Single-tube collection and nucleic acid analysis of clinical samples for SARS-CoV-2 saliva testing


Human RNase P (RPP30) was amplified from the Hs_RPP30_Positive control plasmid (Integrated DNA Technologies, IDT, 10006626). SARS-CoV-2 N1 DNA was amplified from the 2019-nCoV_N_Positive control plasmid (IDT, 10006625). SARS-CoV-2 viral particles and synthetic RNA were provided by the XPRIZE Foundation (Team ID: 3650) and produced by ZeptoMetrix and Twist Biosciences, respectively. Viral particles were stored at 4 °C per manufacturer recommendation. Synthetic RNA was stored at -80 °C per manufacturer recommendation. 

RT-qPCR primer design

Human RNase P (RPP30) primers and probe were synthesized by IDT (oligonucleotides: 25 nmol, standard desalting; hydrolysis probe: 25 nmol, HPLC purified). SARS-CoV-2 N1 and N2 primers and probes were synthesized by IDT (oligonucleotides: 25 nmol, standard desalting; hydrolysis probe: 25 nmol, HPLC purified). Beta-actin (ACTB) primers and probe (IDT, Hs.PT.39a.22214847) were synthesized by IDT (oligonucleotides: 25 nmol, standard desalting; hydrolysis probe: 25 nmol, HPLC purified). Oligonucleotides and probes were resuspended in 10 mM Tris-HCl, pH 7.5 and 0.1 mM EDTA to 100 µM. RNase P and SARS-CoV-2 N1 primers and probes were designed for research-use only by the Centers for Disease Control and Prevention 1 . Primer and probe sequences are listed in Table S1. 

RT-LAMP primer design

N1 RT-LAMP primers were designed by Huang et al. for the SARS-CoV-2 N gene 1 . Oligos were synthesized by IDT (25 nmol, standard desalting). Oligomers were resuspended in 10 mM Tris-HCl, pH 7.5 and 0.1 mM EDTA to 100 µM. RT-LAMP sequences can be found in Table S1. 

Table 1 : RT-qPCR and RT-LAMP primers.

 1 Human RNase P and SARS-CoV-2 primers and probes were designed by the Centers for Disease Control and Prevention 1 .SARS-CoV-2 N2 primers and probe were designed for this study.Human beta-actin (ACTB) primers and probe were designed byIDT (Hs.PT.39a.22214847). SARS-CoV-2 N1 RT-LAMP primers were designed by Huang et al 2 . All primers were purchased and synthesized by IDT. 


	Test	Target	Name	Sequence
	RT-	Human RNase	RNase P	AGATTTGGACCTGCGAGCG
	qPCR	P 1 (RPP30;	Forward	
		NM_006413.5)		
			RNase P	GAGCGGCTGTCTCCACAAGT
			Reverse	
			RNase P	FAM-TTCTGACCTGAAGGCTCTGCGCG-BHQ1
			Probe	
	RT-	SARS-CoV-2	2019-	GACCCCAAAATCAGCGAAAT
	qPCR	N1 1 (N;	nCoV_N1	
		NC_045512.2)	Forward	
			2019-	TCTGGTTACTGCCAGTTGAATCTG
			nCoV_N1	
			Reverse	
			2019-	FAM-ACCCCGCATTACGTTTGGTGGACC-BHQ1
			nCoV_N1	
			Probe	
	RT-	SARS-CoV-2	N2 Forward	TTACAAACATTGGCCGCAA
	qPCR	N2		
			N2 Reverse	GTGACTTCCATGCCAATGC
			N2 Probe	SUN-ACAATTTGCCCCCAGCGCTTCAG-3IABkFQ
	RT-	Human beta-	ACTB	TAACAGATTGATGATGCATGAAATGGG
	qPCR	actin (ACTB;	Forward	
		NM_001101.5)		
			ACTB	CCCATGAGTGGCTCCTAAAGCAGCTGC
			Reverse	
			ACTB Probe (see IDT: Hs.PT.39a.22214847)
	RT-	SARS-CoV-2	F3	TGGACCCCAAAATCAGCG
	LAMP	N1 2		
			B3	GCCTTGTCCTCGAGGGAAT
			FIP	CCACTGCGTTCTCCATTCTGGTAAATGCACCCCGCATTACG
			BIP	CGCGATCAAAACAACGTCGGCCCTTGCCATGTTGAGTGAGA
			LF	TGAATCTGAGGGTCCACCAAA
			LB	GGTTTACCCAATAATACTGCGTCTT
	Supplementary		

Supplementary Information
Kyle H Cole, Alexis Bouin, Caila Ruiz, Bert L Semler, Matthew A Inlay, Andrej Lupták
Single-tube collection and nucleic acid analysis of clinical samples for SARS-CoV-2 saliva testing