Conservation and variability of hepatitis B core at different chronic hepatitis stages

Table 1 Primer design and polymerase chain reaction protocols for each amplified region

	PCR	Primer	Primer sequence (5'- > 3')	Amplified region	Protocol
1st step	PCR1	Forward	TAGGAGGCTGTAGGCATA	1774-2930	95 ºC 5 min; (95ºC 20 s, 49 ºC 20 s, 72 ºC 15 s) × 35 cycles; 72 ºC 3 min
		Reverse	GGAAAGAATCCCAGAGG
2nd step	PCR2 A.1	Forward A.1	GTTGTAAAACGACGGCCAGTTTCAAGCCTCCAAGCTGT	1863-2317	95 ºC 2 min; (95 ºC 20 s, 58 ºC 20 s, 72 ºC 15s ) × 35 cycles; 72 ºC 3 min
		Reverse A.1	CACAGGAAACAGCTATGACCGATAGGGGCATTTGGTGGTCT
	PCR2 A.2	Forward1 A.2	GTTGTAAAACGACGGCCAGTGGTTTCATATTTCTTGCC	2205-2483	95 ºC 2 min; (95 ºC 20 s, 50 ºC 20 s, 72 ºC 15 s) × 35 cycles; 72 ºC 3 min
		Forward2 A.2	GTTGTAAAACGACGGCCAGTGGTTTCACATTTCCTGTC
		Forward3 A.2	GTTGTAAAACGACGGCCAGTGGTTTCACATTTCTTGTC
		Forward4 A.2	GTTGTAAAACGACGGCCAGTGGTTTCACATTTCCTGCC
		Reverse A.2	CACAGGAAACAGCTATGACCTCCCACCTTATGAGTCCAAG
3rd step	PCR3	Forward (specific per sample)	GTTGTAAAACGACGGCCAGT+specific 10 nt MID		95 ºC 2 min; (95 ºC 20 s, 60 ºC 20 s, 72 ºC 15 s) × 20 cycles; 72 ºC 3 min
		Reverse (specific per sample)	CACAGGAAACAGCTATGACC+specific 10 nt MID

Bold nucleotides indicate the M13 sequence. Forward primers in PCR2-A2 were multiplexed at the same concentration to cover all HBV genotypes. The protocols of amplification are reported. A.1: Amplicon 1; A.2: Amplicon 2; PCR: Polymerase chain reaction; MID: Multiplex identifier.