Development of a Novel Multiplex PCR Method for the Rapid Detection of SARS-CoV-2, Influenza A Virus, and Influenza B Virus

Table 1

Primers, probe-target genes, and sequence information.

Virus	Primer and probe	Sequence	Modification
SARS-CoV-2^a	CoV-ORF-F	CCC​TGT​GGG​TTT​TAC​ACT​TAA
	CoV-ORF-P	CCG​TCT​GCG​GTA​TGT​GGA​AAG​GTT​ATG​G	5′Texas RED, 3′BHQ2
	CoV-ORF-R	ACG​ATT​GTG​CAT​CAG​CTG​A
	CoV-NP-F	GGG​GAA​CTT​CTC​CTG​CTA​GAA​T
	CoV-NP-P	TTG​CTG​CTG​CTT​GAC​AGA​TT	5′FAM, 3′BHQ1
	CoV-NP-R	CAG​ACA​TTT​TGC​TCT​CAA​GCT​G
FluA^b	FluA-F	TAA​AGA​CAA​GAC​CAA​TCC​TGT​CAC​C
	FluA-P	ACG​CTC​ACC​GTG​CCC​AGT​GAG​CGA	5′CY5, 3′BHQ2
	FluA-R	TCC​CAT​TTA​GGG​CAT​TTT​GGA​CAA​AGC
FluB^c	FluB-F	AAA​GAT​GGC​CAT​CGG​ATC​CTC
	FluB-P	AAA​GCC​AAT​TCG​AGC​AGC​TGA​AAC​TG	5′CY5.5, 3′BHQ2
	FluB-R	GCT​CTT​GAC​CAA​ATT​GGG​AT
Psrp^d	Psrp-F	GTCCCTTCATCGTCGCTG
	Psrp-P	CAC​CGT​TGC​TGT​TTT​CCT​TAT​CGG​TTA​CGC	5′HEX, 3′BHQ1
	Psrp-R	GGC​GGT​TTG​TCA​AGC​TGA​T

^aThe primer sequences are from “technical guidelines for laboratory testing of novel coronavirus pneumonia” (https://www.chinacdc.cn/jkzt/crb/zl/szkb_11803/jszl_11815/202003/W020200309540843062947.pdf). ^bThe primer sequences have been modified from “National Technical Guidelines for Influenza Surveillance (2017 edition),” (https://ivdc.chinacdc.cn/cnic/zyzx/jcfa/201709/P020170930331067634607.pdf). ^cThe primer sequences have been modified from “Mokkapati, Anupama., Brown, Bradley., Jones, Robert. 2019, methods of detecting influenza, United States, CEPHEID (Sunnyvale, CA, US), 10480036,” (https://www.freepatentsonline.com/10480036.html). ^dThe primer sequences have been designed based on Arabidopsis genome sequences.