RT-qPCR molecular detection and diagnosis

A kit for multiplex polymerase chain reaction (PCR) comprising a reagent mix comprising:

• (i) an oligonucleotide probe for detecting a N3 gene of a 2019-nCoV comprising a probe sequence consisting of the sequence of SEQ ID NO:
• 6, or a sequence having at least about 90% identity to the sequence of SEQ ID NO:
• 6;
• (ii) an oligonucleotide probe for detecting a N2 gene of the 2019-nCoV comprising a probe sequence consisting of the sequence of SEQ ID NO:
• 5, or a sequence having at least about 90% identity to the sequence of SEQ ID NO:
• 5;
• and
• (iii) an oligonucleotide probe for detecting a N1 gene of the 2019-nCoV comprising a probe sequence consisting of the sequence of SEQ ID NO:
• 4, or a sequence having at least about 90% identity to the sequence of SEQ ID NO:
• 4;
• wherein:
• each probe sequence is independently modified at its 5′ terminus with a fluorophore having an emission maximum of from about 500 nm to about 710 nm;
• each probe sequence is independently modified with the same single quencher which is a single compound whose structure is of the following structural formula:
• wherein:
• indicates point of attachment of the moiety to the probe sequence;
• and
• X is a linker;
• and
• the fluorophores are independently-detectable and selected from 6-carboxyfluorescein, 6-carboxy-X-rhodamine, Cy5, 4-(3-(ethoxycarbonyl)-6,8,8-trimethyl-2-oxo-7,8-dihydro-2H-pyrano[3,2-g]quinolin-9(6H)-yl)butanoic acid or 6-carboxy-2′,4,4′,5′,7,7′-hexachlorofluorescein;
• and instructions for using the reagent mix for multiplex PCR detection of 2019-nCoV RNA.