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©The Author(s) 2023.
World J Gastroenterol. Mar 21, 2023; 29(11): 1721-1734
Published online Mar 21, 2023. doi: 10.3748/wjg.v29.i11.1721
Published online Mar 21, 2023. doi: 10.3748/wjg.v29.i11.1721
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 |
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%) |
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 |
- Citation: Kim K, Choi YM, Kim DH, Jang J, Choe WH, Kim BJ. Locked nucleic acid real-time polymerase chain reaction method identifying two polymorphisms of hepatitis B virus genotype C2 infections, rt269L and rt269I. World J Gastroenterol 2023; 29(11): 1721-1734
- URL: https://www.wjgnet.com/1007-9327/full/v29/i11/1721.htm
- DOI: https://dx.doi.org/10.3748/wjg.v29.i11.1721