RT-PCR detection reagent for detecting novel coronavirus, kit and detection method thereof

1 . A (RT)-PCR detection reagent system for detecting a coronavirus, comprising:

a first plurality of primer sequences of a SARS-CoV-2 ORF1lab gene comprising:

a first upstream primer comprising the nucleotide sequence of SEQ ID NO. 1; and

a first downstream primer comprising the nucleotide sequence of SEQ ID NO. 2; and

a second plurality of primer sequences of a SARS-CoV-2 N gene fragment comprising:

a second upstream primer comprising the nucleotide sequence of SEQ ID NO. 3; and

a second downstream primer comprising the nucleotide sequence of SEQ ID NO. 4;

a third plurality of primer sequences of an influenza A (IFV-A) gene comprising:

a third upstream primer comprising the nucleotide sequence of SEQ ID NO. 5; and

a third downstream primer comprising the nucleotide sequence of SEQ ID NO. 6; and

a fourth plurality of primer sequences of an influenza B (IFV-B) gene comprising:

a fourth upstream primer comprising the nucleotide sequence of SEQ ID NO. 7; and

a fourth downstream primer comprising the nucleotide sequence of SEQ ID NO. 8;

a first fluorescent probe sequence of an influenza A (IFV-A) gene comprising the nucleotide sequence of SEQ ID NO. 9;

a second fluorescent probe sequence of an influenza B (IFV-B) gene comprising the nucleotide sequence of SEQ ID NO. 10;

a third fluorescent probe sequence of a SARS-CoV-2 ORF1lab gene comprising the nucleotide sequence of SEQ ID NO. 11;

a fourth fluorescent probe sequence of a SARS-CoV-2 N gene fragment comprising the nucleotide sequence of SEQ ID NO. 12; and

an internal reference gene comprising:

a fifth upstream primer comprising the nucleotide sequence of SEQ ID NO. 13; and

a fifth downstream primer comprising the nucleotide sequence of SEQ ID NO. 14.

2 . The (RT)-PCR detection reagent system according to claim 1 , further comprising:

a plurality of fluorescent probes comprising:

an IFV A-probe: FAM-SEQ ID NO. 16-BHQ1;

an IFV B-probe: HEX-SEQ ID NO. 17-BHQ1;

a first SAR-CoV-2-probe: ROX-SEQ ID NO. 18-BHQ2;

a second SAR-CoV-2 probe: ROX-SEQ ID NO. 19-BHQ; and

an IC-probe: Cy5-SEQ ID NO. 20-BHQ2.

3 . A multiple fluorescent direct (RT)-PCR detection method for detecting coronavirus in a patient using an (RT)-PCR detection reagent system consisting of upstream primers, downstream primers, and fluorescent probes, comprising:

obtaining a sample from the patient;

utilizing the detection regent system consisting of

primers comprising SEQ ID NOS: 1-8 and primers comprising SEQ ID NOS: 13 and 14,

a first IFV-A fluorescent probe comprising SEQ ID NO: 9,

a second IFV-B fluorescent probe comprising SEQ ID NO: 10,

a third SARS-CoV-2 ORF1lab gene fluorescent probe comprising SEQ ID NO: 11, and

a fourth SARS-CoV-2 N gene fluorescent probe comprising SEQ ID NO: 12;

for simultaneous amplification of a SARS-CoV-2 ORF1ab gene fragment, a SARS-CoV-2 N gene fragment, and the internal reference gene of the sample to obtain amplified products without performing a nucleic acid extraction;

subjecting the amplified products to a multiple fluorescence quantitative PCR detection method comprising an in vitro amplification of multiple fluorescent probes; and

detecting the presence of SARS-CoV-2 sequences in the patient sample.

4 . A rapid multiple fluorescent direct (RT)-PCR detection method for detecting coronavirus in a patient using the (RT)-PCR detection reagent system of claim 3 comprising:

obtaining a sample from the patient;

initiating the (RT)-PCR detection method in a container using the detection reagent system for simultaneously amplifying the SARS-CoV-2 ORF1ab gene fragment, the SARS-CoV-2 N gene fragment, and the internal reference gene of the sample to obtain amplified products without performing an RNA extraction;

subjecting the amplified products to a multiple fluorescence quantitative PCR detection method in the container using an in vitro amplification of multiple fluorescent probes comprising SEQ ID NOS. 9-12, and wherein the multiple fluorescent probes further comprise:

an IFV A-probe: FAM-SEQ ID NO. 16-BHQ1;

an IFV B-probe: HEX-SEQ ID NO. 17-BHQ1;

a first SAR-CoV-2-probe: ROX-SEQ ID NO. 18-BHQ2;

a second SAR-CoV-2 probe: ROX-SEQ ID NO. 19-BHQ;

an internal reference probe: Cy5-SEQ ID NO. 20-BHQ2; and

analyzing the results of the multiple fluorescence quantitative PCR detection method; and

determining if the sample includes SARS-CoV-2 sequences, wherein the determination regarding the presence of SARS-CoV-2 sequences is completed with 60 minutes of obtaining the sample from the patient.

5 . A rapid multiple fluorescent direct RT-PCR detection method according to claim 4 , wherein the analyzing operation is conducted on point of care testing (POCT) quantitative PCR (qPCR) equipment.