Method and kit for multiple detection of respiratory virus nucleic acids
Provided in the present invention are a method and a kit for multiple detection of respiratory virus nucleic acids, and in particular, disclosed are a method, a primer, a probe and a kit for detecting a plurality of influenza A viruses such as H1N1(2019), H3N2, H5N1, H1N1 and H7N9, influenza B viruses such as Yamagata and Victoria, 2019-nCoV OFR1ab and N genes, and human internal standard gene GAPDH on the basis of a real-time fluorescent quantitative PCR technical platform.
1 . A kit for multiplex detection of respiratory virus nucleic acid comprising a primer pair set and a probe set,
the primer pair set comprising:
a first primer pair group,
wherein the first primer pair group comprises a forward primer as shown in SEQ ID NO: 5 and a reverse primer as shown in SEQ ID NO: 6;
a second primer pair group,
wherein the second primer pair group comprises a forward primer as shown in SEQ ID NO: 7 and a reverse primer as shown in SEQ ID NO: 8;
a third primer pair group,
wherein the third primer pair group comprises a forward primer as shown in SEQ ID NO: 1 and a reverse primer as shown in SEQ ID NO: 2;
a fourth primer pair group,
wherein the fourth primer pair group comprises a forward primer as shown in SEQ ID NO: 3; and a reverse primer as shown in SEQ ID NO: 4; and
a fifth primer pair group,
wherein the fifth primer pair group comprises a forward primer as shown in SEQ ID NO: 9 and a reverse primer as shown in SEQ ID NO: 10, and
the probe set comprising probes respectively having polynucleotide sequences shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15,
wherein the SEQ ID NO: 11 nucleotide sequence is labeled with FAM at a 5′ end and MGB at a 3′ end, the SEQ ID NO: 12 nucleotide sequence is labeled with TexasRed at a 5′ end and MGB at a 3′ end, the SEQ ID NO: 13 nucleotide sequence is labeled with VIC at a 5′ end and BHQ2 at a 3′ end, the SEQ ID NO: 14 nucleotide sequence is labeled with TAMRA at a 5′ end and BHQ2 at a 3′ end, and the SEQ ID NO: 15 nucleotide sequence is labeled with CY5 at a 5′ end and MGB at a 3′ end.
2 . The kit of claim 1 , further comprising a first container, which comprises a primer-probe mixture,
wherein the primer-probe mixture contains-comprises multiple primers and multiple probes,
the primers respectively having the polynucleotide sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, and
the probes respectively having the polynucleotide sequences shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15.
3 . The kit of claim 2 , wherein the kit further comprises a second container comprising a hot-start enzyme, a reverse transcriptase, and dNTPs.
4 . The kit of claim 3 , wherein the kit further comprises a third container comprising a positive control.
5 . A multiplex PCR method for detecting influenza A virus (IAV), influenza B virus (IBV), and SARS-COV-2 nucleic acids comprising:
extracting a nucleic acid template from a subject test sample;
mixing the nucleic acid template with a PCR reaction system comprising the following components:
an IAV primer pair consisting of an upstream primer consisting of SEQ ID NO: 1 and a downstream primer consisting of SEQ ID NO: 2;
an IBV primer pair consisting of an upstream primer consisting of SEQ ID NO: 3 and a downstream primer consisting of SEQ ID NO: 4;
a 2019-nCOV OFR 1ab upstream primer pair consisting of SEQ ID NO: 5 and a downstream primer consisting of SEQ ID NO: 6;
a 2019-nCOV N upstream primer pair consisting of SEQ ID NO: 7 and a downstream primer SEQ ID NO: 8;
an internal standard GAPDH primer pair consisting of an upstream primer consisting of SEQ ID NO: 9 and a downstream primer consisting of SEQ ID NO: 10; and
labeled detection probes consisting of SEQ ID NOs: 11-15, wherein the SEQ ID NO: 11 is labeled with FAM at a 5′ end and MGB at a 3′ end; the SEQ ID NO: 12 is labeled with TexasRed at a 5′ end and MGB at a 3′ end; the SEQ ID NO: 13 is labeled with VIC at a 5′ end and BHQ2 at a 3′ end; the SEQ ID NO: 14 is labeled with TAMRA at a 5′ end and BHQ2 at a 3′ end; and the SEQ ID NO: 15 is labeled with CY5 at a 5′ end and MGB at a 3′ end;
performing real-time fluorescent PCR; and
detecting amplified DNA using different fluorescent channel curves.
6 . The method of claim 5 , wherein a reaction of the real-time fluorescent PCR comprises:
a first stage of holding at 50° C. for 15 min followed by holding at 95° C. for 15 min for 1 cycle; and
a second stage of holding at 94° C. for 15 s followed by holding at 55° C. for 45 s for 45 cycles.
7 . The kit of claim 4 , wherein the kit further comprises a fourth container comprising a negative control.