TABLE I. Diagnostic, confirmatory and genotyping-polymerase chain reaction (PCR) assays for the molecular analysis of hepatitis B virus (HBV) genome.
A: diagnostic assay
| PCR primers | Primer sequence | Nucleotide position (HBV) | Fragment size (bp) | Tm (C) |
|---|---|---|---|---|
| S gene | ||||
| First round | ||||
| DS-7 (sense) | 5’-TCCTGCTGGTGGCTCCAGTT-3’ | 55-74 | 418 | 60 |
| DS-8 (antisense) | 5’-CAAACGGGCAACATACCTTG-3’ | 474-455 | ||
| Nested | ||||
| MS-1 (sense) | 5’-GGACCCCTGCTCGTGTTACA-3’ | 182-201 | 232 | 60 |
| MS2 (antisense) | 5’-CAGGATGAAGAGGAA(T/G)ATGA-3’ | 415-396 | ||
|
| ||||
| final reaction volume: 25 μL. PCR buffer: 1X MgCl2, 2 mM, dNTPs, 75 μM, Taq DNA polymerase 1 IU (Invitrogen™, Life Technologies™). Primers: 0.5 μM each, 5 μL of DNA sample. PCR cycle: 94ºC 3 min, 94ºC 30 s, 60ºC 30 s, 72ºC 30 s, 72ºC 5 min. Forty and 25 cycles, first and nested PCR, respectively. Analytical sensitivity: 1-10 copies HBV DNA (Sanchez et al. 2002). Tm: melting temperature. | ||||
| B: confirmatory-PCR assay | ||||
|
| ||||
| PCR primers | Primer sequence | Nucleotide position (HBV) | Fragment size (bp) | Tm (C) |
|
| ||||
| S gene | ||||
| First round | ||||
| S1-sense | 5’-CATCAGGATTCCTAGGACCCCT-3’ | 168-189 | 290 | 55 |
| S3-antisense | 5’-AGGACAAACGGGCAACATAC-3’ | 478-458 | ||
| Nested | ||||
| S2-sense | 5’-CTTGTTGACAAGAATCCTCACA-3’ | 214-235 | 228 | 55 |
| S4-antisense | 5’-CCAACAAGAAGATGAGGCATA-3’ | 442-420 | ||
| C gene | ||||
| First round | ||||
| C1-sense | 5’-TCACCTCTGCCTAATCATC-3’ | 1825-1843 | 566 | 55 |
| C3-antisense | 5’-GAGGGAGTTCTTCTTCTAGG-3’ | 2391-2371 | ||
| Nested | ||||
| C2-sense | 5’-TTCAAGCCTCCAAGCTGTGCC-3’ | 1862-1882 | 415 | 68 |
| C4-antisense | 5’-AGGAGTGCGAATCCACACTCC-3’ | 2277-2267 | ||
| P gene | ||||
| First round | ||||
| P1-sense | 5’-CGTCGCAGAAGATCTCAATC-3’ | 2420-2439 | 425 | 60 |
| P3-antisense | 5’-TCTTGTTCCCAAGAATATGGT-3’ | 2845-2825 | ||
| Nested | ||||
| P2-sense | 5´-CCTTGGACTCATAAGGT-3’ | 2463-2479 | 376 | 55 |
| P4-antisense | 5’-TCCCAAGAATATGGTGACCC-3’ | 2839-2820 | ||
| X gene | ||||
| First round | ||||
| X1-sense | 5’-CGCCAACTTACAAGGCCTTTC-3’ | 1100-1120 | 528 | 60 |
| X3-antisense | 5’-GGCGTTCACGGTGGTCTCCAT-3’ | 1628-1608 | ||
| Nested | ||||
| X2-sense | 5’-CCATACTGCGGAACTCCTAG-3’ | 1266-1685 | 274 | 55 |
| X4-antisense | 5’-CGTAAAGAGAGGTGCGCCCC-3’ | 1540-1521 | ||
|
| ||||
| final reaction volume 50 μL. PCR buffer: 1X MgCl2, 2 mM, dNTPs, 75 μM, Taq DNA polymerase 1 IU (Invitrogen™, Life Technologies™). Primers: 0.5 μM each, 5 μL of DNA sample. PCR cycle: 94ºC 3 min, 94ºC 30 s, 30 s, 72ºC 30 s, 72ºC 5 min. Forty and 25 cycles, first and nested PCR, respectively. The analytical sensitivity of OBI primer sets: 1-10 copies HBV-DNA. Tm: melting temperature. | ||||
| C: HVB genotype H-specific PCR-assay | ||||
|
| ||||
| PCR primers | Primer sequence | Nucleotide position (HBV) | Fragment size (bp) | Tm (C) |
|
| ||||
| S gene | ||||
| H1-sense | 5’-CTACAGCATGGGAGCACCTCTCTC(A/C)ACGGC-3´ | 2844-2873 | 279 | 60 |
| H2-antisense | 5’-GTGGATC(G/T)GGTGGCGAGGTTGTCAGAATGC-3’ | 3123-3098 | ||
|
| ||||
| final reaction volume 25 μL. PCR buffer: 1X MgCl2, 1.5 mM, dNTPs, 200 μM, Hot Star Taq polymerase 2.5 IU (Qiagen). Primers: 0.2 μM each, 5 μL of DNA sample. PCR cycle: 95ºC 15 min, 94ºC 1 min, 60ºC 1 min, 72ºC 2 min. Single PCR, 35 cycles. Analytical sensitivity: 1-10 copies HBV-DNA. Tm: melting temperature. | ||||