Primer pool for co-amplification of influenza and novel coronavirus genomes and its library construction method

Abstract

The present invention discloses a primer pool for co-amplification of the whole genome of influenza and novel coronavirus and a library construction method thereof. The primer pool for co-amplification of the whole genome of influenza and novel coronavirus disclosed in the present invention consists of 78 single-stranded DNAs shown in SEQ ID No.1-78 in the sequence table. Using the primer set of the present invention, a multiple long PCR tube co-amplification is used to capture the virus, and a PCR free high-throughput sequencing library is constructed by one-step DNA shearing, end repair & adding dA and adding a linker, and high-throughput sequencing analysis is performed. On the one hand, the relative content of the virus can be obtained by adding a certain number of copies of external references, and on the other hand, the whole genome sequence data and mutation information of influenza and novel coronavirus can be obtained at the same time, which has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a primer pool for co-amplification of the whole genomes of influenza and novel coronavirus and a library construction method thereof. Background Art

[0002] Respiratory viral infection is a major global health problem. It is estimated that the annual seasonal epidemic of influenza will infect 5% to 10% of the world's population, resulting in 3 million to 5 million hospitalizations and 290,000 to 650,000 deaths. There are three types of influenza viruses that infect humans: type A, type B, and type C. The more common and serious ones are usually type A and type B. SARS-CoV-2, a new coronavirus that broke out in late December 2019, is also a very harmful respiratory virus. Its infection and transmission capabilities are significantly stronger than those of SARS and MERS. Respiratory infectious diseases caused by influenza and the new coronavirus pose a huge threat to human health, so the detection, monitoring and real-time tracking of influenza viruses and the new coronavirus have become extremely important and urgent.

[0003] Studies have shown that among patients with positive COVID-19 nucleic acid, there is a certain proportion of patients who are co-infected with influenza pathogens, that is, patients are infected with influenza virus and COVID-19 at the same time. In clinical practice, the manifestations of pneumonia caused by influenza and COVID-19 are somewhat similar and difficult to distinguish directly. Therefore, a method with high sensitivity and accuracy that can detect both influenza and COVID-19 is very necessary to help quickly diagnose the disease. However, it is far from enough to simply identify whether a person has COVID-19 or influenza infection. Both influenza and COVID-19 viruses are RNA pathogens, with highly variable antigens and rapid transmission. If you want to conduct regular prevention and treatment of COVID-19 and influenza viruses, you also need to obtain the full genome sequence of the pathogen and monitor the mutation of the virus at all times.

[0004] At present, the conventional detection method of respiratory pathogens is based on real-time fluorescence quantitative PCR detection. The principle of its detection is mainly to design one or several pairs of primers for amplification of certain target sequences in the viral genome. Its advantages are convenience and rapidity, and it plays a pivotal role in the diagnosis of the population. However, this method has low detection throughput and can only detect pathogens but cannot obtain the whole genome of the virus. When facing rapidly mutating influenza and new coronavirus, this method is prone to false negatives and cannot meet the needs of monitoring viral mutations. Compared with traditional PCR technology, high-throughput sequencing methods can obtain full-length information of the virus in the case of trace samples. On the basis of typing the pathogen, it can judge the distance of its homology and distinguish different evolutionary routes, which can track or prevent the outbreak of epidemic diseases. At present, pathogen detection based on genome sequencing technology (NGS) mainly includes metagenomic sequencing technology (mNGS), whole genome targeted sequencing based on probe capture, and whole genome targeted sequencing based on multiplex PCR. mNGS has outstanding advantages in the discovery of unknown viral pathogens, but the amount of sequencing data required is large, the proportion of truly meaningful gene fragment data is low, and the method has low sensitivity, which is easy to lead to missed detection. The probe capture method first builds a library for the sample, then enriches the virus in a targeted manner, and then sequences it. The operation process is very cumbersome and takes a long time, which is not conducive to virus prevention and control. The whole genome capture method based on multiplex PCR uses specific primers to enrich the target virus by a million times and build a library, and then sequences to obtain the whole genome data of the virus. This method is simple to operate and has very high sensitivity. It can also sensitively detect weak positive samples, and is a more ideal technical means. However, due to the large number of influenza virus typing types, only the first and last sequences of the long fragment are conserved regions, and primers can only be designed in the conserved region for long PCR amplification. In addition, if the new crown and influenza are to be co-amplified in one tube, the new coronavirus also needs to use the same long-fragment PCR method so that the continuous region of the new coronavirus can also be amplified in one tube. It can be seen that it is very technically difficult to co-amplify and build a library for influenza and the new coronavirus in one tube through the multiplex PCR capture method, and the primer design requirements for the two types of viruses are very high. At present, there is no corresponding primer pool or product on the market that can meet the demand. Summary of the invention

[0005] The technical problem to be solved by the present invention is how to screen patients infected with both influenza virus and novel coronavirus.

[0006] To solve the above technical problems, the present invention first provides a primer set, which includes a forward primer and a reverse primer;

[0007] Influenza A-01F, Influenza A-02F, InfluenzaA-03F, Influenza A-04F, Influenza A-05F, Influenza A-06F, Influenza A-07F、influenza A-08F、influenza A-09F、influenza B-01F、influenza B-02F、influenza B-03F、influenza B-04F、influenza B-05F、influenza B-06F、influenza B-07F, influenzaB-08F, SARS-CoV-2-01F, SARS-CoV-2-02F, SARS-CoV-2-03F, SARS-CoV-2-04F, SARS-CoV-2-05F, SARS-CoV-2-06F, SARS-CoV-2-07F, SARS-CoV-2-08F, SARS-CoV-2-09F, SARS-C oV-2-10F, SARS-CoV-2-11F, SARS-CoV-2-12F, SARS-CoV-2-13F, SARS-CoV-2-14F, SARS-CoV-2-1 5F SARS-CoV-2-16F SARS-CoV-2-17F SARS-CoV-2-18F SARS-CoV-2-19F SARS-CoV-2-20F

[0008] The reverse primers include names of influenza A-01R, influenza A-02R, influenzaA-03R, influenza A-04R, influenza A-05R, influenza A-06R, influenza A-07R, influenza A-08R, influenza A-09R, influenza B-01R, influenza B-02R, influenza B-03R, influenza B-04R, influenza B-05R, influenza B-06R, influenza B-07R, influenzaB-08R, C-SARS-CoV-2-01R, C-SARS-CoV-2-02R, C-SARS-CoV-2-03R, C-SARS-CoV-2-04R, C- SARS-CoV-2-05R, C-SARS-CoV-2-06R, C-SARS-CoV-2-07R, C-SARS-CoV-2-08R, C-SARS-CoV-2-09R, C-SARS-C oV-2-10R, C-SARS-CoV-2-11R, C-SARS-CoV-2-12R, C-SARS-CoV-2-13R, C-SARS-CoV-2-14R, C-SARS-CoV-2-1 Primers for 5R, C-SARS-CoV-2-16R, C-SARS-CoV-2-17R, C-SARS-CoV-2-18R, C-SARS-CoV-2-19R and C-SARS-CoV-2-20R;

[0009] The influenza A-01F is any single-stranded DNA of the following a11) to a14):

[0010] a11) single-stranded DNA corresponding to positions 8 to 23 of SEQ ID No. 1 in the sequence list;

[0011] a12) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of a11);

[0012] a13) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in a11) or a12);

[0013] a14) a single-stranded DNA that hybridizes with the single-stranded DNA defined in a11) or a12) under stringent conditions;

[0014] The influenza A-02F is any single-stranded DNA of the following a21) to a24):

[0015] a21) the single-stranded DNA corresponding to positions 8 to 24 of SEQ ID No. 2 in the sequence list;

[0016] a22) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of a21);

[0017] a23) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in a21) or a22);

[0018] a24) a single-stranded DNA that hybridizes with the single-stranded DNA defined in a21) or a22) under stringent conditions;

[0019] The influenza A-03F is any single-stranded DNA of the following a31) to a34):

[0020] a31) single-stranded DNA corresponding to positions 8 to 25 of SEQ ID No. 3 in the sequence list;

[0021] a32) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of a31);

[0022] a33) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in a31) or a32);

[0023] a34) a single-stranded DNA that hybridizes with the single-stranded DNA defined in a31) or a32) under stringent conditions;

[0024] The influenza A-04F is any single-stranded DNA of the following a41) to a44):

[0025] a41) the single-stranded DNA corresponding to positions 8 to 21 of SEQ ID No. 4 in the sequence list;

[0026] a42) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of a41);

[0027] a43) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in a41) or a42);

[0028] a44) a single-stranded DNA that hybridizes with the single-stranded DNA defined in a41) or a42) under stringent conditions;

[0029] The influenza A-05F is any single-stranded DNA of the following a51) to a54):

[0030] a51) the single-stranded DNA corresponding to positions 8 to 24 of SEQ ID No. 5 in the sequence list;

[0031] a52) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of a51);

[0032] a53) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in a51) or a52);

[0033] a54) a single-stranded DNA that hybridizes with the single-stranded DNA defined in a51) or a52) under stringent conditions;

[0034] The influenza A-06F is any single-stranded DNA of the following a61) to a64):

[0035] a61) the single-stranded DNA corresponding to positions 8 to 22 of SEQ ID No. 6 in the sequence list;

[0036] a62) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of a61);

[0037] a63) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in a61) or a62);

[0038] a64) a single-stranded DNA that hybridizes with the single-stranded DNA defined in a61) or a62) under stringent conditions;

[0039] The influenza A-07F is any single-stranded DNA of the following a71) to a74):

[0040] a71) the single-stranded DNA corresponding to positions 8 to 24 of SEQ ID No. 7 in the sequence list;

[0041] a72) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of a71);

[0042] a73) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in a71) or a72);

[0043] a74) a single-stranded DNA that hybridizes with the single-stranded DNA defined in a71) or a72) under stringent conditions;

[0044] The influenza A-08F is any single-stranded DNA of the following a81) to a84):

[0045] a81) the single-stranded DNA corresponding to positions 8 to 24 of SEQ ID No. 8 in the sequence list;

[0046] a82) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of a81);

[0047] a83) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in a81) or a82);

[0048] a84) a single-stranded DNA that hybridizes with the single-stranded DNA defined in a81) or a82) under stringent conditions;

[0049] The influenza A-09F is any single-stranded DNA of the following a91) to a94):

[0050] a91) the single-stranded DNA corresponding to positions 8 to 22 of SEQ ID No. 9 in the sequence list;

[0051] a92) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of a91);

[0052] a93) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in a91) or a92);

[0053] a94) a single-stranded DNA that hybridizes with the single-stranded DNA defined in a91) or a92) under stringent conditions;

[0054] The influenza B-01F is any single-stranded DNA of the following b11) to b14):

[0055] b11) the single-stranded DNA corresponding to positions 8 to 22 of SEQ ID No. 10 in the sequence list;

[0056] b12) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of b11);

[0057] b13) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in b11) or b12);

[0058] b14) a single-stranded DNA that hybridizes with the single-stranded DNA defined in b11) or b12) under stringent conditions;

[0059] The influenza B-02F is any single-stranded DNA of the following b21) to b24):

[0060] b21) the single-stranded DNA corresponding to positions 8 to 22 of SEQ ID No. 11 in the sequence list;

[0061] b22) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of b21);

[0062] b23) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in b21) or b22);

[0063] b24) a single-stranded DNA that hybridizes with the single-stranded DNA defined in b21) or b22) under stringent conditions;

[0064] The influenza B-03F is any single-stranded DNA of the following b31) to b34):

[0065] b31) the single-stranded DNA corresponding to positions 8 to 21 of SEQ ID No. 12 in the sequence list;

[0066] b32) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of b31);

[0067] b33) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in b31) or b32);

[0068] b34) a single-stranded DNA that hybridizes with the single-stranded DNA defined in b31) or b32) under stringent conditions;

[0069] The influenza B-04F is any single-stranded DNA of the following b41) to b44):

[0070] b41) the single-stranded DNA corresponding to positions 8-27 of SEQ ID No.13 in the sequence list;

[0071] b42) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of b41);

[0072] b43) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in b41) or b42);

[0073] b44) a single-stranded DNA that hybridizes with the single-stranded DNA defined in b41) or b42) under stringent conditions;

[0074] The influenza B-05F is any single-stranded DNA of the following b51) to b54):

[0075] b51) the single-stranded DNA corresponding to positions 8-21 of SEQ ID No.14 in the sequence list;

[0076] b52) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of b51);

[0077] b53) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in b51) or b52);

[0078] b54) a single-stranded DNA that hybridizes with the single-stranded DNA defined in b51) or b52) under stringent conditions;

[0079] The influenza B-06F is any single-stranded DNA of the following b61) to b64):

[0080] b61) the single-stranded DNA corresponding to positions 8-21 of SEQ ID No.15 in the sequence list;

[0081] b62) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of b61);

[0082] b63) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in b61) or b62);

[0083] b64) a single-stranded DNA that hybridizes with the single-stranded DNA defined in b61) or b62) under stringent conditions;

[0084] The influenza B-07F is any single-stranded DNA of the following b71) to b74):

[0085] b71) the single-stranded DNA corresponding to positions 8 to 21 of SEQ ID No. 16 in the sequence list;

[0086] b72) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of b71);

[0087] b73) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in b71) or b72);

[0088] b74) a single-stranded DNA that hybridizes with the single-stranded DNA defined in b71) or b72) under stringent conditions;

[0089] The influenza B-08F is any single-stranded DNA of the following b81) to b84):

[0090] b81) the single-stranded DNA corresponding to positions 8 to 25 of SEQ ID No. 17 in the sequence list;

[0091] b82) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of b81);

[0092] b83) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in b81) or b82);

[0093] b84) a single-stranded DNA that hybridizes with the single-stranded DNA defined in b81) or b82) under stringent conditions;

[0094] The SARS-CoV-2-01F is any single-stranded DNA of the following c11) to c14):

[0095] c11) the single-stranded DNA corresponding to positions 8 to 30 of SEQ ID No. 18 in the sequence list;

[0096] c12) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of c11);

[0097] c13) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in c11) or c12);

[0098] c14) a single-stranded DNA that hybridizes with the single-stranded DNA defined in c11) or c12) under stringent conditions;

[0099] The SARS-CoV-2-02F is any single-stranded DNA of the following c21) to c24):

[0100] c21) the single-stranded DNA corresponding to positions 8 to 29 of SEQ ID No. 19 in the sequence list;

[0101] c22) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of c21);

[0102] c23) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in c21) or c22);

[0103] c24) a single-stranded DNA that hybridizes with the single-stranded DNA defined in c21) or c22) under stringent conditions;

[0104] The SARS-CoV-2-03F is any single-stranded DNA of the following c31) to c34):

[0105] c31) the single-stranded DNA corresponding to positions 8 to 27 of SEQ ID No. 20 in the sequence list;

[0106] c32) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of c31);

[0107] c33) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in c31) or c32);

[0108] c34) a single-stranded DNA that hybridizes with the single-stranded DNA defined in c31) or c32) under stringent conditions;

[0109] The SARS-CoV-2-04F is any single-stranded DNA of the following c41) to c44):

[0110] c41) the single-stranded DNA corresponding to positions 8 to 26 of SEQ ID No. 21 in the sequence list;

[0111] c42) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of c41);

[0112] c43) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in c41) or c42);

[0113] c44) a single-stranded DNA that hybridizes with the single-stranded DNA defined in c41) or c42) under stringent conditions;

[0114] The SARS-CoV-2-05F is any single-stranded DNA of the following c51) to c54):

[0115] c51) the single-stranded DNA corresponding to positions 8-39 of SEQ ID No.22 in the sequence list;

[0116] c52) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of c51);

[0117] c53) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in c51) or c52);

[0118] c54) a single-stranded DNA that hybridizes with the single-stranded DNA defined in c51) or c52) under stringent conditions;

[0119] The SARS-CoV-2-06F is any single-stranded DNA of the following c61) to c64):

[0120] c61) the single-stranded DNA corresponding to positions 8-26 of SEQ ID No.23 in the sequence list;

[0121] c62) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of c61);

[0122] c63) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in c61) or c62);

[0123] c64) a single-stranded DNA that hybridizes with the single-stranded DNA defined in c61) or c62) under stringent conditions;

[0124] The SARS-CoV-2-07F is any single-stranded DNA of the following c71) to c74):

[0125] c71) the single-stranded DNA corresponding to positions 8 to 25 of SEQ ID No. 24 in the sequence list;

[0126] c72) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of c71);

[0127] c73) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in c71) or c72);

[0128] c74) a single-stranded DNA that hybridizes with the single-stranded DNA defined in c71) or c72) under stringent conditions;

[0129] The SARS-CoV-2-08F is any single-stranded DNA of the following c81) to c84):

[0130] c81) the single-stranded DNA corresponding to positions 8 to 28 of SEQ ID No. 25 in the sequence list;

[0131] c82) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of c81);

[0132] c83) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in c81) or c82);

[0133] c84) a single-stranded DNA that hybridizes with the single-stranded DNA defined in c81) or c82) under stringent conditions;

[0134] The SARS-CoV-2-09F is any single-stranded DNA of the following c91) to c94):

[0135] c91) the single-stranded DNA corresponding to positions 8-37 of SEQ ID No.26 in the sequence list;

[0136] c92) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of c91);

[0137] c93) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in c91) or c92);

[0138] c94) a single-stranded DNA that hybridizes with the single-stranded DNA defined in c91) or c92) under stringent conditions;

[0139] The SARS-CoV-2-10F is any single-stranded DNA of the following c101) to c104):

[0140] c101) the single-stranded DNA corresponding to positions 8-35 of SEQ ID No.27 in the sequence list;

[0141] c102) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of c101);

[0142] c103) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in c101) or c102);

[0143] c104) a single-stranded DNA that hybridizes with the single-stranded DNA defined in c101) or c102) under stringent conditions;

[0144] The SARS-CoV-2-11F is any single-stranded DNA of the following c111) to c114):

[0145] c111) the single-stranded DNA corresponding to positions 8-36 of SEQ ID No.28 in the sequence list;

[0146] c112) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of c111);

[0147] c113) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in c111) or c112);

[0148] c114) a single-stranded DNA that hybridizes with the single-stranded DNA defined in c111) or c112) under stringent conditions;

[0149] The SARS-CoV-2-12F is any single-stranded DNA of the following c121) to c124):

[0150] c121) the single-stranded DNA corresponding to positions 8-36 of SEQ ID No.29 in the sequence list;

[0151] c122) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of c121);

[0152] c123) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in c121) or c122);

[0153] c124) a single-stranded DNA that hybridizes with the single-stranded DNA defined in c121) or c122) under stringent conditions;

[0154] The SARS-CoV-2-13F is any single-stranded DNA of the following c131) to c134):

[0155] c131) the single-stranded DNA corresponding to positions 8-27 of SEQ ID No.30 in the sequence list;

[0156] c132) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of c131);

[0157] c133) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in c131) or c132);

[0158] c134) a single-stranded DNA that hybridizes with the single-stranded DNA defined in c131) or c132) under stringent conditions;

[0159] The SARS-CoV-2-14F is any single-stranded DNA of the following c141) to c144):

[0160] c141) the single-stranded DNA corresponding to positions 8-39 of SEQ ID No.31 in the sequence list;

[0161] c142) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of c141);

[0162] c143) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in c141) or c142);

[0163] c144) a single-stranded DNA that hybridizes with the single-stranded DNA defined in c141) or c142) under stringent conditions;

[0164] The SARS-CoV-2-15F is any single-stranded DNA of the following c151) to c154):

[0165] c151) the single-stranded DNA corresponding to positions 8-40 of SEQ ID No.32 in the sequence list;

[0166] c152) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of c151);

[0167] c153) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in c151) or c152);

[0168] c154) a single-stranded DNA that hybridizes with the single-stranded DNA defined in c151) or c152) under stringent conditions;

[0169] The SARS-CoV-2-16F is any single-stranded DNA of the following c161) to c164):

[0170] c161) the single-stranded DNA corresponding to positions 8-29 of SEQ ID No.33 in the sequence list;

[0171] c162) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of c161);

[0172] c163) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in c161) or c162);

[0173] c164) a single-stranded DNA that hybridizes with the single-stranded DNA defined in c161) or c162) under stringent conditions;

[0174] The SARS-CoV-2-17F is any single-stranded DNA of the following c171) to c174):

[0175] c171) the single-stranded DNA corresponding to positions 8-28 of SEQ ID No.34 in the sequence list;

[0176] c172) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of c171);

[0177] c173) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in c171) or c172);

[0178] c174) a single-stranded DNA that hybridizes with the single-stranded DNA defined in c171) or c172) under stringent conditions;

[0179] The SARS-CoV-2-18F is any single-stranded DNA of the following c181) to c184):

[0180] c181) the single-stranded DNA corresponding to positions 8-26 of SEQ ID No.35 in the sequence list;

[0181] c182) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of c181);

[0182] c183) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in c181) or c182);

[0183] c184) a single-stranded DNA that hybridizes with the single-stranded DNA defined in c181) or c182) under stringent conditions;

[0184] The SARS-CoV-2-19F is any single-stranded DNA of the following c191) to c194):

[0185] c191) the single-stranded DNA corresponding to positions 8 to 34 of SEQ ID No. 36 in the sequence list;

[0186] c192) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of c191);

[0187] c193) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in c191) or c192);

[0188] c194) a single-stranded DNA that hybridizes with the single-stranded DNA defined in c191) or c192) under stringent conditions;

[0189] The SARS-CoV-2-20F is any single-stranded DNA of the following c201) to c204):

[0190] c201) the single-stranded DNA corresponding to positions 8 to 36 of SEQ ID No. 37 in the sequence list;

[0191] c202) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of c201);

[0192] c203) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in c201) or c202);

[0193] c204) a single-stranded DNA that hybridizes with the single-stranded DNA defined in c201) or c202) under stringent conditions;

[0194] The influenza A-01R is any single-stranded DNA of the following d11) to d14):

[0195] d11) the single-stranded DNA corresponding to positions 8 to 25 of SEQ ID No. 40 in the sequence listing;

[0196] d12) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of d11);

[0197] d13) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in d11) or d12);

[0198] d14) a single-stranded DNA that hybridizes with the single-stranded DNA defined in d11) or d12) under stringent conditions;

[0199] The influenza A-02R is any single-stranded DNA of the following d21) to d24):

[0200] d21) the single-stranded DNA corresponding to positions 8 to 24 of SEQ ID No. 41 in the sequence listing;

[0201] d22) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of d21);

[0202] d23) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in d21) or d22);

[0203] d24) a single-stranded DNA that hybridizes with the single-stranded DNA defined in d21) or d22) under stringent conditions;

[0204] The influenza A-03R is any single-stranded DNA of the following d31) to d34):

[0205] d31) the single-stranded DNA corresponding to positions 8-25 of SEQ ID No.42 in the sequence listing;

[0206] d32) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of d31);

[0207] d33) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in d31) or d32);

[0208] d34) a single-stranded DNA that hybridizes with the single-stranded DNA defined in d31) or d32) under stringent conditions;

[0209] The influenza A-04R is any single-stranded DNA of the following d41) to d44):

[0210] d41) the single-stranded DNA corresponding to positions 8-24 of SEQ ID No.43 in the sequence listing;

[0211] d42) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of d41);

[0212] d43) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in d41) or d42);

[0213] d44) a single-stranded DNA that hybridizes with the single-stranded DNA defined in d41) or d42) under stringent conditions;

[0214] The influenza A-05R is any single-stranded DNA of the following d51) to d54):

[0215] d51) the single-stranded DNA corresponding to positions 8-25 of SEQ ID No.44 in the sequence listing;

[0216] d52) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of d51);

[0217] d53) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in d51) or d52);

[0218] d54) a single-stranded DNA that hybridizes with the single-stranded DNA defined in d51) or d52) under stringent conditions;

[0219] The influenza A-06R is any one of the following single-stranded DNAs d61) to d64):

[0220] d61) the single-stranded DNA corresponding to positions 8-24 of SEQ ID No.45 in the sequence listing;

[0221] d62) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of d61);

[0222] d63) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in d61) or d62);

[0223] d64) a single-stranded DNA that hybridizes with the single-stranded DNA defined in d61) or d62) under stringent conditions;

[0224] The influenza A-07R is any one of the following single-stranded DNAs d71) to d74):

[0225] d71) the single-stranded DNA corresponding to positions 8-25 of SEQ ID No.46 in the sequence listing;

[0226] d72) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of d71);

[0227] d73) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in d71) or d72);

[0228] d74) a single-stranded DNA that hybridizes with the single-stranded DNA defined in d71) or d72) under stringent conditions;

[0229] The influenza A-08R is any single-stranded DNA of the following d81) to d84):

[0230] d81) the single-stranded DNA corresponding to positions 8-25 of SEQ ID No.47 in the sequence listing;

[0231] d82) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of d81);

[0232] d83) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in d81) or d82);

[0233] d84) a single-stranded DNA that hybridizes with the single-stranded DNA defined in d81) or d82) under stringent conditions;

[0234] The influenza A-09R is any single-stranded DNA of the following d91) to d94):

[0235] d91) the single-stranded DNA corresponding to positions 8-25 of SEQ ID No.48 in the sequence listing;

[0236] d92) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of d91);

[0237] d93) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in d91) or d92);

[0238] d94) a single-stranded DNA that hybridizes with the single-stranded DNA defined in d91) or d92) under stringent conditions;

[0239] The influenza B-01R is any single-stranded DNA of the following e11) to e14):

[0240] e11) the single-stranded DNA corresponding to positions 8 to 25 of SEQ ID No. 49 in the sequence list;

[0241] e12) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of e11);

[0242] e13) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in e11) or e12);

[0243] e14) a single-stranded DNA that hybridizes with the single-stranded DNA defined in e11) or e12) under stringent conditions;

[0244] The influenza B-02R is any single-stranded DNA of the following e21) to e24):

[0245] e21) the single-stranded DNA corresponding to positions 8 to 25 of SEQ ID No. 50 in the sequence list;

[0246] e22) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of e21);

[0247] e23) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in e21) or e22);

[0248] e24) a single-stranded DNA that hybridizes with the single-stranded DNA defined in e21) or e22) under stringent conditions;

[0249] The influenza B-03R is any single-stranded DNA of the following e31) to e34):

[0250] e31) the single-stranded DNA corresponding to positions 8-25 of SEQ ID No.51 in the sequence list;

[0251] e32) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of e31);

[0252] e33) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in e31) or e32);

[0253] e34) a single-stranded DNA that hybridizes with the single-stranded DNA defined in e31) or e32) under stringent conditions;

[0254] The influenza B-04R is any single-stranded DNA of the following e41) to e44):

[0255] e41) the single-stranded DNA corresponding to positions 8-25 of SEQ ID No.52 in the sequence list;

[0256] e42) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of e41);

[0257] e43) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in e41) or e42);

[0258] e44) a single-stranded DNA that hybridizes with the single-stranded DNA defined in e41) or e42) under stringent conditions;

[0259] The influenza B-05R is any single-stranded DNA of the following e51) to e54):

[0260] e51) the single-stranded DNA corresponding to positions 8-25 of SEQ ID No.53 in the sequence list;

[0261] e52) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of e51);

[0262] e53) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in e51) or e52);

[0263] e54) a single-stranded DNA that hybridizes with the single-stranded DNA defined in e51) or e52) under stringent conditions;

[0264] The influenza B-06R is any single-stranded DNA of the following e61) to e64):

[0265] e61) the single-stranded DNA corresponding to positions 8-25 of SEQ ID No.54 in the sequence list;

[0266] e62) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of e61);

[0267] e63) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in e61) or e62);

[0268] e64) a single-stranded DNA that hybridizes with the single-stranded DNA defined in e61) or e62) under stringent conditions;

[0269] The influenza B-07R is any single-stranded DNA of the following e71) to e74):

[0270] e71) the single-stranded DNA corresponding to positions 8-25 of SEQ ID No.55 in the sequence list;

[0271] e72) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of e71);

[0272] e73) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in e71) or e72);

[0273] e74) a single-stranded DNA that hybridizes with the single-stranded DNA defined in e71) or e72) under stringent conditions;

[0274] The influenza B-08R is any single-stranded DNA of the following e81) to e84):

[0275] e81) the single-stranded DNA corresponding to positions 8-25 of SEQ ID No.56 in the sequence list;

[0276] e82) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of e81);

[0277] e83) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in e81) or e82);

[0278] e84) a single-stranded DNA that hybridizes with the single-stranded DNA defined in e81) or e82) under stringent conditions;

[0279] The C-SARS-CoV-2-01R is any single-stranded DNA of the following f11) to f14):

[0280] f11) the single-stranded DNA corresponding to positions 8 to 35 of SEQ ID No. 57 in the sequence list;

[0281] f12) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of f11);

[0282] f13) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in f11) or f12);

[0283] f14) a single-stranded DNA that hybridizes with the single-stranded DNA defined in f11) or f12) under stringent conditions;

[0284] The C-SARS-CoV-2-02R is any single-stranded DNA of the following f21) to f24):

[0285] f21) the single-stranded DNA corresponding to positions 8 to 35 of SEQ ID No. 58 in the sequence list;

[0286] f22) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of f21);

[0287] f23) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in f21) or f22);

[0288] f24) a single-stranded DNA that hybridizes with the single-stranded DNA defined in f21) or f22) under stringent conditions;

[0289] The C-SARS-CoV-2-03R is any single-stranded DNA of the following f31) to f34):

[0290] f31) the single-stranded DNA corresponding to positions 8 to 40 of SEQ ID No. 59 in the sequence list;

[0291] f32) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of f31);

[0292] f33) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in f31) or f32);

[0293] f34) a single-stranded DNA that hybridizes with the single-stranded DNA defined in f31) or f32) under stringent conditions;

[0294] The C-SARS-CoV-2-04R is any single-stranded DNA of the following f41) to f44):

[0295] f41) the single-stranded DNA corresponding to positions 8-39 of SEQ ID No.60 in the sequence list;

[0296] f42) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of f41);

[0297] f43) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in f41) or f42);

[0298] f44) a single-stranded DNA that hybridizes with the single-stranded DNA defined in f41) or f42) under stringent conditions;

[0299] The C-SARS-CoV-2-05R is any single-stranded DNA of the following f51) to f54):

[0300] f51) the single-stranded DNA corresponding to positions 8-42 of SEQ ID No.61 in the sequence list;

[0301] f52) a single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of f51);

[0302] f53) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in f51) or f52);

[0303] f54) a single-stranded DNA that hybridizes with the single-stranded DNA defined in f51) or f52) under stringent conditions;

[0304] The C-SARS-CoV-2-06R is any single-stranded DNA of the following f61) to f64):

[0305] f61) the single-stranded DNA corresponding to positions 8-33 of SEQ ID No.62 in the sequence list;

[0306] f62) A single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of f61);

[0307] f63) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in f61) or f62);

[0308] f64) a single-stranded DNA that hybridizes with the single-stranded DNA defined in f61) or f62) under stringent conditions;

[0309] The C-SARS-CoV-2-07R is any single-stranded DNA of the following f71) to f74):

[0310] f71) the single-stranded DNA corresponding to positions 8-35 of SEQ ID No.63 in the sequence list;

[0311] f72) A single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of f71);

[0312] f73) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in f71) or f72);

[0313] f74) a single-stranded DNA that hybridizes with the single-stranded DNA defined in f71) or f72) under stringent conditions;

[0314] The C-SARS-CoV-2-08R is any single-stranded DNA as follows f81) to f84):

[0315] f81) the single-stranded DNA corresponding to positions 8-38 of SEQ ID No.64 in the sequence list;

[0316] f82) A single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of f81);

[0317] f83) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in f81) or f82);

[0318] f84) ​​a single-stranded DNA that hybridizes with the single-stranded DNA defined in f81) or f82) under stringent conditions;

[0319] The C-SARS-CoV-2-09R is any single-stranded DNA of the following f91) to f94):

[0320] f91) the single-stranded DNA corresponding to positions 8-42 of SEQ ID No.65 in the sequence list;

[0321] f92) A single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of f91);

[0322] f93) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in f91) or f92);

[0323] f94) a single-stranded DNA that hybridizes with the single-stranded DNA defined in f91) or f92) under stringent conditions;

[0324] The C-SARS-CoV-2-10R is any single-stranded DNA of the following f101) to f104):

[0325] f101) the single-stranded DNA corresponding to positions 8-40 of SEQ ID No.66 in the sequence list;

[0326] f102) A single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of f101);

[0327] f103) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in f101) or f102);

[0328] f104) a single-stranded DNA that hybridizes with the single-stranded DNA defined in f101) or f102) under stringent conditions;

[0329] The C-SARS-CoV-2-11R is any single-stranded DNA of the following f111) to f114):

[0330] f111) the single-stranded DNA corresponding to positions 8-41 of SEQ ID No.67 in the sequence list;

[0331] f112) A single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of f111);

[0332] f113) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in f111) or f112);

[0333] f114) a single-stranded DNA that hybridizes with the single-stranded DNA defined in f111) or f112) under stringent conditions;

[0334] The C-SARS-CoV-2-12R is any single-stranded DNA of the following f121) to f124):

[0335] f121) the single-stranded DNA corresponding to positions 8-35 of SEQ ID No.68 in the sequence list;

[0336] f122) A single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of f121);

[0337] f123) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in f121) or f122);

[0338] f124) a single-stranded DNA that hybridizes with the single-stranded DNA defined in f121) or f122) under stringent conditions;

[0339] The C-SARS-CoV-2-13R is any single-stranded DNA of the following f131) to f134):

[0340] f131) the single-stranded DNA corresponding to positions 8-34 of SEQ ID No.69 in the sequence list;

[0341] f132) A single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of f131);

[0342] f133) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in f131) or f132);

[0343] f134) a single-stranded DNA that hybridizes with the single-stranded DNA defined in f131) or f132) under stringent conditions;

[0344] The C-SARS-CoV-2-14R is any single-stranded DNA of the following f141) to f144):

[0345] f141) the single-stranded DNA corresponding to positions 8-41 of SEQ ID No.70 in the sequence list;

[0346] f142) A single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of f141);

[0347] f143) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in f141) or f142);

[0348] f144) a single-stranded DNA that hybridizes with the single-stranded DNA defined in f141) or f142) under stringent conditions;

[0349] The C-SARS-CoV-2-15R is any single-stranded DNA of the following f151) to f154):

[0350] f151) the single-stranded DNA corresponding to positions 8-40 of SEQ ID No.71 in the sequence list;

[0351] f152) A single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of f151);

[0352] f153) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in f151) or f152);

[0353] f154) a single-stranded DNA that hybridizes with the single-stranded DNA defined in f151) or f152) under stringent conditions;

[0354] The C-SARS-CoV-2-16R is any single-stranded DNA of the following f161) to f164):

[0355] f161) the single-stranded DNA corresponding to positions 8-38 of SEQ ID No.72 in the sequence list;

[0356] f162) A single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of f161);

[0357] f163) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in f161) or f162);

[0358] f164) a single-stranded DNA that hybridizes with the single-stranded DNA defined in f161) or f162) under stringent conditions;

[0359] The C-SARS-CoV-2-17R is any single-stranded DNA as follows f171) to f174):

[0360] f171) the single-stranded DNA corresponding to positions 8-31 of SEQ ID No.73 in the sequence list;

[0361] f172) A single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of f171);

[0362] f173) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in f171) or f172);

[0363] f174) a single-stranded DNA that hybridizes with the single-stranded DNA defined in f171) or f172) under stringent conditions;

[0364] The C-SARS-CoV-2-18R is any single-stranded DNA as follows f181) to f184):

[0365] f181) the single-stranded DNA corresponding to positions 8-31 of SEQ ID No.74 in the sequence list;

[0366] f182) A single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of f181);

[0367] f183) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in f181) or f182);

[0368] f184) a single-stranded DNA that hybridizes with the single-stranded DNA defined in f181) or f182) under stringent conditions;

[0369] The C-SARS-CoV-2-19R is any single-stranded DNA of the following f191) to f194):

[0370] f191) the single-stranded DNA corresponding to positions 8-36 of SEQ ID No.75 in the sequence list;

[0371] f192) A single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of f191);

[0372] f193) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in f191) or f192);

[0373] f194) a single-stranded DNA that hybridizes with the single-stranded DNA defined in f191) or f192) under stringent conditions;

[0374] The C-SARS-CoV-2-20R is any single-stranded DNA of the following f201) to f204):

[0375] f201) the single-stranded DNA corresponding to positions 8-26 of SEQ ID No.76 in the sequence list;

[0376] f202) A single-stranded DNA obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of f201);

[0377] f203) a single-stranded DNA having 70% or more identity with the single-stranded DNA defined in f201) or f202);

[0378] f204) A single-stranded DNA that hybridizes with the single-stranded DNA defined in f201) or f202) under stringent conditions.

[0379] In the above primer set, the single-stranded DNAs a12), a22), a32), a42), a52), a62), a72), a82), and a92) are the single-stranded DNAs corresponding to SEQ ID No. 1-9 in the sequence table respectively;

[0380] b12), b22), b32), b42), b52), b62), b72), and b82) are the single-stranded DNAs corresponding to SEQ ID No. 10-17 in the sequence list;

[0381] c12), c22), c32), c42), c52), c62), c72), c82), c92), c102), c112), c122), c132), c142), c152), c162), c172), c182), c192), and c202) are the single-stranded DNAs corresponding to SEQ ID No.18-37 in the sequence list;

[0382] The single-stranded DNAs described in d12), d22), d32), d42), d52), d62), d72), d82), and d92) are the single-stranded DNAs corresponding to SEQ ID No.40-48 in the sequence list;

[0383] e12), e22), e32), e42), e52), e62), e72), and e82) are the single-stranded DNAs corresponding to SEQ ID No. 49-56 in the sequence list;

[0384] The single-stranded DNAs described in the invention are respectively the single-stranded DNAs corresponding to SEQ ID No. 57-76 in the sequence list.

[0385] Influenza A-01F, Influenza A-02F, Influenza A-03F, Influenza A-04F, Influenza A-05F, Influenza A-06F、influenza A-07F、influenza A-08F、influenza A-09F、influenza B-01F、influenza B-02F、influenzaB-03F、influenza B-04F、influenza B-05F、influenza B-06F、flu B-07F、flu B-08F, SARS-CoV-2-01F, SARS-CoV-2-02F, SARS-CoV-2-03F, SARS-CoV-2-04F, SARS-CoV-2- 05F, SARS-CoV-2-06F, SARS-CoV-2-07F, SARS-CoV-2-08F, SARS-CoV-2-09F, SARS-CoV-2-1 F, SARS-CoV-2-11F, SARS-CoV-2-12F, SARS-CoV-2-13F, SARS-CoV-2-14F, SARS-CoV-2-15F, SARS-CoV-2-16F, SARS-CoV-2-17F, SARS-CoV-2-18F, SARS-CoV-2-19F, SARS-CoV-2-20F,The reverse primer may only consist of influenza A-01R, influenza A-02R, influenza A-03R, influenza A-04R, influenza A-05R, influenza A-06R, influenza A-07R, influenza A-08R, influenza A-09R, influenza B-01R, influenza B-02R, influenzaB-03R, influenza B-04R, influenza B-05R, influenza B-06R, influenza B-07R, influenza B-08R, C-SARS-CoV-2-01R, C-SARS-CoV-2-02R, C-SARS-CoV-2-03R, C-SARS-CoV-2-04R, C-SARS-CoV-2- 05R, C-SARS-CoV-2-06R, C-SARS-CoV-2-07R, C-SARS-CoV-2-08R, C-SARS-CoV-2-09R, C-SARS-CoV-2-10 R, C-SARS-CoV-2-11R, C-SARS-CoV-2-12R, C-SARS-CoV-2-13R, C-SARS-CoV-2-14R, C-SARS-CoV-2-15R, Composed of C-SARS-CoV-2-16R, C-SARS-CoV-2-17R, C-SARS-CoV-2-18R, C-SARS-CoV-2-19R and C-SARS-CoV-2-20R. ,

[0386] The primer set may consist of only the forward primer and the reverse primer, or may consist of the forward primer, the reverse primer and a primer of an external reference gene (ie, an external reference primer).

[0387] In one embodiment of the present invention, the primer sequence of the external reference gene is SEQ ID No. 38, 39, 77 and / or 78 in the sequence listing.

[0388] In the above primer set, the forward primer, the reverse primer and the primer of the external reference gene may contain a tag sequence (such as GGGTGGG).

[0389] Each single-stranded DNA in the primer set can be packaged independently or together. The molar number of each single-stranded DNA in the primer set is equal.

[0390] The primer set may have any one or more of the following uses:

[0391] X1. Detect or assist in detecting influenza virus and / or novel coronavirus;

[0392] X2. Prepare products for detecting or assisting in detecting influenza viruses and / or novel coronaviruses;

[0393] X3. Screening or assisting in screening of patients infected with influenza virus and / or novel coronavirus;

[0394] X4. Prepare products for screening or assisting in screening of patients infected with influenza virus and / or novel coronavirus;

[0395] X5. Detect or assist in detecting whether the sample to be tested contains influenza virus and / or novel coronavirus;

[0396] X6. Prepare products for testing or assisting in testing whether the sample to be tested contains influenza virus and / or novel coronavirus;

[0397] X7. Quantitative detection or auxiliary detection of influenza virus and / or novel coronavirus content in the sample to be tested;

[0398] X8. Prepare products for quantitative detection or auxiliary detection of influenza virus and / or novel coronavirus content in samples to be tested;

[0399] X9. Detect or assist in detecting influenza virus and / or novel coronavirus mutations;

[0400] X10. Prepare products for detecting or assisting in detecting influenza virus and / or novel coronavirus mutations;

[0401] X11, influenza virus and / or novel coronavirus genome library construction;

[0402] X12. Prepare influenza virus and / or novel coronavirus genome library products.

[0403] In the above primer set, the quantification may be relative quantification (eg, relative to the content of an external reference gene).

[0404] The present invention also provides a kit, which contains the primer set.

[0405] The kit may also contain other reagents required for library construction (such as reagents required for MGI or NGS library construction) and other reagents required for high-throughput sequencing (such as reagents required for sequencing on MGISEQ-2000 or MGI or Illumina or Proton platforms).

[0406] The kit may have any one or more of the following uses:

[0407] X1. Detect or assist in detecting influenza virus and / or novel coronavirus;

[0408] X2. Prepare products for detecting or assisting in detecting influenza viruses and / or novel coronaviruses;

[0409] X3. Screening or assisting in screening of patients infected with influenza virus and / or novel coronavirus;

[0410] X4. Prepare products for screening or assisting in screening of patients infected with influenza virus and / or novel coronavirus;

[0411] X5. Detect or assist in detecting whether the sample to be tested contains influenza virus and / or novel coronavirus;

[0412] X6. Prepare products for testing or assisting in testing whether the sample to be tested contains influenza virus and / or novel coronavirus;

[0413] X7. Quantitative detection or auxiliary detection of influenza virus and / or novel coronavirus content in the sample to be tested;

[0414] X8. Prepare products for quantitative detection or auxiliary detection of influenza virus and / or novel coronavirus content in samples to be tested;

[0415] X9. Detect or assist in detecting influenza virus and / or novel coronavirus mutations;

[0416] X10. Prepare products for detecting or assisting in detecting influenza virus and / or novel coronavirus mutations;

[0417] X11, influenza virus and / or novel coronavirus genome library construction;

[0418] X12. Prepare influenza virus and / or novel coronavirus genome library products.

[0419] The primer set or any of the following applications of the kit also falls within the protection scope of the present invention:

[0420] X1. Detect or assist in detecting influenza virus and / or novel coronavirus;

[0421] X2. Prepare products for detecting or assisting in detecting influenza viruses and / or novel coronaviruses;

[0422] X3. Screening or assisting in screening of patients infected with influenza virus and / or novel coronavirus;

[0423] X4. Prepare products for screening or assisting in screening of patients infected with influenza virus and / or novel coronavirus;

[0424] X5. Detect or assist in detecting whether the sample to be tested contains influenza virus and / or novel coronavirus;

[0425] X6. Prepare products for testing or assisting in testing whether the sample to be tested contains influenza virus and / or novel coronavirus;

[0426] X7. Quantitative detection or auxiliary detection of influenza virus and / or novel coronavirus content in the sample to be tested;

[0427] X8. Prepare products for quantitative detection or auxiliary detection of influenza virus and / or novel coronavirus content in samples to be tested;

[0428] X9. Detect or assist in detecting influenza virus and / or novel coronavirus mutations;

[0429] X10. Prepare products for detecting or assisting in detecting influenza virus and / or novel coronavirus mutations;

[0430] X11, influenza virus and / or novel coronavirus genome library construction;

[0431] X12. Prepare influenza virus and / or novel coronavirus genome library products.

[0432] In the present invention, the influenza virus may be influenza A virus (such as any subtype of HA (1-16) or NA (1-9)) and / or influenza B virus (such as BV or BY subtype).

[0433] The primer set of the present invention is compatible with primers for multiple influenza types and covers specific primers for the entire genome, as well as the entire genome of the new coronavirus, and can obtain the full length and variation information of the virus; a 5-15bp identical tag sequence with a high Tm value is introduced at the 5' end of all primers, which, on the one hand, increases the Tm value of the influenza primer, allowing the influenza and new coronavirus primers to anneal at the same high temperature, thereby improving specificity; on the other hand, it inhibits the short-fragment advantage amplification of adjacent new coronavirus primers in the overlapping amplification region, thereby realizing one-tube long PCR amplification of a continuous region.

[0434] The primer set of the present invention is used to amplify the captured virus by multiple long PCR tubes, and a PCR free high-throughput sequencing library is constructed by one-step DNA shearing, end repair & adding dA and adding adapters for high-throughput sequencing analysis. On the one hand, the relative content of the virus can be obtained by adding a certain number of copies of external references, and on the other hand, the whole genome sequence data and mutation information of influenza and new coronavirus can be obtained at the same time.

[0435] The primer set of the present invention combines reverse transcription and multiple long PCR technology into one step in amplification, thereby reducing the risk of viral RNA degradation and reducing experimental steps; and combines DNA shearing and end repair & dA addition into one step in library construction, thereby further reducing operation steps and shortening library construction time. BRIEF DESCRIPTION OF THE DRAWINGS

[0436] Figure 1 The experimental flow chart is shown in Figure 2.

[0437] Figure 2This is a schematic diagram of the principle of Tag sequence inhibition of primer dimer amplification and short fragment amplification of the overlapping region of the new coronavirus. DETAILED DESCRIPTION

[0438] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0439] The experimental methods in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, instruments, etc. used in the following examples are all commercially available unless otherwise specified. The quantitative tests in the following examples were repeated three times, and the results were averaged.

[0440] Influenza A standard product GWA: Influenza A H1N1 RNA quality control product (Gene Gene, catalog number CA0959).

[0441] Fluorovirus B standard GWB: Fluorovirus B Victoria RNA quality control product (Gene Gene, catalog number CA0959).

[0442] Novel coronavirus standard GWS: COVID-19 RNA quality control product (high copy) -L (Jingliang Gene, product number CA0959).

[0443] Example 1. Preparation of primer sets for detecting influenza virus and novel coronavirus

[0444] This embodiment provides a primer set (denoted as an influenza novel coronavirus detection primer set) that can detect influenza A virus, influenza B virus, and novel coronavirus, which consists of a forward primer set and a reverse primer set, the forward primer set consists of 39 single-stranded DNAs shown in sequence 1-39 in the sequence table, and the reverse primer set consists of 39 single-stranded DNAs shown in sequence 40-78 in the sequence table. The molar number of each primer in the influenza novel coronavirus detection primer set is equal. The primer information of each primer set is shown in Table 1 and Table 2. In Tables 1 and 2, Influenza A is influenza A virus, Influenza B is influenza B virus, and SARS-CoV-2 is a novel coronavirus; lambda-1F, lambda-2F, lambda-1R, and lambda-2R are all external reference primers; in each primer sequence, GGGTGGG is a tag sequence, R represents A or G, Y represents C or T, S represents G or C, and M represents A or C.

[0445] Table 1. Forward primer set sequence information

[0446]

[0447]

[0448] Table 2. Reverse primer set sequence information

[0449]

[0450]

[0451] Example 2: Sample detection

[0452] Sample type 1: For single virus samples, the dry powders of influenza A standard GWA, influenza B standard GWB and novel coronavirus standard GWS were dissolved in 1 mL of nuclease-free water, and then diluted 10-fold in a gradient of 10 2 ,10 3 ,10 4 ,10 5 , and 10 6 After the amplification, a portion of the sample was used as the test sample, and another portion was used for multiple RT-PCR amplification, library construction and NGS sequencing. Water was used as a negative sample control.

[0453] Sample type 2: To simulate a mixed virus sample, dilute 10 3 The influenza A, influenza B and COVID-19 standards were mixed in equal volumes in pairs as the test samples for multiple RT-PCR amplification, library construction and NGS sequencing. Water was used as a negative sample control.

[0454] A certain amount of lambda DNA molecules ( Standard 2 in dsDNA HS AssayKit, Invitrogen, catalog number Q32851) was used as an external reference gene. The sample did not need to be quantified, and multiple RT-PCR amplification and NGS library construction were directly performed. The number of virus and external reference reads was obtained by sequencing analysis, and the relative content of the virus was calculated according to Formula 1. The whole genome data of the virus was obtained by information analysis and comparison, and mutation detection was performed.

[0455] The steps for QPCR verification of influenza A virus standard GWA using BGI's universal nucleic acid detection kit are as follows: prepare the nucleic acid reaction system and internal reference reaction system separately according to the following table:

[0456] Components volume Samples to be tested 10μl Nucleic acid reaction solution or internal reference reaction solution 19.7μl Reverse transcriptase 0.3μl

[0457] The obtained reaction system is reacted according to the following procedure to obtain the CT value:

[0458]

[0459] The steps for QPCR verification of influenza B virus nucleic acid detection kit of BGI on influenza B standard GWB are as follows: Prepare the reaction system according to the following table:

[0460] Components volume Samples to be tested 10μl Nucleic acid reaction solution 24.7μl Reverse transcriptase 0.3μl

[0461] The obtained reaction system is reacted according to the following procedure to obtain the CT value:

[0462]

[0463] The steps for QPCR validation of the novel coronavirus standard GWS using BGI's 2019-nCoV nucleic acid detection kit are as follows: Prepare the reaction system according to the following table:

[0464] Components volume Samples to be tested 10μl 2019-nCoV reaction fluid 18.5μl 2019-nCoV Detection Enzyme Solution 1.5μl

[0465] The obtained reaction system is reacted according to the following procedure to obtain the CT value:

[0466]

[0467] The principle of Tag sequence inhibition of primer dimer amplification and short fragment amplification in the overlapping region of COVID-19 is as follows Figure 2 The experimental process is as shown in Figure 1 As shown, the specific steps are as follows:

[0468] 1. Reverse transcription and long PCR amplification

[0469] One-step RT-PCR amplification capture was performed using MGI's Alpha Reverse Transcriptase (Cat. No. 1000014383), RNase inhibitor (Cat. No. 1000015146), and 2X PCR reaction mixture Multiplex PCR Readymix (Cat. No. 1000014386) or other reagents with equivalent functions.

[0470] Place a 0.2 ml PCR tube on an ice box and prepare the reaction system according to the following table:

[0471]

[0472]

[0473] The forward primer pool was obtained by diluting the forward primer set in Example 1 with nuclease-free water, wherein the concentration of each primer was 20 μM; the reverse primer pool was obtained by diluting the reverse primer set in Example 1 with nuclease-free water, wherein the concentration of each primer was 20 μM.

[0474] The obtained reaction system is reacted according to the following procedure:

[0475]

[0476] After the reaction, the multiple RT-PCR amplification products were obtained, and 40 μl of XP beads were added thereto for purification (Agencourt AMPure XP magnetic beads from Beckman Company, catalog number A63881). The purified products were dissolved in 30 μl of TE and the concentration was quantified to obtain the RT-PCR purified products.

[0477] 2. PCR free sequencing library construction

[0478] The above RT-PCR purified products were used for high-throughput library construction, and the library construction operation was performed using MGI's MGIEasy Fast PCR-FREE restriction enzyme library preparation reagent kit (catalog number 940-000019-00).

[0479] Place a 0.2 ml PCR tube on an ice box and prepare the reaction system according to the following table:

[0480] Components volume RT-PCR purified product (500ng) Xμl Fast FS Buffer 10μl Fast FS Enzyme 5μl Nuclease-free water 45-Xμl total 60μl

[0481] The obtained reaction system is reacted according to the following procedure:

[0482]

[0483]

[0484] After the reaction is completed, the fragmented product is obtained, and the obtained fragmented product is directly connected to the sequencing adapter primer. The connection adapter primer system is prepared according to the following table:

[0485] Components volume Previous step fragmentation product 45μl Adapter Primer 5μl Fast Ligation Buffer 23μl Ad Ligase 5μl Ligation Enhancer 2μl total 80μl

[0486] The sequencing adapter is a bubble-type adapter primer (Ad153) from MGI, which contains long-chain and short-chain sequences.

[0487] Long chain sequence: / 5Phos / AGTCGGAGGCCAAGCGGTCTTAGGAAGACAANNNNNNNNNNCAACTCCTTGGCTCACA-3′, where N is the tag sequence and the 5′ end of the primer is phosphorylated, which is used to connect the adapter primer to the double-stranded DNA with the A protruding at the 3′ end in the previous step.

[0488] Short chain sequence: / 5Phos / TTGTCTTCCTAAGGAACGACATGGCTACGATCCGACTT-3′, the 5′ end of the primer is phosphorylated for the next step of circularization.

[0489] For different sequencing platforms, adapters for different platforms can be added.

[0490] Set the reaction program: 25℃10min; 4℃hold, react;

[0491] The obtained product of the ligated adapter was purified by adding 20 μl of En-TE and 20 μl of En-beads, and the product was dissolved in 30 μl of TE to obtain a high-throughput sequencing library.

[0492] Sequencing on the machine:

[0493] The obtained high-throughput sequencing libraries were mixed with equal mass and sequenced in parallel. The sequencing platform was MGISEQ-2000 and the sequencing type was PE100.

[0494] 3. Data Analysis

[0495] The analysis steps include filtering low-quality sequences and adapter primer sequences, data alignment and other basic steps to obtain basic information.

[0496] The total number of reads of the influenza A virus genome, influenza B virus genome, novel coronavirus genome and external reference genome (lambda DNA) obtained by sequencing was counted, as shown in Tables 3 to 6. The sequences aligned to the virus were assembled into the whole genome, and the coverage and variation of the genes were obtained.

[0497] result:

[0498] 1. Virus detection and relative quantification

[0499]

[0500] Among them, Nv represents the number of viral reads, and Ne represents the number of external reference reads.

[0501] According to Formula 1, the content Pct% of the new coronavirus relative to the external reference can be obtained; as shown in Tables 1, 2, 3 and 4.

[0502] When it is a single virus sample, the five concentration gradients of influenza A virus (i.e., influenza A virus) gave virus contents Pct% relative to the external reference lambda of 99.41%, 96.72%, 84.43%, 25.85%, and 8.15%, respectively. As the dilution factor increased, the virus content Pct% gradually decreased, and in the negative sample water, it was 0.13%, and in the last 10 6 The CT value of the qPCR test was significantly different from that of the 8.15% dilution gradient sample, which ensures that the results obtained by this method are reliable. 5 and 10 6 The gradient samples had no CT value and no virus was detected, while the method of the invention detected a certain amount of virus, indicating that the method has a high sensitivity (as shown in Table 3); similarly, the five concentration gradients of influenza B (i.e., influenza B virus) obtained virus contents Pct% relative to the external reference lambda were 99.67%, 97.74%, 85.27%, 28.55%, and 6.76%, respectively, while 0.06% was obtained in the negative sample water, and the QPCR detection was diluted 10 5 and 10 6 The gradient samples had no CT value and no virus was detected, while the method of the present invention detected a certain amount of virus (as shown in Table 4); for the five gradient dilution samples of the new coronavirus, the Pct% of the virus relative to the external reference lambda was 79.09%, 66.43%, 46.28%, 6.37%, and 1.97%, respectively, while 0.03% was obtained in the negative sample water, and the dilution of 10 for QPCR detection 6 The gradient sample had no CT value and no virus was detected, but the method of the present invention detected a certain amount of virus content (as shown in Table 5). The above proves that the method of the present invention has high sensitivity and the results are credible.

[0503] When it is a mixed virus sample, 1. The Pct% of the virus content relative to the external reference lambda obtained from the mixed samples of influenza A and B viruses are 77.9% and 80.5%, respectively, while the new coronavirus is only 0.4%, which is significantly different. Therefore, it can be explained that the accuracy of the virus detection method and the results obtained are credible; 2. The Pct% of the virus content relative to the external reference lambda obtained from the mixed samples of influenza A and new coronavirus are 38.8% and 32.1%, respectively, while the influenza B virus is only 0.1%, which is significantly different; 3. The Pct% of the virus content relative to the external reference lambda obtained from the mixed samples of influenza B and new coronavirus are 51.7% and 41.7%, respectively, while the influenza B virus is only 0.1%, which is significantly different (as shown in Table 6).

[0504] 2. Virus genome coverage and variation

[0505] The five concentration gradient dilution samples of influenza A, influenza B and new coronavirus can all obtain certain virus sequence data. When the dilution factor is less than 10 4 When the virus 100x reads coverage is > 95%, the full-length sequence of the virus can be obtained;

[0506] When the sample was a mixed virus sample, all viruses were correctly detected, and the 100x reads coverage of the detected viruses was > 95%, and the full-length sequence of the virus could be obtained.

[0507] No mutations were detected in all gradient diluted influenza A, influenza B and COVID-19 sample libraries, and the sequence consistency with the standard was 100%.

[0508] Table 3. Results of influenza A virus testing

[0509]

[0510]

[0511] Table 4. Results of influenza B virus testing

[0512]

[0513] Table 5. Novel coronavirus test results

[0514]

[0515] Table 6. Mixed virus detection results

[0516]

[0517]

[0518] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims. <110> Shenzhen MGI Technology Co., Ltd. <120> Primer pool for co-amplification of influenza and novel coronavirus genomes and its library construction method <160> 78 <170> PatentIn version 3.5 <210> 1 <211> twenty three <212> DNA <213> Artificial sequence <400> 1 gggtgggagc raaagcaggt caa 23 <210> 2 <211> twenty four <212> DNA <213> Artificial sequence <400> 2 gggtgggagc raaagcaggc aaac 24 <210> 3 <211> 25 <212> DNA <213> Artificial sequence <400> 3 gggtggggagc raaagcaggt actga 25 <210> 4 <211> twenty one <212> DNA <213> Artificial sequence <400> 4 gggtgggagc raaagcaggg g 21 <210> 5 <211> twenty four <212> DNA <213> Artificial sequence <400> 5 gggtgggagc raaagcaggg taga 24 <210> 6 <211> twenty two <212> DNA <213> Artificial sequence <400> 6 gggtgggagc aaaagcagga gt 22 <210> 7 <211> twenty four <212> DNA <213> Artificial sequence <400> 7 gggtgggagc raaagcaggt agat 24 <210> 8 <211> twenty four <212> DNA <213> Artificial sequence <400> 8 gggtgggagc raaagcaggg tgac 24 <210> 9 <211> twenty two <212> DNA <213> Artificial sequence <400> 9 gggtgggagc gaaagcaggt gc 22 <210> 10 <211> twenty two <212> DNA <213> Artificial sequence <400> 10 gggtgggagc agaagcggag cc 22 <210> 11 <211> twenty two <212> DNA <213> Artificial sequence <400> 11 gggtgggagc agaagcggag cs 22 <210> 12 <211> twenty one <212> DNA <213> Artificial sequence <400> 12 gggtgggagc agaagcggtg c 21 <210> 13 <211> 27 <212> DNA <213> Artificial sequence <400> 13 gggtggggagc agaagcagag cattttc 27 <210> 14 <211> twenty one <212> DNA <213> Artificial sequence <400> 14 gggtgggagc agaagyacag c 21 <210> 15 <211> twenty one <212> DNA <213> Artificial sequence <400> 15 gggtgggagc agaagcagag c 21 <210> 16 <211> twenty one <212> DNA <213> Artificial sequence <400> 16 gggtgggagc agaagcacgc a 21 <210> 17 <211> 25 <212> DNA <213> Artificial sequence <400> 17 gggtggggagc agaagcagag sattt 25 <210> 18 <211> 30 <212> DNA <213> Artificial sequence <400> 18 gggtgggatt aaaggtttat accttcccag 30 <210> 19 <211> 29 <212> DNA <213> Artificial sequence <400> 19 gggtgggtcg taagggtggt cgcactatt 29 <210> 20 <211> 27 <212> DNA <213> Artificial sequence <400> 20 gggtgggtgt ggcagatgct gtcataa 27 <210> twenty one <211> 26 <212> DNA <213> Artificial sequence <400> twenty one gggtggggag gcaaagacag tgctta 26 <210> twenty two <211> 39 <212> DNA <213> Artificial sequence <400> twenty two gggtgggttt acttacaaag tcctcagaat acaaaggtc 39 <210> twenty three <211> 26 <212> DNA <213> Artificial sequence <400> twenty three gggtgggctt agttgcagag tggttt 26 <210> twenty four <211> 25 <212> DNA <213> Artificial sequence <400> twenty four gggtggggtt tgcctggcac gatat 25 <210> 25 <211> 28 <212> DNA <213> Artificial sequence <400> 25 gggtgggcca tctggtgttt accaatgt 28 <210> 26 <211> 37 <212> DNA <213> Artificial sequence <400> 26 gggtgggttc aaactcaaca ttaaattgtt gggtgtt 37 <210> 27 <211> 35 <212> DNA <213> Artificial sequence <400> 27 gggtgggcca catagatcat ccaaatccta aagga 35 <210> 28 <211> 36 <212> DNA <213> Artificial sequence <400> 28 gggtgggtat aatctaccaa caatgtgtga tatcag 36 <210> 29 <211> 36 <212> DNA <213> Artificial sequence <400> 29 gggtggggtc acagatgtga ctcaacttta cttagg 36 <210> 30 <211> 27 <212> DNA <213> Artificial sequence <400> 30 gggtggggct gtagcctcaa agatttt 27 <210> 31 <211> 39 <212> DNA <213> Artificial sequence <400> 31 gggtgggata tagattatgt accactaaag tctgctacg 39 <210> 32 <211> 40 <212> DNA <213> Artificial sequence <400> 32 gggtggggtc gcaaaatata ctcaactgtg tcaatattta 40 <210> 33 <211> 29 <212> DNA <213> Artificial sequence <400> 33 gggtggggaa ccattggtag atttgccaa 29 <210> 34 <211> 28 <212> DNA <213> Artificial sequence <400> 34 gggtgggtca actgaatgca gcaatctt 28 <210> 35 <211> 26 <212> DNA <213> Artificial sequence <400> 35 gggtgggtgc tgtatgacca gttgct 26 <210> 36 <211> 34 <212> DNA <213> Artificial sequence <400> 36 gggtgggcag aataaattgg atcaccggtg gaat 34 <210> 37 <211> 36 <212> DNA <213> Artificial sequence <400> 37 gggtggggtt cgttctatga agacttttta gagtat 36 <210> 38 <211> 26 <212> DNA <213> Artificial sequence <400> 38 gggtgggacg ctcaccatga agcgtt 26 <210> 39 <211> twenty four <212> DNA <213> Artificial sequence <400> 39 gggtgggatt cacaagcaat gcgt 24 <210> 40 <211> 25 <212> DNA <213> Artificial sequence <400> 40 gggtggggagt agaaacaagg tcgtt 25 <210> 41 <211> twenty four <212> DNA <213> Artificial sequence <400> 41 gggtgggagt agaaacaagg catt 24 <210> 42 <211> 25 <212> DNA <213> Artificial sequence <400> 42 gggtggggagt agaaacaagg trctt 25 <210> 43 <211> twenty four <212> DNA <213> Artificial sequence <400> 43 gggtgggagt agaaacaagg gtgt 24 <210> 44 <211> 25 <212> DNA <213> Artificial sequence <400> 44 gggtggggagt agaaacaagg gtatt 25 <210> 45 <211> twenty four <212> DNA <213> Artificial sequence <400> 45 gggtgggagt agaaacaagg agtt 24 <210> 46 <211> 25 <212> DNA <213> Artificial sequence <400> 46 gggtggggagt agaaacaagg tagtt 25 <210> 47 <211> 25 <212> DNA <213> Artificial sequence <400> 47 gggtggggagt agaaacaagg gtgtt 25 <210> 48 <211> 25 <212> DNA <213> Artificial sequence <400> 48 gggtggggagt agaaacaagg gtstt 25 <210> 49 <211> 25 <212> DNA <213> Artificial sequence <400> 49 gggtgggagt agaaacacga gcctt 25 <210> 50 <211> 25 <212> DNA <213> Artificial sequence <400> 50 gggtgggagt agaaacacga gcmtt 25 <210> 51 <211> 25 <212> DNA <213> Artificial sequence <400> 51 gggtggggagt agaaacacgt gcatt 25 <210> 52 <211> 25 <212> DNA <213> Artificial sequence <400> 52 gggtgggagt agtaacaaga gcatt 25 <210> 53 <211> 25 <212> DNA <213> Artificial sequence <400> 53 gggtgggagt agaaacaaca gcatt 25 <210> 54 <211> 25 <212> DNA <213> Artificial sequence <400> 54 gggtgggagt agtaacaaga gcatt 25 <210> 55 <211> 25 <212> DNA <213> Artificial sequence <400> 55 gggtggggagt agaaacaacg cactt 25 <210> 56 <211> 25 <212> DNA <213> Artificial sequence <400> 56 gggtgggagt agtaacaaga ggatt 25 <210> 57 <211> 35 <212> DNA <213> Artificial sequence <400> 57 gggtgggctt gttatggcaa ccaacataag agaac 35 <210> 58 <211> 35 <212> DNA <213> Artificial sequence <400> 58 gggtgggtgg tgtaagtaat tcagatactg gttgc 35 <210> 59 <211> 40 <212> DNA <213> Artificial sequence <400> 59 gggtggggat aatagatggt agaatgtaaa aggcactttt 40 <210> 60 <211> 39 <212> DNA <213> Artificial sequence <400> 60 gggtggggta actgttttct ttgtagaaaa catccgtaa 39 <210> 61 <211> 42 <212> DNA <213> Artificial sequence <400> 61 gggtgggagt acatagaaaa acctagtgaa aagaatat gc 42 <210> 62 <211> 33 <212> DNA <213> Artificial sequence <400> 62 gggtgggcaa aaagtcacca ttagttgtgc gta 33 <210> 63 <211> 35 <212> DNA <213> Artificial sequence <400> 63 gggtgggccc ttatagtga aattggggcct catag <210> 64 <211> 38 <212> DNA <213> Individual sequence(Artificial sequence) <400> 64 38. gggtgggatt ttagactgta cagtggctac tttgatac <210> 65 <211> 42 <212> DNA <213> Individual sequence(Artificial sequence) <400> 65 gggtgggttg taggtatttg tacatactta ccttttaagt approx <210> 66 <211> 40 <212> DNA <213> Individual sequence(Artificial sequence) <400> 66 gggtgggtac ttatcaacaa cttcaactac aaatagtagt <210> 67 <211> 41 <212> DNA <213> Individual sequence(Artificial sequence) <400> 67 gggtggggtg gtttatgtga tttacaataa tagctcatac c <210> 68 <211> 35 <212> DNA <213> Individual sequence(Artificial sequence) <400> 68 gggtgggatg aatcaacagt ttgagttggt agtcc <210> 69 <211> 34 <212> DNA <213> Artificial sequence <400> 69 gggtgggcac cacctaaatt gcaacgtgtt atac 34 <210> 70 <211> 41 <212> DNA <213> Artificial sequence <400> 70 gggtgggact ctcatattat agggtacagc taatgttaat g 41 <210> 71 <211> 40 <212> DNA <213> Artificial sequence <400> 71 gggtgggagt aaagtttgaa acctagtgat gttaatacct 40 <210> 72 <211> 38 <212> DNA <213> Artificial sequence <400> 72 gggtggggtt taattgtgta caaaaactgc catattgc 38 <210> 73 <211> 31 <212> DNA <213> Artificial sequence <400> 73 gggtggggaa caacagccct tgagacaact a 31 <210> 74 <211> 31 <212> DNA <213> Artificial sequence <400> 74 gggtgggcct acaagacaag ccattgcgat a 31 <210> 75 <211> 36 <212> DNA <213> Artificial sequence <400> 75 gggtggggat gaaatctaaa acaacacgaa cgtcat 36 <210> 76 <211> 26 <212> DNA <213> Artificial sequence <400> 76 gggtgggtgt cattctccta agaagc 26 <210> 77 <211> 27 <212> DNA <213> Artificial sequence <400> 77 gggtggggag cgttcataca caatggt 27 <210> 78 <211> 25 <212> DNA <213> Artificial sequence <400> 78 gggtgggaat gcagccatac cgaat 25

Claims

1. Primer set, including forward primer and reverse primer; The forward primers include names such as Influenza A-01F, Influenza A-02F, Influenza A-03F, Influenza A-04F, Influenza A-05F, Influenza A-06F, Influenza A-07F, InfluenzaA-08F, Influenza A-09F, Influenza B-01F, Influenza B-02F、influenza B-03F、influenza B-04F、influenza B-05F、influenza B-06F、influenza B-07F、influenza B-08F, SARS-CoV-2-01F, SARS-CoV-2-02F, SARS-CoV-2-03F, SARS-CoV-2-04F, SARS-CoV-2-05F, SARS-CoV-2-06F, SARS-CoV-2-07F, SARS-CoV-2-08F, SARS-CoV-2-09F, SARS-CoV-2-10 F, primers for SARS-CoV-2-11F, SARS-CoV-2-12F, SARS-CoV-2-13F, SARS-CoV-2-14F, SARS-CoV-2-15F, SARS-CoV-2-16F, SARS-CoV-2-17F, SARS-CoV-2-18F, SARS-CoV-2-19F, SARS-CoV-2-20F; The reverse primers include names of influenza A-01R, influenza A-02R, influenza A-03R, influenza A-04R, influenza A-05R, influenza A-06R, influenza A-07R, influenzaA-08R, influenza A-09R, influenza B-01R, influenza B-02R, influenza B-03R, influenza B-04R, influenza B-05R, influenza B-06R, influenza B-07R, influenza B-08R, C-SARS-CoV-2-01R, C-SARS-CoV-2-02R, C-SARS-CoV-2-03R, C-SARS-CoV-2-04R, C-SARS-CoV-2- 05R, C-SARS-CoV-2-06R, C-SARS-CoV-2-07R, C-SARS-CoV-2-08R, C-SARS-CoV-2-09R, C-SARS-CoV-2-10 R, C-SARS-CoV-2-11R, C-SARS-CoV-2-12R, C-SARS-CoV-2-13R, C-SARS-CoV-2-14R, C-SARS-CoV-2-15R, Primers for C-SARS-CoV-2-16R, C-SARS-CoV-2-17R, C-SARS-CoV-2-18R, C-SARS-CoV-2-19R and C-SARS-CoV-2-20R; The influenza A-01F to the influenza A-09F correspond to the single-stranded DNA shown in SEQ ID No. 1 to SEQ ID No. 9 in the sequence list, respectively; The influenza B-01F to the influenza B-08F correspond to the single-stranded DNA shown in SEQ ID No. 10 to SEQ ID No. 17 in the sequence list, respectively; The SARS-CoV-2-01F to the SARS-CoV-2-20F correspond to the single-stranded DNA shown in SEQ ID No.18 to SEQ ID No.37 in the sequence list, respectively; The influenza A-01R to the influenza A-09R correspond to the single-stranded DNA shown in SEQ ID No. 40 to SEQ ID No. 48 in the sequence list, respectively; The influenza B-01R to the influenza B-08R correspond to the single-stranded DNA shown in SEQ ID No. 49 to SEQ ID No. 56 in the sequence list, respectively; The C-SARS-CoV-2-01R to the C-SARS-CoV-2-20R correspond to the single-stranded DNA shown in SEQ ID No. 57 to SEQ ID No. 76 in the sequence list, respectively.

2. The primer set according to claim 1, characterized in that: The primer set further includes primers for external reference genes.

3. The primer set according to claim 2, characterized in that: The primer sequence of the external reference gene is SEQ ID No.38, SEQ ID No.39, SEQ ID No.77 or SEQ ID No.

78.

4. The primer set according to claim 1 or 2, characterized in that: The primer set has one or more of the following uses: X1. Detect or assist in detecting influenza virus and / or novel coronavirus; X2. Prepare products for detecting or assisting in detecting influenza viruses and / or novel coronaviruses; X3. Screening or assisting in screening of patients infected with influenza virus and / or novel coronavirus; X4. Prepare products for screening or assisting in screening of patients infected with influenza virus and / or novel coronavirus; X5. Detect or assist in detecting whether the sample to be tested contains influenza virus and / or novel coronavirus; X6. Prepare products for testing or assisting in testing whether the sample to be tested contains influenza virus and / or novel coronavirus; X7. Quantitative detection or auxiliary detection of influenza virus and / or novel coronavirus content in the sample to be tested; X8. Prepare products for quantitative detection or auxiliary detection of influenza virus and / or novel coronavirus content in samples to be tested; X9. Detect or assist in detecting influenza virus and / or novel coronavirus mutations; X10. Prepare products for detecting or assisting in detecting influenza virus and / or novel coronavirus mutations; X11, influenza virus and / or novel coronavirus genome library construction; X12. Preparation of influenza virus and / or novel coronavirus genome library products; The influenza virus is influenza A virus and / or influenza B virus.

5. A kit comprising the primer set according to claim 1 or 2.

6. The kit according to claim 5, characterized in that: The kit has one or more of the following uses: X1. Detect or assist in detecting influenza virus and / or novel coronavirus; X2. Prepare products for detecting or assisting in detecting influenza viruses and / or novel coronaviruses; X3. Screening or assisting in screening of patients infected with influenza virus and / or novel coronavirus; X4. Prepare products for screening or assisting in screening of patients infected with influenza virus and / or novel coronavirus; X5. Detect or assist in detecting whether the sample to be tested contains influenza virus and / or novel coronavirus; X6. Prepare products for testing or assisting in testing whether the sample to be tested contains influenza virus and / or novel coronavirus; X7. Quantitative detection or auxiliary detection of influenza virus and / or novel coronavirus content in the sample to be tested; X8. Prepare products for quantitative detection or auxiliary detection of influenza virus and / or novel coronavirus content in samples to be tested; X9. Detect or assist in detecting influenza virus and / or novel coronavirus mutations; X10. Prepare products for detecting or assisting in detecting influenza virus and / or novel coronavirus mutations; X11, influenza virus and / or novel coronavirus genome library construction; X12. Preparation of influenza virus and / or novel coronavirus genome library products; The influenza virus is influenza A virus and / or influenza B virus.

7. An application of the primer set according to claim 1 or 2 or the kit according to claim 5 for non-disease diagnosis and treatment purposes, comprising one or more of the following applications: X1. Prepare products for detecting or assisting in detecting influenza viruses and / or novel coronaviruses; X2. Prepare products for screening or assisting in screening of patients infected with influenza virus and / or novel coronavirus; X3. Prepare products for testing or assisting in testing whether the sample to be tested contains influenza virus and / or novel coronavirus; X4. Prepare products for quantitative detection or auxiliary detection of influenza virus and / or novel coronavirus content in samples to be tested; X5. Prepare products for detecting or assisting in detecting influenza virus and / or novel coronavirus mutations; X6. Construction of influenza virus and / or novel coronavirus genome libraries; X7. Prepare influenza virus and / or novel coronavirus genome library products; The influenza virus is influenza A virus and / or influenza B virus.

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