Locked nucleic acid real-time polymerase chain reaction method identifying two polymorphisms of hepatitis B virus genotype C2 infections, rt269L and rt269I

Table 1 Primers and locked nucleic acid probes developed for the identification of the hepatitis B virus L/I/L2 variants by multiprobe locked nucleic acid real-time polymerase chain reaction

Primer/probe	Sequence (5’ to 3’)1	Tm (°C)2	Target	CH
Primers (product: 128 bp)
Forward	ATGGGATATGTAATTGGAAGtTGGGG	65-67	HBV P gene
Reverse	CCCACAATTCttTGACATACTTTCCAATCAATAGG	67-69	HBV P gene
LNA Probes
L_CTC	5’ 6-FAM-AAA+C+T+CAAR+CA+ATGT - 3’ IABkFQ	61-64	L (WT)	FAM
I_ATC	5’ HEX-AAA+A+T+CAAR+CAA+T+GT - 3’ IABkFQ	61-64	I	HEX
L2_CTA	5’ CY5-AAA+C+T+AAAR+CAA+T+GT - 3’ IABkFQ	60-63	L2	CY5

¹Locked nucleic acid nucleotides are written +A, +C, +T or +G.

²Primer T_m was calculated by using LC PDS software version 2.0, and probe T_m was calculated by https://www.exiqon.com/ls/pages/exiqontmpredictiontool.aspx.

T_m: Melting temperature; CH: Channel; HBV: Hepatitis B virus; WT: Wild type; LNA: Locked nucleic acid.

Table 2 Measurement of melting temperatures of the L/I/L2 variants by multiprobe locked nucleic acid real-time polymerase chain reaction

Genotype of positive control DNA [copies, (4.0E + 00)-(4.0E + 08)]	Target sequence1	Measured Tm (°C) in channel2
		Min3	Max3	mean ± SD (detection)3	Min4	Max4		mean ± SD (detection)4	Min5	Max5	mean ± SD (detection) 5
L (n = 34)	AAACTCAAGCAATGTT	61.6	63.1	62.4 ± 0.4 (34, 100%)	-	-		- (0, 0%)	55.2	56.2	55.8 ± 0.2 (26, 76.4%)
L’ (n = 34)	AAACTCAAACAATGTT	57.8	58.7	58.0 ± 0.2 (34, 100%)	-		-	- (0, 0%)	-	-	- (0, 0%)
I (n = 42)	AAAATCAAGCAATGTT	-	-	- (0, 0%)	59.0		61.6	60.2 ± 0.7 (42, 100%)	-	-	- (0, 0%)
I’ (n = 34)	AAAATCAAACAATGTT	-	-	- (0, 0%)	56.3		57.1	56.6 ± 0.2 (34, 100%)	-	-	- (0, 0%)
L2 (n = 34)	AAACTAAAGCAATGTT	-	-	- (0, 0%)	-		-	- (0, 0%)	64.4	64.8	64.6 ± 0.1 (26, 100%)
L2’ (n = 18)	AAACTAAAACAATGTT	-	-	- (0, 0%)	-	-		- (0, 0%)	60.9	61.4	61.2 ± 0.2 (18, 100%)

¹Bold, target codon; underline, A/G polymorphism.

²Bold, target specific melting temperature.

³FAM.

⁴Hex.

⁵Cy5.

Bold words represent genotype-specific T_ms. T_m: Melting temperature; -: No significant melting temperature; SD: Standard deviation.

Table 3 Rates of positive detection of the hepatitis B virus L/I/L2 variants in a total of 94 clinical samples by locked nucleic acid real-time polymerase chain reaction

Type of detection	No. of samples	Percentage
Clinical samples	94	100
Single	63	67.0
L	55	58.5
I	8	8.5
Mixed	24	24.5
L + I (1:1)	7	7.4
L dominant	13	4.3
I dominant	4	12.8
Unidentified	7	7.4
Inconsistent with direct sequencing	1	1.1

Table 4 Samples which cannot be identified by locked nucleic acid real-time polymerase chain reaction assay

No.	Patients	Direct sequencing (AAACTCAARCAATGT)	Type	LNA-RT-PCR
1	SNU3 30 HCC	AAAATCAAGCACTGT	I	Not detected
2	SNU3 70 HCC	AAAATTAAGCAATGT	I	Not detected
3	SNU3 82 CH	AAAATCAAACTATGT	I	Not detected
4	SNU3 123 HCC	AAACTTAAGCAATGT	L	Not detected
5	SNU3 31 CH	AAAATCCAGCAATGT	I	Not detected
6	SNU3 355 LC	AAAATTAAGCAATG	I	Not detected
7	SNU3 388 LC	AAACTTAAGCAATGT	L	Not detected

Bases in bold indicate the different ones from the target probe sequence. LNA-RT-PCR: Locked nucleic acid real-time polymerase chain reaction; HCC: Hepatocellular carcinoma; CH: Chronic hepatitis; LC: Liver cirrhosis.