A multiplex RPA-CRISPR/Cas12a-based POCT technique and its application in human papillomavirus (HPV) typing assay

Table 1 Sequences of primers

	Primer	Sequences (5′–3′)	Length, nt
Forward	HF5-1-1*	AATAAACCTTATTGGTTACAACGTGCACAGGG	32
	HF5-1-2	AATAAACCTTATTGGTTACAACGAGCGCAGGG	32
	HF5-1-3*	AATAAACCTTATTGGTTACAACGTGCTCAGGG	32
	HF5-2-1	AATCAGCCATATTGGCTACATAAGGCACAGGG	32
	HF5-2-2*	AATCAGCCATATTGGCTACATAAGGCCCAGGG	32
	HF5-3-1	AATAAACCTTATTGGTTGCAACGTGCACAAGG	32
	HF5-3-2*	AATAAACCTTATTGGTTACAACGTGCACAAGG	32
	HF5-18Y*	ACCATATTGGTTACATAAGGCACAGGGTCA	30
Reverse	HR4*	TAAACTGTAAATCATATTCCTCACCATGTC	30
	HR4-1	TAAACTGTAATTCATACTCTTCACCATGCC	30
	HR4-2	TAAATTGTAAATCATACTCCTCCACGTGCC	30
	HR4-3	TAGACTGTAAATCATACTCTTCCCCATGCC	30
	HR4-18Q2*	AATAAACTGCAAATCATATTCCTCAACATG	30
	HR4-33Q2*	AACAAACTGTAGATCATATTCTTCAACATG	30

The above primers were designed with high specificity for various HPV subtypes based on the combination of HF5 and HR4 primers and their corresponding amplification regions. Sensitivity screening was conducted using plasmids of HPV types 16, 18, 31, 33, 35, and 45 as templates. Plasmids for the remaining four HPV types were prepared based on the NCBI HPV full genome sequences (HPV31 reference sequence number LR862053.1; HPV33 reference sequence number LR862077.1; HPV35 reference sequence number LR862022.1; HPV45 reference sequence number LR862061.1); * The primers comprising the final multiplex primer pool

ISSN: 1689-1392