation sequencing

[0084] (1) Primer preparation: 10 μl primer, 5 μl F and 5 μl R for each system, with a final concentration of 0.25 μM each. 0.25 μM × 25 μl / 5 μl = 1.25 μM; required volume: 12 × 5 μl = 60 μl, 24 × 5 μl = 120 μl; 1.25 μM (1.25 pmol / μl) primer preparation: dilute single primer to 100 μM, take 2.5 μl of each primer, add 197.5 μl of water to obtain 200 μl of primer (directly in a 96-well plate); (2) Prepare the second round of amplification system according to Table 9, and perform PCR reaction under the same conditions as the first round of amplification reaction. Centrifuge and store the product.

[0085] Table 9 Second-round amplification system

[0086] 6. Magnetic bead purification: The PCR products were purified using Beckman AMpure PB beads for subsequent library construction. The required PCR product for library construction was 500-1000 ng / 50 μl (concentration ≥10-20 ng / μl).

[0087] Add magnetic beads (90 μl) at a ratio of 0.6×, vortex for 1-2 minutes, let stand for 5 minutes, place on a magnetic rack, and adsorb for 3-5 minutes to remove all the supernatant.

[0088] Add 200 μl of 75% ethanol, wash and repeat, air dry, add 20 μl of elution buffer, vortex for 1 min, let stand for 5-10 min, place on a magnetic rack, and transfer the supernatant nucleic acid. After quantifying the elution product using Qubit, it can be directly used for second-generation (Miseq, Illumina) or third-generation library construction and sequencing (SEQUEL, PacBio).

[0089] 7. Sequencing results After sequencing samples with high, medium and low viral load using primer set 5, the coverage was close to 100%, and it was applicable to both second-generation and third-generation sequencing platforms. Furthermore, primer set 5 performed better than primer sets 1-4.

[0090] Table 10 Average Coverage Results of Second-Generation Sequencing and Third-Generation Sequencing

[0091] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A primer combination for enriching the whole genome of metapneumovirus, characterized in that, The primer combination includes primer pool A and primer pool B; Preferably, the nucleotide sequence of primer pool A is shown in SEQ ID NO:1-9; Preferably, the nucleotide sequence of primer pool B is shown in SEQ ID NO:10-21; Preferably, the primer combination further includes nucleotide sequences as shown in SEQ ID NO:74-75 or SEQ ID NO:76-139.

2. The primer combination according to claim 1, characterized in that, The primer combination includes degenerate bases; Preferably, the degenerate bases include R, W, H or Y, wherein R includes A or G, W includes A or T, H includes A, C or T, and Y includes C or T.

3. A method for amplifying the complete genome sequence of a metapneumovirus, characterized in that, The amplification method includes performing an amplification reaction using the primer combination described in claim 1 or 2.

4. The amplification method according to claim 3, characterized in that, The amplification method includes: performing a first round of amplification using primer pool A and primer pool B respectively with pneumovirus cDNA as a template; and performing a second round of amplification using the primers shown in SEQ ID NO:74-75 or SEQ ID NO:76-139 with the first round amplification product as a template. Preferably, the amplification method further includes extracting metapneumovirus nucleic acid from the sample to be tested; Preferably, the amplification method further includes reverse transcription of viral nucleic acid to obtain cDNA; Preferably, the amplification method further includes the steps of purifying and quantifying the amplification products; Preferably, the amplification method further includes the step of constructing a library using the second-round amplification products for sequencing.

5. The amplification method according to claim 3, characterized in that, The amplification reaction is performed using methods including LCR, NASBA, SDA, TMA, bDNA, and PCR. Preferably, the amplification reaction is performed by PCR. Preferably, the system for the first round of amplification consists of 25 μl PrimeSTAR Max, 20 μl primer pool A or primer pool B, and 5 μl cDNA template; Preferably, the second round of amplification consists of 25 μl PrimeSTAR Max, 5 μl primers shown in SEQ ID NO:74-75 or SEQ ID NO:76-77, 5 μl of the first round amplification product, and 15 μl ddH2O. Preferably, the PCR amplification reaction conditions are: (98°C 10s→50°C 15s→72°C 40s) 5 cycles, (98°C 10s→55°C 5s→72°C 20s) 30 cycles, 72°C 5min 1 cycle, 4°C Hold.

6. The amplification method according to claim 4, characterized in that, The samples to be tested include nasopharyngeal swabs, throat swabs, saliva, sputum, and bronchoalveolar lavage fluid; Preferably, the purification is magnetic bead purification; Preferably, the sequencing includes second-generation sequencing and third-generation sequencing.

7. A product for detecting metapneumovirus or amplifying the whole genome sequence of metapneumovirus, characterized in that, The product comprises the primer combination as described in claim 1 or 2; Preferably, the product includes a chip, a nucleic acid membrane strip, and a reagent kit.

8. The product according to claim 7, characterized in that, The kit also includes acceptable adjuvants; Preferably, the acceptable auxiliaries include PCR amplification buffer and amplification enzyme; Preferably, the amplification enzyme includes DNA polymerase or RNA polymerase; Preferably, the DNA polymerase includes PrimeSTAR Max DNA polymerase, Taq DNA polymerase, Pfu DNA polymerase, KOD DNA polymerase, Q5 high-fidelity DNA polymerase, Q5 hot-start ultra-fidelity DNA polymerase, Phanta high-fidelity DNA polymerase, and VAHTS HiFi DNA polymerase. Preferably, the acceptable auxiliaries further include a reverse transcription reaction system; Preferably, the reverse transcription reaction system includes primers, reverse transcription buffer, reverse transcriptase, ribonuclease inhibitor, DTT, and dNTPs; Preferably, the primers are random primers; Preferably, the reverse transcription buffer includes M-MLV reverse transcription buffer, Hifair® III Reverse Transcriptase Buffer, Hifair® V Buffer, and SSIV Buffer; Preferably, the reverse transcriptase includes M-MLV RT enzyme, HIV RT enzyme, ASLV RT enzyme, RSV RT enzyme, AMV RT enzyme, REV-A RT enzyme, RAV RT enzyme, MAV RT enzyme, and SSIV RT enzyme; Preferably, the kit also includes instructions.

9. The use of the primer combination according to claim 1 or 2 in the preparation of products for detecting metapneumovirus or for the preparation of products for whole-genome amplification and / or sequencing of metapneumovirus; Preferably, the metapneumovirus includes metapneumovirus A and / or metapneumovirus B.

10. The application of the primer combination of claim 1 or 2 or the product of claim 7 or 8 in metapneumovirus typing, tracing or evolutionary analysis; Preferably, the metapneumovirus includes metapneumovirus A and / or metapneumovirus B.

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